Vasorelaxation Effect of Estrone Derivate EA204 in Rabbit Aorta.

Estrogen and its derivatives exert vascular protective effects, but the underlying mechanisms remain to be studied fully. Objective. To investigate the vasorelaxation effect and related mechanisms of an estrone derivate EA204[3-(2-piperidin-1-yl)-ethoxy-estra-1, 3, 5 (10)-trien-17-one] on isolated arterial preparation from rabbit thoracic aorta. Methods. Aortic rings from rabbit thoracic aorta were prepared and held in small organ bath filled with Krebs solution; tension change was recorded by a multichannel physiological signal collection and handling system. Results. EA204 (10(-5) to 10(-3) M) induced a concentration-dependent relaxation of aortic rings with endothelium and without endothelium. In denuded arterial preparations, EA204 had a potent relaxing effect on isolated arterial preparations contracted with phenylephrine, norepinephrine, and high-K(+) solution or BaCl2. Mechanism study indicates that EA204 relaxes aortic rings by inhibiting Ca(2+) channels (both receptor-operating Ca(2+) channels and the voltage-dependent Ca(2+) channels were involved) to decrease extracellular Ca(2+) influx and intracellular Ca(2+) release. EA204 is different from verapamil, which is a noncompetitive inhibitor of Ca(2+) channels. In addition, K(+) channels opening may contribute to this vasorelaxation effect. Conclusion. EA204 had a potent endothelium-independent relaxing effect on isolated arterial preparation by inhibiting Ca(2+) channels and opening K(+) channels. The results suggest that EA204 is a potential compound for treatment of cardiovascular diseases in postmenopausal women.

Resveratrol inhibits oligomeric Aß-induced microglial activation via NADPH oxidase.

Microglia­mediated neuroinflammation is key in the pathogenesis of Alzheimer's disease (AD). Several studies have suggested that NADPH oxidase contributes to microglia­mediated neuroinflammation. Resveratrol, which is a natural polyphenolic compound, exerts neuroprotective effects in AD due to its anti­inflammatory properties. The present study aimed to investigate the effects of resveratrol on the activation of oligomeric amyloid ß (oAß)­induced BV­2 microglia, and to determine the role of NADPH oxidase in these effects. Microglial proliferation was measured by high­content screening cell counting and using a bromodeoxyuridine incorporation assay. In addition, the levels of reactive oxygen species (ROS), nitric oxide (NO), tumor necrosis factor (TNF)­α and interleukin (IL)­1ß were assessed. The results of the present study demonstrated that resveratrol inhibited the proliferation of oAß­induced microglia and the production of pro­inflammatory factors, including ROS, NO, TNF­α and IL­1ß. Subsequent mechanistic investigations demonstrated that resveratrol inhibited the oAß­induced mRNA and protein expression levels of p47phox and gp91phox. These results suggested that NADPH oxidase may be a potential target for AD treatment, and resveratrol may be a valuable natural product possessing therapeutic potential against AD.

Iron-catalyzed aminolysis of ß-carbonyl 1,3-dithianes: synthesis of stereodefined ß-enaminones and 3,4-disubstituted pyrazoles.

A novel iron-catalyzed aminolysis of ß-carbonyl 1,3-dithianes with various amines including ammonia, primary and secondary amines, as well as hydrazine hydrate has been developed, leading to the synthesis of stereodefined ß-enaminones and 3,4-disubstituted pyrazoles in good to high yields.

A multicenter blinded study to evaluate KRAS mutation testing methodologies in the clinical setting.

Evidence that activating mutations of the KRAS oncogene abolish the response to anti-epidermal growth factor receptor therapy has revolutionized the treatment of advanced colorectal cancer. This has resulted in the urgent demand for KRAS mutation testing in the clinical setting to aid choice of therapy. The aim of this study was to evaluate six different KRAS mutation detection methodologies on two series of primary colorectal cancer samples. Two series of 80 frozen and 74 formalin-fixed paraffin-embedded tissue samples were sourced and DNA was extracted at a central site before distribution to seven different testing sites. KRAS mutations in codons 12 and 13 were assessed by using single strand conformation polymorphism analysis, pyrosequencing, high resolution melting analysis, dideoxy sequencing, or the commercially available TIB Molbiol (Berlin, Germany) or DxS Diagnostic Innovations (Manchester, UK) kits. In frozen tissue samples, concordance in KRAS status (defined as consensus in at least five assays) was observed in 66/80 (83%) cases. In paraffin tissue, concordance was 46/74 (63%) if all assays were considered or 71/74 (96%) using the five best performing assays. These results demonstrate that a variety of detection methodologies are suitable and provide comparable results for KRAS mutation analysis of clinical samples.

Terpenes from Eupatorium adenophorum and their allelopathic effects on Arabidopsis seeds germination (dagger).

