Semi-quantitative RT-PCR Assay Research Articles

Abstract 814: Identification of PCDHB3 as a potential tumor suppressor gene in colorectal cancer by exome sequencing.

Abstract Colorectal cancer (CRC) is one of the most common cancers in worldwide. Previous studies have identified hundreds of recurrent mutations in CRC. To gain a comprehensive view of the genetic alterations underlying colorectal tumorigenesis, we sequenced the exome in colorectal cancer from 10 patients without chemo-/radio- therapy histories. In total, we identified 378 novel somatic alterations, in which 341 missense, 41 small indels, and 7 splice-site mutations, 9 genes with more than two novel alterations were discovered in the list. Sequenom MassARRAY validation reached a positive rate of 85%. Two PCDHB family members which totally mutated in 4 sites attracted our attentions. We focused on PCDHB3, which had three missense mutations in 3 individuals and has never been investigated before. We re-sequenced another 104 cases of CRC genomic DNA through PCR arrays focused on the PCDHB3 exon and reached a final mutation rate of 7.02%. A semi-quantitative RT-PCR assay was performed to compare the PCDHB3 mRNA levels among 98 pairs of CRC tissues. A statistical significant decrease of PCDHB3 level was observed in tumor tissues compared with normal intestinal epithelium mucosa (P<0.001). IHC results showed that PCDHB3 expressed less and weaker in tumor. Furthermore, reduced PCDHB3 expression is associated with poor clinical outcome (overall survival: P=0.028; progression free survival: P=0.004) in advanced CRC patients with stage III and IV. It indicates that tumor tissues with higher and stronger expression of PCDHB3 are less aggressive in inducing metastasis and leads to longer survival of patients. In vitro studies, over-expressed PCDHB3 in DLD1, a CRC cell line, showed a decreased number of cells capable of passing through the filter in a transwell migration assay, whereas the SW480 cell line with decreased PCDHB3 passed through more compared to its control. In the MTT assays, HT29/DLD1 cells with higher PCDHB3 levels showed a lower ratio of proliferative performance compared to their negative controls; and SW480 with less PCDHB3 had an advantage over its control. In vivo assays of nude mice confirmed that with higher expression of wild-type PCDHB3, tumor developed significantly smaller than the control group, as well as the mutant-type group. It comes to a conclusion that PCDHB3 may function as a potential tumor suppressor gene in CRC, and we made a hypothesis that there are two mechanisms underlying PCDHB3-related CRC tumorigenesis: by mutations affecting amino acid sequence or by down-regulated expression of amino acid. In summary, our study provides a catalog of genetic alterations in colorectal cancer from 10 patients. The discovery of PCDHB3 as a potential tumor suppressor gene and the elucidation of mutated genes involved in pathways known to be relevant with colorectal cancer has broadened our understanding of molecular basis in colorectal tumorigenesis. Citation Format: Wen Ye, Jiangxue Wu, Ranyi Liu, Wenlin Huang. Identification of PCDHB3 as a potential tumor suppressor gene in colorectal cancer by exome sequencing. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 814. doi:10.1158/1538-7445.AM2013-814

Vacuolar invertases in potato (Solanum tuberosum L.): molecular cloning, characterization, sequence comparison, and analysis of gene expression in the cultivars

