Topoisomer gel retardation: detection of anti-Z-DNA antibodies bound to Z-DNA within supercoiled DNA minicircles.

Small DNA fragments of approximately 350 bp in length, either with or without d(CG)n tracts, are ligated into underwound DNA minicircles to generate topoisomeric rings with different topological linking numbers, Lk. These minicircles, differing by an Lk of one, can be separated by acrylamide gel electrophoresis. Furthermore, electrophoresis can be used to reveal DNA double helix conformational changes that are induced by supercoiling, such as left-handed Z-DNA. When anti-Z-DNA antibodies are added to such minicircles, their binding leads to a selective retardation of the electrophoretic migration of the Z-DNA containing circles. This effect is not seen with relaxed minicircles and those with insufficient torsional stress to induce a conformational transition. Thus the technique of 'topoisomer gel retardation' presents a very sensitive assay for the identification of proteins that selectively bind to DNA conformations stabilized by negative DNA supercoiling.     

Alu-PCR: characterization of a chromosome 6-specific hybrid mapping panel and cloning of chromosome-specific markers.

The Alu-polymerase chain reaction (Alu-PCR) was applied to selectively amplify DNA sequences from human chromosome 6 using a single primer (A1) directed to the human Alu consensus sequence. A specific amplification pattern was demonstrated for a panel of eight somatic cell hybrids containing different portions of chromosome 6. This PCR pattern permits the identification of submicroscopic DNA alterations and can be utilized as a reference for additional chromosome 6-specific hybrids. To obtain new chromosome 6-specific markers we established two libraries from PCR-amplified sequences using two somatic cell hybrids (MCH381.2D and 640-5A). Out of a total of 109 clones that were found to be chromosome 6 specific, 13 clones were regionally assigned. We also included a procedure that allows the isolation of chromosome 6-specific markers from hybrids that contain human chromosomes other than 6. Our results will contribute to the molecular characterization of chromosome 6 by fostering characterization of somatic cell hybrids and by the generation of new regionally assigned DNA markers.     

Smoking alters thromboxane metabolism in man

Chronic smoking is a major risk factor of atherosclerosis and coronary heart disease. The measurement of three major thromboxane A2 metabolites, 11-dehydrothromboxane B2, 2,3-dinorthromboxane B2 and thromboxane B2, in the urines of 13 apparently healthy smokers (average 39 years, range 27-56 years) showed significantly elevated excretion rates for all thromboxane A2 metabolites as compared to 10 apparently healthy age-matched non-smokers (average 37 years, range 26-56 years). Importantly, characteristic alterations in the thromboxane A2 metabolite pattern were found in the urines of smokers. The contribution of 2,3-dinorthromboxane B2 to total measured excretion of thromboxane A2 metabolites was 59.2% in smokers (404.0 +/- 53.0 pg/mg creatinine) versus 19.4% in non-smokers (85.2 +/- 8.3 pg/mg creatinine), that of 11-dehydrothromboxane B2 35.7% in smokers (673.2 +/- 88.9 pg/mg creatinine) as compared to 75.5% in non-smokers (332.6 +/- 30.9 pg/mg creatinine). The contribution of thromboxane B2 (57.5 +/- 7.7 pg/mg creatinine in smokers versus 21.9 +/- 1.5 pg/mg creatinine in non-smokers) was similar at 5.1%. The excretion of cotinine, the major urinary metabolite of nicotine that correlates well with the reported daily cigarette consumption (r = 0.97, P less than 0.0001), showed a good correlation to thromboxane A2 metabolite excretion (2,3-dinorthromboxane B2: r = 0.92, P less than 0.0001; 11-dehydrothromboxane B2; r = 0.87, P less than 0.0001).     

Large-scale physical mapping within the region 22q12.3-13.1 in meningioma

The lack of physical mapping data strongly restricts the analysis of the meningioma chromosomal region that was assigned to the bands 22q12.3-qter. Recently, we reported a new marker D22S16 for chromosome 22 that was assigned to the region 22q13-qter by in situ hybridization. Utilizing somatic cell hybrids we now sublocalized the marker D22S16 within the band region 22q12-13.1, thus placing it in the vicinity of the gene for the platelet derived growth factor (PDGFB). A physical map was established for the regions surrounding the PDGFB gene and the D22S16 marker. By means of pulsed-field gel electrophoresis (PFGE) D22S16 and PDGFB were found to be physically linked within 900 kb. We also identified two CpG clusters bordering the PDGFB gene. For the enzyme NotI, a variation of the PDGFB restriction pattern was found between different individuals. PFGE analysis of the two loci (PDFGB and D22S16) failed to identify major rearrangements in meningioma.     

Curcumin Intake Affects miRNA Signature in Murine Melanoma with mmu-miR-205-5p Most Significantly Altered