The invasive plant Eupatorium adenophorum Spreng. (or Ageratina adenophora (Spreng.) King and Robinson) (Compositae) has caused great economic loss in China, especially the southwestern region, and is gravely threatening the native biodiversity. The aerial part of this plant was phytochemically investigated for its allelochemicals. Eleven terpenes (2 monoterpenes and 9 sesquiterpenes) were isolated and identified, which include a new monoterpene, (-)-(1R*,2S,*4R*,5S*)-3,3-dimethyl-5-hydroxybicyclo[2,2,1]hept-2-ylmethanol (1), two new cadinane sesquiterpenes, (-)-(5S*,6S*,7S*,9R*,10S*)-7-hydroxy-5,7-epidioxycadinan-3-ene-2-one (2) and (+)-(5S*,6R*,9R*,10S*)-5,6-dihydroxycadinan-3-ene-2,7-dione (3), and eight known terpene compounds (4, 6-12). The new structures were established by spectroscopic studies such as 1D- and 2D-NMR and MS analyses. Meanwhile, the potential allelopathic effects of these compounds on the Arabidopsis seeds germination were tested. Compounds 3 and 7 retarded the Arabidopsis seeds germination at 0.5 mM and 1.0 mM concentrations, respectively, while other compounds showed no obvious inhibitory effects.

BRAF mutation is associated with the CpG island methylator phenotype in colorectal cancer from young patients.

This study investigated the relationship between BRAF mutation, the CpG island methylator phenotype (CIMP+) and APC methylation in colorectal cancer (CRC) from young patients. The V600E BRAF mutation was found in 7% of cases and was strongly associated with the tumour features of proximal site, advanced stage and poor histological grade. More than half (53%) the tumours with BRAF mutation were also CIMP+ as evaluated by a standard panel of markers, compared to only 4% of tumours with wildtype BRAF (P<0.0001). In contrast to CIMP+, APC methylation was inversely correlated with BRAF mutation (P=0.02). BRAF mutation and CIMP+ are therefore likely to be involved in an alternate, albeit rare, pathway to APC inactivation during the development of CRC in younger patients.

Population-based detection of Lynch syndrome in young colorectal cancer patients using microsatellite instability as the initial test.

Approximately 1-2% of colorectal cancers (CRC) arise because of germline mutations in DNA mismatch repair genes, referred to as Lynch syndrome. These tumours show microsatellite instability (MSI) and loss of expression of mismatch repair proteins. Pre-symptomatic identification of mutation carriers has been demonstrated to improve survival; however, there is concern that many are not being identified using current practices. We evaluated population-based MSI screening of CRC in young patients as a means of ascertaining mutation carriers. CRC diagnosed in patients aged <60 years were identified from pathology records. No prior information was available on family history of cancer. PCR techniques were used to determine MSI in the BAT-26 mononucleotide repeat and mutation in the BRAF oncogene. Loss of MLH1, MSH2, MSH6 and PMS2 protein expression was evaluated in MSI+ tumours by immunohistochemistry. MSI+ tumours were found in 105/1,344 (7.8%) patients, of which 7 were excluded as possible Lynch syndrome because of BRAF mutation. Of the 98 "red flag" cases that were followed up, 25 were already known as mutation carriers or members of mutation carrier families. Germline test results were obtained for 35 patients and revealed that 22 showed no apparent mutation, 11 showed likely pathogenic mutations and 2 had unclassified variants. The proportion of MSI+ cases in different age groups that were estimated to be mutation carriers was 89% (<30 years), 83% (30-39), 68% (40-49) and 17% (50-59). We recommend MSI as the initial test for population-based screening of Lynch syndrome in younger CRC patients, regardless of family history.

[Dok6 promote neurite outgrowth of tropomyosin-related kinase C overexpressed PC12 cells in stimulation of neurotrophin-3].

OBJECTIVE: To study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells. METHODS: Series of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3. RESULTS: Each fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect. CONCLUSION: Dok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Detection of BRAF V600E mutation by pyrosequencing.

INTRODUCTION: Detection of the V600E hotspot mutation in BRAF oncogene is extremely useful for the screening of hereditary non-polyposis colorectal cancer (Lynch's syndrome) and for the prediction of sensitivity to MEK inhibitors. Here we describe a method for detecting this mutation based upon pyrosequencing technology. METHODS: The efficiency of pyrosequencing for detecting BRAF V600E mutations was compared with the conventional dideoxy sequencing method in 12 tumour cell lines and in 108 colorectal tumours. RESULTS: The results from pyrosequencing were 100% concordant with those from dideoxy sequencing. This method was capable of detecting BRAF V600E mutations at a much lower ratio of mutant to wild-type alleles (1:50) than dideoxy sequencing (1:5) while being considerably faster and less expensive. CONCLUSIONS: Pyrosequencing offers a specific, sensitive, rapid and cost-effective alternative to dideoxy sequencing for the detection of BRAF V600E mutations in clinical tumour specimens.