In plants, vacuolar invertase (β-fructofuranosidase, EC 3.2.1.26) is known to play as a key modulator for hexose accumulation and cell expansion. In this study, two cDNA clones (2,013 and 1,945 bp, with 99 % sequence identity) encoding vacuolar invertase isoforms were isolated from a commercially important Indian potato cultivar, Kufri Chipsona-1 by RT-PCR. The corresponding predicted proteins consisted of 635 amino acids (designated as KC-VIN1, lacking a few amino acids at N-terminus) and 639 amino acids (designated as KC-VIN2), respectively. They showed 99 % identity, and found to vary at several locations with mostly non-conservative substitutions. Multiple sequence alignment of vacuolar invertase homologs covering four Solanaceae family members revealed some notable distinguishing sequence features (signature-type sequences). A consensus sequence was predicted using 45 vacuolar invertase sequences from 27 taxonomically different plant species, and a phylogenetic tree was generated to know the evolutionary relation between them. Hydrophobic characters were predicted, and compared in different plant species. All these data are presented in a comprehensive manner which were not documented in the earlier reports. As a preliminary study, vacuolar invertase expression patterns in the tubers of some Indian potato cultivars were analyzed by semi-quantitative RT-PCR and extractable enzyme assay. In all the potato cultivars, the overall expression level of invertase was found to be considerably higher after storage at low temperature as compared to the freshly harvested tubers.

In silico Expression Profile of Genes in Response to Drought in Peanut

Drought, one of the most important abiotic stresses, usually causes adverse effects on the productivity and quality of crops. In this study, a drought-resistant variety Fenghua 5 was used to analyse leaf cDNA library of peanut treated with 15% PEG by Solexa high-throughput technology, and detect the differentially expressed genes under drought stress. The results of Solexa sequencing indicated the gene expression in peanut transcriptome presented strong nonhomogeneity and redundancy. The se- quenced tags less than 10 copies accounted for 73.1% of the total tag types, however its expression level only accounted for 9.0% of the total. A total of 935 differentially expressed genes were screened out based on the reference tags, of which 64.5% were down-regulated. These differentially expressed genes were involved in metabolisms of carbohydrate, protein, nucleic acid, lipid and energy, and secondary metabolism. Gene expression analysis of peanut also showed that nine transcripts related to flavonoid metabolism significantly up-regulated under drought stress, including four encoding flavonoid biosynthesis enzymes, three en- coding methyltransferase and two encoding MYB transcription factor. The gene expression analysis using semi-quantitative RT-PCR assays indicated that AhPAL was induced significantly by 15% PEG treatment for 6 h. The result showed that flavonoid

A Small Molecule Swertisin fromEnicostemma littoraleDifferentiates NIH3T3 Cells into Islet-Like Clusters and Restores Normoglycemia upon Transplantation in Diabetic Balb/c Mice

Aim. Stem cell therapy is one of the upcoming therapies for the treatment of diabetes. Discovery of potent differentiating agents is a prerequisite for increasing islet mass. The present study is an attempt to screen the potential of novel small biomolecules for their differentiating property into pancreatic islet cells using NIH3T3, as representative of extra pancreatic stem cells/progenitors. Methods. To identify new agents that stimulate islet differentiation, we screened various compounds isolated from Enicostemma littorale using NIH3T3 cells and morphological changes were observed. Characterization was performed by semiquantitative RT-PCR, Q-PCR, immunocytochemistry, immunoblotting, and insulin secretion assay for functional response in newly generated islet-like cell clusters (ILCC). Reversal of hyperglycemia was monitored after transplanting ILCC in STZ-induced diabetic mice. Results. Among various compounds tested, swertisin, an isolated flavonoid, was the most effective in differentiating NIH3T3 into endocrine cells. Swertisin efficiently changed the morphology of NIH3T3 cells from fibroblastic to round aggregate cell cluster in huge numbers. Dithizone (DTZ) stain primarily confirmed differentiation and gene expression studies signified rapid onset of differentiation signaling cascade in swertisin-induced ILCC. Molecular imaging and immunoblotting further confirmed presence of islet specific proteins. Moreover, glucose induced insulin release (in vitro) and decreased fasting blood glucose (FBG) (in vivo) in transplanted diabetic BALB/c mice depicted functional maturity of ILCC. Insulin and glucagon expression in excised islet grafts illustrated survival and functional integrity. Conclusions. Rapid induction for islet differentiation by swertisin, a novel herbal biomolecule, provides low cost and readily available differentiating agent that can be translated as a therapeutic tool for effective treatment in diabetes.