Melanoma is the most aggressive form of skin cancer with estimated 48,000 deaths per year worldwide. The polyphenol curcumin derived from the plant Curcuma longa is well known for its anti-inflammatory and anti-cancerogenic properties. Accordingly, dietary intake of this compound may be suitable for melanoma prevention. However, how this compound affects basic cellular mechanisms in developing melanoma still remains elusive. Therefore, the aim of this study was to investigate for the first time the impact of oral curcumin administration on the miRNA signature of engrafting melanoma. For this purpose, the effects of a 4% curcumin diet were tested on melanoma, which were established by injection of murine B78H1 cells in the flank of C57BL/6 mice. Curcumin diet or standard chow (control) was administered two weeks prior to injection of tumor cells until termination of the experiment. High throughput chip-based array analysis was deployed to detect alterations in the miRNA signature of the tumors. Curcumin treatment significantly reduced the growth of the flank tumors. Furthermore the miRNA expression signature in tumors was substantially altered by curcumin intake with mmu-miR-205-5p over 100 times higher expressed when compared to controls. The expression levels of identified key miRNAs in the tumor samples were confirmed by quantitative real-time polymerase chain reaction (qRT-PCR). A comparable expression pattern of these miRNAs was also detected in other curcumin-treated melanoma cell lines under in vitro conditions. Putative targets of curcumin-induced up-regulated miRNAs were enriched in ‘o-glycan biosynthesis’, ‘endoplasmatic reticulum protein processing’ and different cancer-related pathways. Western Blot analyses revealed that of these targets anti-apoptotic B-cell CLL/lymphoma 2 (Bcl-2) and proliferating cell nuclear antigen (PCNA) were significantly down-regulated in curcumin-treated tumors. These findings demonstrate a profound alteration of the miRNA expression signature in engrafting curcumin-treated melanoma with mmu-miR-205-5p being up-regulated most significantly.     

EDISON-WMW: Exact Dynamic Programing Solution of the Wilcoxon–Mann–Whitney Test

In many research disciplines, hypothesis tests are applied to evaluate whether findings are statistically significant or could be explained by chance. The Wilcoxon–Mann–Whitney(WMW) test is among the most popular hypothesis tests in medicine and life science to analyze if two groups of samples are equally distributed. This nonparametric statistical homogeneity test is commonly applied in molecular diagnosis. Generally, the solution of the WMW test takes a high combinatorial effort for large sample cohorts containing a significant number of ties. Hence, P value is frequently approximated by a normal distribution. We developed EDISON-WMW, a new approach to calculate the exact permutation of the two-tailed unpaired WMW test without any corrections required and allowing for ties. The method relies on dynamic programing to solve the combinatorial problem of the WMW test efficiently. Beyond a straightforward implementation of the algorithm, we presented different optimization strategies and developed a parallel solution. Using our program,the exact P value for large cohorts containing more than 1000 samples with ties can be calculated within minutes. We demonstrate the performance of this novel approach on randomly-generated data, benchmark it against 13 other commonly-applied approaches and moreover evaluate molecular biomarkers for lung carcinoma and chronic obstructive pulmonary disease(COPD). We foundthat approximated P values were generally higher than the exact solution provided by EDISONWMW. Importantly, the algorithm can also be applied to high-throughput omics datasets, where hundreds or thousands of features are included. To provide easy access to the multi-threaded version of EDISON-WMW, a web-based solution of our algorithm is freely available at http://www.ccb.uni-saarland.de/software/wtest/.     

Expression of CaT-like, a novel calcium-selective channel, correlates with the malignancy of prostate cancer

The regulation of intracellular Ca(2+) plays a key role in the development and growth of cells. Here we report the cloning and functional expression of a highly calcium-selective channel localized on the human chromosome 7. The sequence of the new channel is structurally related to the gene product of the CaT1 protein cloned from rat duodenum and is therefore called CaT-like (CaT-L). CaT-L is expressed in locally advanced prostate cancer, metastatic and androgen-insensitive prostatic lesions but is undetectable in healthy prostate tissue and benign prostatic hyperplasia. Additionally, CaT-L is expressed in normal placenta, exocrine pancreas, and salivary glands. New markers with well defined biological function that correlate with aberrant cell growth are needed for the molecular staging of cancer and to predict the clinical outcome. The human CaT-L channel represents a marker for prostate cancer progression and may serve as a target for therapeutic strategies.     

Identification of a nuclear variant of MGEA5, a cytoplasmic hyaluronidase and a beta-N-acetylglucosaminidase

MGEA5 was originally identified to be a novel human hyaluronidase, which is immunogenic in meningioma patients. Recently an N-acetylglucosaminidase was reported with identical sequence. Here, we define the origin of a splice variant by determining the genomic organization of the mgea5 gene. We find the splice variant missing a putative acetyltransferase domain of MGEA5. As for evolutionary analysis, we show that the MGEA5 is highly conserved in higher eukaryotes. As for expression analysis, we find both mRNA variants ubiquitously expressed in various human tissues and throughout mouse development. We generated polyclonal antibodies against MGEA5s/5 and identified proteins of 75 and 130 kDa, indicating posttranslational modifications of the larger protein. Cell fractionation revealed the cytoplasmic/cytoskeletal localization of the 130-kDa protein and the nuclear localization of the 75-kDa protein. We propose a model in which MGEA5 functions both as a hyaluronidase and an N-acetylglucosaminidase.     

The MGEA6 multigene family has an active locus on 14q and at least nine pseudogenes on different chromosomes

The meningioma expressed antigen-6 (MGEA6) was originally identified as an immunogenic antigen in meningioma patients. Somatic hybrid panel mapping and fluorescence in situ hybridization revealed MGEA6-related sequences on different human chromosomes. Here we carry out database analysis to investigate the complexity of the MGEA6-related sequences and demonstrate the existence of a multigene family. We localized the active gene (spanning over 83 kb) to chromosome 14q and elucidated its exon/intron structure. We identified and characterized 9 processed pseudogenes on 9 different chromosomes including chromosomes 2, 3, 6, 7, 9, 10, 12, 13, and 18. We performed phylogenetic analysis and concluded that the MGEA6 pseudogenes may result from more than one retrotransposition event; we calculated divergence times of the pseudogenes to be between 21.5 and 28.9 million years ago.