Detection of epidermal growth factor receptor variations by partially denaturing HPLC.

BACKGROUND: Epidermal growth factor receptor gene (EGFR) variants may be useful markers for identifying responders to gefitinib and erlotinib, small-molecule tyrosine kinase inhibitors of EGFR; therefore, sensitive and cost-effective assays are needed to detect EGFR variants in routine clinical samples. We have developed a partially denaturing HPLC (pDHPLC) assay that is superior to direct sequencing with respect to detection limits, costs, and time requirements. METHODS: Primers, temperatures, and buffer conditions were optimized for PCR-pDHPLC analysis of EGFR exons 18-21. We evaluated the detection limits of pDHPLC and direct sequencing by analyzing mixtures of wild-type and variant EGFR DNA and screened 192 lung cancer samples to examine the diversity of pDHPLC-detectable variants. To assess amenability to routine analysis, we tested lung and pleural tissue specimens from 14 lung cancer patients treated with gefitinib. RESULTS: The detection limits for variant alleles were 1:100 for pDHPLC and 1:5 for direct sequencing. pDHPLC analysis detected 26 unique EGFR variants, including the common deletions in exon 19 and substitutions in codons 787 and 858. Direct sequencing could not identify 30% (18 of 60) of the variant amplicons identified by pDHPLC. We identified these 18 amplicons by fraction collection after pDHPLC analysis. Analysis of a limited series of lung biopsy samples detected EGFR variants more frequently in gefitinib responders than in nonresponders. pDHPLC analysis was 56% less expensive and 39% faster than direct sequencing. CONCLUSIONS: pDHPLC-based analysis detects EGFR variations in routine clinical samples with a better detection limit and lower cost and time requirement than direct sequencing.

BRAF mutations are associated with distinctive clinical, pathological and molecular features of colorectal cancer independently of microsatellite instability status.

BACKGROUND: BRAF is a member of RAF family of serine/threonine kinases and mediates cellular responses to growth signals through the RAS-RAF-MAP kinase pathway. Activating mutations in BRAF have recently been found in about 10% of colorectal cancers, with the vast majority being a V600E hotspot mutation. The aim of the present study was to evaluate the clinical, pathological and molecular phenotype of colorectal tumors with BRAF mutations. RESULTS: Mutations in BRAF were identified in 8% (23/275) of colorectal cancers. They were 5-10-fold more frequent in tumors with infiltrating lymphocytes, location in the proximal colon, poor histological grade and mucinous appearance (P < 0.002 for each). Tumors with BRAF mutation were also 10-fold more likely to show microsatellite instability and frequent DNA methylation (P < 0.0001) compared to tumors without this mutation. The characteristic morphological features of tumors with BRAF mutation (infiltrating lymphocytes, poor grade, mucinous) remained after stratification according to microsatellite instability and methylator phenotypes. Mutations in BRAF were mutually exclusive with mutations in KRAS but showed no clear association with the presence of TP53 mutation. CONCLUSION: BRAF mutation identifies a colorectal cancer subgroup with distinctive phenotypic properties independent of microsatellite instability status and thus could be a valuable marker for studies into the clinical properties of these tumors.

Genetic polymorphisms in the MMP-2 and MMP-9 genes and breast cancer phenotype.

The matrix metalloproteinases (MMPs) have been shown to play important roles in cancer progression. In this study we examined whether common genetic variants in two key MMPs are associated with phenotypic features of breast cancers and patient outcome. A single nucleotide polymorphism in the promoter region of MMP-2 (-1306 C-->T) abolishes Sp1 binding and is associated with lower transcriptional activity, while another in the promoter region of MMP-9 (-1562 C-->T) increases the transcription of this gene. MMP-2 TT homozygous patients had smaller tumors (p=0.006) and contained lower concentrations of estrogen receptor (ER; p=0.002) compared to patients with the MMP-2 CC or CT genotype. Homozygosity for the MMP-2 -1306 T allele was associated with markedly different patient survival depending upon tumor ER status. For patients with ER negative tumors, the MMP-2 TT genotype was associated with poor survival (2/8 patients alive at end of study, 25%) compared to the CC or CT genotypes (59/70, 84%; p < 0.001). For patients with ER positive tumors, the MMP-2 TT genotype was associated with a trend for very good survival (10/10, 100%) compared to the CC or CT genotypes (130/157, 83%; p=0.16). The MMP-9 -1562 T allele was associated with features of good prognosis including non-ductal type histology, positive ER status and the absence of TP53 mutation. Patients with MMP-9 -1562 CT or TT genotypes showed marginally better prognosis compared to CC homozygotes (p=0.06). These findings suggest that breast cancer phenotype and outcome can be influenced by common functional polymorphisms in MMP genes.