Construction and analysis of an SSH cDNA library of early heat-induced genes ofVigna aconitifoliavariety RMO-40

Moth bean ( Vigna aconitifolia (Jacq.) Marechal) is an important grain legume crop grown in rain fed areas of hot desert regions of Thar, India, under scorching sun rays with very little supplementation of water. An SSH cDNA library was generated from leaf tissues of V. aconitifolia var. RMO-40 exposed to an elevated temperature of 42°C for 5min to identify early-induced genes. A total of 488 unigenes (114 contigs and 374 singletons) were derived by cluster assembly and sequence alignment of 738 ESTs; out of 206 ESTs (28%) of unknown proteins, 160 ESTs (14%) were found to be novel to moth bean. Only 578 ESTs (78%) showed significant BLASTX similarity (<1 × 10(-6)) in the NCBI non-redundant database. Gene ontology functional classification terms were retrieved for 479 (65%) sequences, and 339 sequences were annotated with 165 EC codes and mapped to 68 different KEGG pathways. Four hundred and fifty-two ESTs were further annotated with InterProScan (IPS), and no IPS was assigned to 153 ESTs. In addition, the expression level of 27 ESTs in response to heat stress was evaluated through semiquantitative RT-PCR assay. Approximately 20 different signaling genes and 16 different transcription factors have been shown to be associated with heat stress in moth bean for the first time.

Renal tubular epithelial cell injury, apoptosis and inflammation are involved in melamine-related kidney stone formation

The objective of this study is to understand pathogenesis of melamine-related kidney stone formation. We investigated the characterization of renal tubular cell under exposure to a mixture of melamine and cyanuric acid in vivo. Male Sprague-Dawley rats were separated into two experimental groups. Treatment group was administered daily with a standard commercial diet mixing with melamine and cyanuric acid, and control group was given a normal diet. Rat kidney specimens were stained with hematoxylin/eosin and the crystals were examined using a polarizing microscope. Renal tubular epithelial cells were observed by transmission electron microscopy. Semiquantitative RT-PCR assay was performed to determine monocyte chemoattractant protein-1 (MCP-1) mRNA expression, a protein in response to various proinflammatory stimuli. Apoptotic cells were examined by TUNEL assay. Melamine-associated crystals formed in glomerulus and wide renal tubule segment including proximal convoluted renal tubules, distal convoluted renal tubules, the limb loops of Henle and medullary collecting ducts in the cortex and medulla. Light microscopy results showed that the crystals lead to tubular lumen dilatation and tubular epithelial cell necrosis. It was observed that nucleus of renal tubular epithelial cells became irregular outlines and condensed, lysosomal-related structures increased, and integrity of renal tubule was deficient under electron microscopy. Apoptotic cells were noted widely in cortex and medulla. MCP-1 mRNA expression was significantly increased in the melamine and cyanuric acid-administrated group. Renal tubular epithelial cell injury, apoptosis and inflammation are involved in melamine-related kidney stone formation. Our findings are important for understanding pathogenesis of melamine-related kidney stone formation and estimating its clinical prognosis.

Pro-Inflammatory Cytokine Responses of A549 Epithelial Cells to Antimicrobial Peptide Brevinin-2R

Brevinin-2R is an antimicrobial peptide which has been isolated from the skin of the frog Rana ridibunda. The purpose of the present study was to examine the cellular cytotoxicity and inflammatory effects of brevinin-2R (B2R) on human lung epithelial adenocarcinoma cell line A549. The effects of different concentrations (5, 10, and 20 μg/ml) of B2R on the expression levels of pro-inflammatory cytokines such as IL-1β, and IL-8 in A549 cells were evaluated by semi-quantitative RT-PCR and real-time PCR assays in a dose- and time-dependent manner. Based on the results of MTT assay, B2R showed a moderate cytotoxicity effect in a dose-dependent manner up to 20 % suppression of the cell growth. Moreover, gene expression results demonstrated that B2R up-regulates the IL-1β and IL-8 expression levels in A549 cells in a dose- and time-dependent manner. Our results suggested that brevinin-2R antimicrobial peptide has potentially a regulatory effect on triggering the inflammatory processes.

Cellular and biochemical characterization of two closely related triosephosphate isomerases fromTrichomonas vaginalis

The glycolytic enzyme triosephosphate isomerase catalyses the isomerization between glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Here we report that Trichomonas vaginalis contains 2 fully functional tpi genes. Both genes are located in separated chromosomal context with different promoter regulatory elements and encode ORFs of 254 amino acids; the only differences between them are the character of 4 amino acids located in α-helices 1, 2 and 8. Semi-quantitative RT-PCR assays showed that tpi2 transcript is approximately 3·3-fold more abundant than tpi1. Using an anti-TvTIM2 polyclonal antibody it was demonstrated that TIM proteins have a cytoplasmic localization and both enzymes are able to complement an Escherichia coli strain carrying a deletion of its endogenous tpi gene. Both TIM proteins assemble as dimers and their secondary structure assessment is essentially identical to TIM from Saccharomyces cerevisiae. The kinetic catalytic constants of the recombinant enzymes using glyceraldehyde-3-phosphate as substrate are similar to the catalytic constants of TIMs from other organisms including parasitic protozoa. As T. vaginalis depends on glycolysis for ATP production, we speculate 2 possible reasons to maintain a duplicated tpi copy on its genome: an increase in gene dosage or an early event of neofunctionalization of TIM as a moonlighting protein.

Expression and role of the inhibitor of apoptosis protein livin in chemotherapy sensitivity of ovarian carcinoma

Ovarian cancer has the highest mortality rate of all gynecological malignancies. Livin, a novel member of the inhibitor of apoptosis protein family, has been found to be expressed in various malignancies and is suggested to have poor prognostic significance. However, no data are available concerning the significance of livin in ovarian cancer. In the present study, we detected the expression of livin isoforms in human epithelial ovarian cancer (EOC) tissues using semi-quantitative RT-PCR and western blot assays. The data indicated that livin expression positive ratio was much higher in cancer tissues compared to those in benign ovarian tumors and normal ovarian specimens. To determine the role of livin in the process of ovarian cancer growth, RNA interference mediated by recombinant lentivirus vectors expressing livin shRNA was applied to induce a long-lasting downregulation of livin gene expression in SKOV3 human ovarian cancer cell line. Cell apoptosis and chemosensitivity were evaluated by MTT assay and flow cytometry, respectively, following downregulation of livin expression, and the cleavage of molecular markers of the mitochondrial apoptotic signaling pathway was investigated by immunoblotting. Livin knockdown with siRNA enhanced spontaneous and drug-induced apoptosis in SKOV3 cells. The inhibition of livin resulted in profound pro-apoptotic and antiproliferative effects, and was associated with the activation of caspase signaling. In conclusion, these data suggested that livin plays an important role in inhibiting the apoptosis of ovarian cancer cells. Specific silencing of livin expression could promote cell apoptosis, enhance chemotherapy sensitivity and may be a promising target for further research in clinical chemotherapy of epitheliod ovarian cancer.

Influence of Culture Medium Composition on Relative mRNA Abundances in Domestic Cat Embryos

Different culture conditions have been used to produce domestic cat embryos. As part of the in vitro procedures, the medium composition significantly affects the quality of the embryo development also. Quality assessments based on cleavage kinetics and blastomere symmetry are useful, but embryos also can differ in their relative gene expression patterns despite similar morphological characteristics. The aim of this study was to compare cat embryos produced with two different in vitro culture systems routinely used in two different laboratories [Smithsonian Conservation Biology Institute, Washington D.C., USA (SCBI) and Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany (IZW)]. Specifically, relative mRNA expression patterns of critical genes for pre-implantation embryo development were assessed in both conditions. Embryos were produced in parallel in both culture systems by IVF using frozen-thawed ejaculated semen in the United States and fresh epididymal sperm in Germany. Success of embryo development in vitro was recorded as well as relative mRNA abundances [DNA methyltransferases 1 and 3A (DNMT1, DNMT3A), gap junction protein alpha 1 (GJA1), octamer-binding transcription factor 4 [OCT4], insulin-like growth factors 1 and 2 receptors (IGF1R, IGF2R), beta-actin (ACTB)] in pools of days 4-5 morulae by semi-quantitative RT-PCR assay. Percentages of cleaved embryos were similar (p > 0.05) between both culture systems, regardless of the location. OCT4 mRNA abundance was higher (p < 0.05) in embryos derived in the SCBI culture system compared with those from the IZW system when epididymal sperm was used for IVF. No clear correlation between the expression pattern and the culture system could be found for all other genes. It is suggested that OCT4 expression might be affected by the media composition in some conditions and can be the indicator of a better embryo quality.

FOXM1 expression correlates with tumor invasion and a poor prognosis of colorectal cancer

FOXM1, a member of the Forkhead Box (Fox) family of transcription factors, plays a critical role in tumor development and metastasis. The aim of this study was to elucidate its role in colorectal cancer (CRC), particularly prognosis and metastasis. Semi-quantitative RT-PCR and Western blot assays were used to measure the expression levels of FOXM1 mRNA and protein in 15 CRC and adjacent normal mucosa tissues. Immunohistochemical assay was performed to detect FOXM1 protein expression in 112 CRC tissues and further determine its clinicopathological and prognostic significance. RNA interference (RNAi) was used to knockdown endogenous FOXM1 expression in CRC cell lines and to analyze the effects of FOXM1 knockdown on migration and invasion of CRC cells. The relative expression levels of FOXM1 mRNA and protein were significantly higher in CRC tissues than in adjacent normal mucosa tissues (P<0.01). In addition, the immunostaining of FOXM1 protein was stronger in CRC tissues than in adjacent normal mucosa tissues. By statistical analysis, we showed that high FOXM1 expression was closely correlated with the presence of lymph node metastasis, incidence of liver metastasis, and advanced TNM stage. Moreover, the cumulative 5-year survival rate of CRC patients with high FOXM1 expression was lower than that of those with low FOXM1 expression (P=0.0047). Multivariate analysis showed that the status of FOXM1 expression was an independent prognostic factor for CRC patients (P=0.025). Furthermore, RNAi-mediated FOXM1 knockdown could significantly inhibit growth, migration and invasion of CRC cells. Our results showed that FOXM1 over-expression is a molecular marker predicting increased invasive/metastatic potential of CRC and a poorer prognosis.

Molecular cloning and characterization of a novel RING zinc-finger protein gene up-regulated under in vitro salt stress in cassava

Cassava (Manihot esculenta Crantz) is one of the world's most important food crops. It is cultivated mainly in developing countries of tropics, since its root is a major source of calories for low-income people due to its high productivity and resistance to many abiotic and biotic factors. A previous study has identified a partial cDNA sequence coding for a putative RING zinc finger in cassava storage root. The RING zinc finger protein is a specialized type of zinc finger protein found in many organisms. Here, we isolated the full-length cDNA sequence coding for M. esculenta RZF (MeRZF) protein by a combination of 5' and 3' RACE assays. BLAST analysis showed that its deduced amino acid sequence has a high level of similarity to plant proteins of RZF family. MeRZF protein contains a signature sequence motif for a RING zinc finger at its C-terminal region. In addition, this protein showed a histidine residue at the fifth coordination site, likely belonging to the RING-H2 subgroup, as confirmed by our phylogenetic analysis. There is also a transmembrane domain in its N-terminal region. Finally, semi-quantitative RT-PCR assays showed that MeRZF expression is increased in detached leaves treated with sodium chloride. Here, we report the first evidence of a RING zinc finger gene of cassava showing potential role in response to salt stress.

Dangkwisoo-san, an herbal medicinal formula, ameliorates acute lung inflammation via activation of Nrf2 and suppression of NF-κB

Ethnopharmacological relevanceDangkwisoo-san (DS), an herbal medicinal formula, has long been used in Korea for the treatment of inflammatory complications caused by physical trauma. Although the therapeutic effect of DS is likely associated with anti-inflammatory activity, the precise underlying mechanisms are largely unknown. Here we sought to elucidate the possible mechanisms of anti-inflammatory activity of DS. Materials and methodsThe water extract of DS was orally fed to C57BL/6 mice for 14 days prior to LPS intranasal instillation for lung inflammation. The effects of DS on lung inflammation were determined by differential cell counting, lung histology, and semi-quantitative RT-PCR of lung sections. The effects of DS on the activities of Nrf2 and NF-κB were assessed by western blotting, semi-quantitative RT-PCR, and luciferase reporter assays in RAW 264.7, an NF-κB reporter cell line, and HEK 293 transfected with an NF-κB reporter construct. ResultsMice that were treated with a water extract of DS showed significant attenuation of lung inflammation induced by intranasal lipopolysaccharide (LPS) compared to control mice treated with vehicle. In vitro experiments show that DS activated Nrf2, an anti-oxidant transcription factor that protects from various inflammatory diseases, and induced Nrf2-regulated genes including GCLC, NQO-1 and HO-1. In addition, DS suppressed NF-κB activity and reduced the production of pro-inflammatory cytokines. Transfection experiment indicates that inhibition of NF-κB likely occurred upstream of IKK complex. Furthermore, DS enhanced the expression of HO-1 and suppressed that of IL-1β and TNF-α in inflamed mouse lungs. ConclusionsThese results suggest that the therapeutic effects of DS are related with suppression of inflammation, which is, at least in part, mediated by activation of anti-inflammatory factor Nrf2 and inhibition of pro-inflammatory factor NF-κB.

P4-02-05: The Regulation of Artemin Signalling by IGF-1 in Mammary Carcinoma Cells.

Abstract Artemin is a neurotrophic signalling factor which belongs to the glial-derived neurotrophic factor (GDNF) family of ligands. Artemin acts as a survival, proliferation and migration factor for a number of neurological cell types, by signalling through the RET (rearranged during transfection) receptor and, in most cases, the GDNF receptor (GFR)-a3 co-receptor. Recently, a number of published studies have implicated Artemin as a potential oncogene in several cell types, including mammary carcinoma cells. Other studies further indicate that Artemin may influence cancer progression and tamoxifen resistance in some breast cancers. Available clinical data has demonstrated that increased Artemin expression is correlated with decreased overall patient survival in breast cancer patients and a poor outcome in tamoxifen treated breast cancer patients. Here we investigate interaction between the Artemin and the insulin-like growth factor-1 (IGF-1) signal transduction pathways. Using mammary carcinoma cell lines, we demonstrate that IGF-1 treatment increases the endogenous expression of both Artemin and its endogenous receptors, RET and GFRa3. Semi-quantitative RT-PCR assays demonstrated that IGF-1 stimulated mRNA expression of Artemin as well as RET and GFRa3 in wild-type MCF-7 and ZR-75-1 cells in a time-dependent and dose-dependent manner. The same effect was not observed in wild-type T47D cells where IGF-1 did not increase Artemin mRNA expression. We also demonstrated that forced expression of Artemin in MCF-7 cells consistently enhanced the response of these cells to IGF-1 in a number of cell function assays. Forced expression of Artemin significantly enhanced IGF-1-mediated stimulation of total cell number in MCF-7 cells. Consistent with this, Artemin enhanced IGF-1-mediated stimulation of S-phase entry and cell survival. In a soft agar assay, forced expression of Artemin also enhanced IGF-1-mediated stimulation of colony formation. Conversely, depletion of Artemin expression using siRNA abrogated the response to IGF-1 stimulation in MCF-7 cells. Artemin depletion significantly decreased IGF-1-stimulated increase in total cell number by decreasing IGF-1-stimulated cell proliferation and protection from apoptotic cell death. In addition, forced expression of Artemin in MCF-7 cells reduced cell sensitivity to the IGF-1 receptor small molecule inhibitor, AG1024. In conclusion, we have demonstrated that IGF-1 increases Artemin mRNA and protein expression in the breast cancer cell lines MCF-7 and ZR-75-1 and have identified potential cross-talk between the Artemin and IGF-1 signalling pathways in MCF-7 cells. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-02-05.

Short-term response of wild grapevines (Vitis vinifera L. ssp. sylvestris) to NaCl salinity exposure: changes of some physiological and molecular characteristics

The physiological and molecular response to salt stress was studied in two wild grapevine (Vitis vinifera L. ssp. sylvestris or Vitis sylvestris) accessions “Khedhayria” and “Houamdia”, previously identified as salt-tolerant and salt-sensitive pair wise. Plants from both accessions were subjected to a progressive salt stress by the use of a nutritional solution containing up to 150 mM NaCl for 2 weeks. Salt stress adversely affected growth and water potential since the first day of exposure to 150 mM NaCl. However, chlorophyll fluorescence parameters were unchanged until 14 days of salt exposure. At that time point the predawn water potential (ΨPD), the non-photochemical quenching of fluorescence (NPQ) and the coefficient of photochemical quenching (qp) were significantly less altered in the tolerant accession. At the molecular level semi-quantitative RT-PCR assays revealed a differential expression of (Vs α-gal/SIP and Vs DHN) genes within these contrasting accessions after exposure to 24 h and 14 days of salt. Comparably, the Vs RD22 gene had increased slightly after only 14 days of treatment in both accessions. These results were the first pieces of information reported on the early and late regulation of salt response genes in wild grapevines. Furthermore, genotype-dependent parameters such as NPQ, qp, mRNA levels of Vs α-gal/SIP and Vs DHN could be used to screen salt-tolerant wild grapevine genotypes.

Overexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancer

Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P=0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326–3.557; P=0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G2/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback loop. Further, inhibition of polo-like kinase 1 could enhance the sensitivity of non-small cell lung cancer cells to taxanes or irradiation. Thus, polo-like kinase 1 might be a prognostic marker and a chemo- or radiotherapeutic target for non-small cell lung cancer.

Clinicopathological significance of Polo-like kinase 1 (PLK1) expression in human malignant glioma

Polo-like kinase 1 (PLK1), a variety of serine/threonine-protein kinase, has been reported to play important roles in malignant transformation. The purpose of this study was to investigate the clinicopathological significance of PLK1 expression in malignant glioma. A semi-quantitative RT-PCR assay was performed to detect the expression of PLK1 mRNA in 68 cases of glioma tissues and corresponding non-cancerous brain tissues. Additionally, the correlation of PLK1 mRNA expression with clinicopathological factors or prognosis of glioma patients was statistically analyzed. Multivariate analysis of prognostic factors was performed using the Cox proportional hazard model. Small interfering RNA was used to knockdown PLK1 expression in a glioma cell line and analyze the effects of PLK1 inhibition on growth, cell cycle, apoptosis and chemo- or radiosensitivity of glioma cells. Results showed that the expression of PLK1 mRNA was significantly higher in glioma tissues than in corresponding normal brain tissues. The expression of PLK1 mRNA was closely correlated with WHO grade, KPS and tumor recurrence of glioma patients (P=0.022, 0.030 and 0.041, respectively). Meanwhile, the disease-free and overall survival rates of patients with high PLK1 mRNA expression were obviously lower than those of patients with low PLK1 mRNA expression. Multivariate analysis showed that high PLK1 mRNA expression was a poor prognostic factor for glioma patients (P=0.028). The expression of PLK1 mRNA and protein was significantly down-regulated in stably transfected U251-S cells. PLK1 down-regulation could inhibit growth, induce cell arrest in G2/M phase of cell cycle and apoptosis enhancement in glioma cells. Further, PLK1 down-regulation could enhance the sensitivity of glioma cells to cisplatin or irradiation. Thus, the status of PLK1 mRNA expression might be an independent prognostic factor for glioma patients and targeting PLK1 could be a novel strategy for chemo- or radiosensitization of human malignant gliomas.

Up-Regulation of Lipoxygenase, Phospholipase, and Oxylipin-Production in the Induced Chemical Defense of the Red Alga Gracilaria chilensis against Epiphytes

The red alga Gracilaria chilensis is commercially farmed for the production of agar hydrocolloids, but some susceptible algae in farms suffer from intense epiphyte growth. We investigated the induced chemical defense response of G. chilensis against epiphytes and demonstrated that an extract of an epiphyte-challenged alga can trigger a defense response. The hormonally active metabolites were purified by RP-HPLC. Treatment with the extract or the purified fraction changed the chemical profile of the alga and increased resistance against epiphyte spores. Semi-quantitative RT-PCR and enzyme assays demonstrated that this metabolic response occurs after an increase in lipoxygenase and phospholipase A2 activity. Although this suggests the involvement of regulatory oxylipins, neither jasmonic acid nor the algal metabolite prostaglandin E2 triggers comparable defense responses.

Identification of sequences expressed during compatible black pepper—Fusarium solani f. sp. piperis interaction

Black pepper (Piper nigrum L.) is one of the most widely used spices in the world. Root rot disease is induced by Fusarium solani f. sp. piperis and causes severe yield losses of this crop in the Amazon region. In this work we used the suppression subtractive hybridization to identify differentially expressed sequences in roots of black pepper infected by F. solani f. sp. piperis. Sequences coding for putative proteins related to oxidative burst and defense response, such as superoxide dismutase, cytochrome p450, and alpha-amylase inhibitors/lipid transfer protein, comprised 28.4% of SSH clones according to computational analyses. Furthermore, semi-quantitative RT-PCR assays showed accumulation of putative cysteine proteinase inhibitor and pathogenesis-related protein 4 transcripts at late stage of infection that can help to explain the success of this pathogen in causing root rot disease in black pepper. The results obtained here contribute to improve our understanding about this plant–pathogen interaction.

Effects of Thai Medicinal Herb Extracts with Anti-Psoriatic Activity on the Expression on NF-κB Signaling Biomarkers in HaCaT Keratinocytes

Psoriasis is a chronic inflammatory skin disorder characterized by rapid proliferation of keratinocytes and incomplete keratinization. Discovery of safer and more effective anti-psoriatic drugs remains an area of active research at the present time. Using a HaCaT keratinocyte cell line as an in vitro model, we had previously found that ethanolic extracts from three Thai medicinal herbs, namely Alpinia galanga, Curcuma longa and Annona squamosa, possessed anti-psoriatic activity. In the current study, we aimed at investigating if these Thai medicinal herb extracts played a molecular role in suppressing psoriasis via regulation of NF-κB signaling biomarkers. Using semi-quantitative RT-PCR and report gene assays, we analyzed the effects of these potential herbal extracts on 10 different genes of the NF-κB signaling network in HaCaT cells. In accordance with our hypothesis, we found that the extract derived from Alpinia galanga significantly increased the expression of TNFAIP3 and significantly reduced the expression of CSF-1 and NF-κB2. Curcuma longa extract significantly decreased the expression of CSF-1, IL-8, NF-κB2, NF-κB1 and RelA, while Annona squamosa extract significantly lowered the expression of CD40 and NF-κB1. Therefore, this in vitro study suggested that these herbal extracts capable of functioning against psoriasis, might exert their activity by controlling the expression of NF-κB signaling biomarkers.