Identification of New Differentially Methylated Genes That Have Potential Functional Consequences in Prostate Cancer

Many differentially methylated genes have been identified in prostate cancer (PCa), primarily using candidate gene-based assays. Recently, several global DNA methylation profiles have been reported in PCa, however, each of these has weaknesses in terms of ability to observe global DNA methylation alterations in PCa. We hypothesize that there remains unidentified aberrant DNA methylation in PCa, which may be identified using higher resolution assay methods. We used the newly developed Illumina HumanMethylation450 BeadChip in PCa (n = 19) and adjacent normal tissues (n = 4) and combined these with gene expression data for identifying new DNA methylation that may have functional consequences in PCa development and progression. We also confirmed our methylation results in an independent data set. Two aberrant DNA methylation genes were validated among an additional 56 PCa samples and 55 adjacent normal tissues. A total 28,735 CpG sites showed significant differences in DNA methylation (FDR adjusted P<0.05), defined as a mean methylation difference of at least 20% between PCa and normal samples. Furthermore, a total of 122 genes had more than one differentially methylated CpG site in their promoter region and a gene expression pattern that was inverse to the direction of change in DNA methylation (e.g. decreased expression with increased methylation, and vice-versa). Aberrant DNA methylation of two genes, AOX1 and SPON2, were confirmed via bisulfate sequencing, with most of the respective CpG sites showing significant differences between tumor samples and normal tissues. The AOX1 promoter region showed hypermethylation in 92.6% of 54 tested PCa samples in contrast to only three out of 53 tested normal tissues. This study used a new BeadChip combined with gene expression data in PCa to identify novel differentially methylated CpG sites located within genes. The newly identified differentially methylated genes may be used as biomarkers for PCa diagnosis.     

The disaccharide L-alpha-D-heptose1-->7-L-alpha-D-heptose1-->of the inner core domain of Salmonella lipopolysaccharide is accessible to antibody and is the epitope of a broadly reactive monoclonal antibody.

We generated a panel of mAb containing at least one specificity against each of the known chemotypes of the Salmonella LPS core domain and used them to investigate the accessibility of core determinants in smooth LPS. Most of the mAb were reactive with at the most three chemotypes of the core as determined by enzyme immunoassay and failed to bind smooth LPS or any of the complete cores of E. coli. One mAb, MASC1-MM3 (MM3), reacted with six different Salmonella core chemotypes, the R2 core of Escherichia coli and a variety of smooth LPS. This mAb reacted equally well with live and heat-killed bacteria. It bound to 123 of 126 clinical isolates of Salmonella and 11 of 73 E. coli strains in a dot-immunoblot assay. Typical ladder-like patterns of bands were observed after immunoblotting of this mAb against electrophoretically resolved smooth LPS from the five major serogroups of Salmonella species (A, B, C1, D1, and E). MM3 had no reactivity with BSA conjugates of O-Ag polysaccharides from the above serogroups confirming specificity for a core epitope. Polysaccharides derived from or synthetic saccharides representative of the various chemotypes of Salmonella LPS core were tested as competitive inhibitors of the binding of MM3 to LPS. The results led to a conclusion that MM3 recognizes the structure, L-alpha-D-Heptose1-->7-L-alpha-D-Heptose1-->disaccharide present as a branch in the Ra, Rb1, Rb2, Rb3 and Rc but lacking in the Rd1, Rd2, and Re chemotypes of the Salmonella LPS core. This disaccharide seems free and accessible on the basis of the previously calculated conformations of the Salmonella (Ra) and E. coli complete cores (R1, R2, R3, R4, and K12). It therefore defines or contains an epitope within the inner core subdomain of Salmonella LPS that is accessible to antibody in long-chained LPS and in intact bacteria with complete LPS.     

1-Methylguanine and 7-methylguanine increase cell agglutinability

Summary 1-Methylguanine and 7-methylguanine, both metabolic products of tRNA degradation, are known to induce transformation of Chinese hamster fibroblasts in culture. The effects of these compounds on the cell membrane have been studied by the method of Concanavalin A-mediated hemadsorption. 1-Methylguanine or 7-methylguanine induced a 50% increase of Con A-mediated hemadsorption within 20 hours of exposure of the cells to the agent at a concentration of 10^-5 M. This alteration was reversed within 13 days when the cells were grown in the control medium. Prolonged treatment with 1-methylguanine or 7-methylguanine resulted in changes which were only slowly reversed during growth of the cells in the control medium. The effect of the methylated purines on the cell membrane could be completely inhibited by simultaneous addition of dibutyryl-cAMP at a concentration of 10^-5 M. The possible mechanism of cell membrane alteration by methylated purines and its relevance to transformation in vitro are discussed.     

Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB

The Salmonella enterica virulence-associated protein SpvB was recently shown to contain a carboxy-terminal mono(ADP-ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full-length SpvB modifies a 43 kDa protein from macrophage-like J774-A.1 and epithelial MDCK cells as shown by label transfer from [32P]-nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two-dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325-infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]-NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB-mediated modification, most probably ADP-ribosylation, and that the modification of G actin interferes with actin polymerization.     

SHPS-1 induces aggregation of Ba/F3 pro-B cells via an interaction with CD47

SHPS-1 (SH2-domain bearing protein tyrosine phosphatase (SHP) substrate-1), a member of the inhibitory-receptor superfamily that is abundantly expressed in macrophages and neural tissue, appears to regulate intracellular signaling events downstream of receptor protein-tyrosine kinases and integrin-extracellular matrix molecule interactions. To investigate the function of SHPS-1 in a hematopoietic cell line, SHPS-1 was expressed in Ba/F3 cells, an IL-3-dependent pro-B-cell line that lacks endogenous SHPS-1 protein. Interestingly, expression of either SHPS-1, or a mutant lacking the intracellular domain of SHPS-1 (DeltaCT SHPS-1), resulted in the rapid formation of macroscopic Ba/F3 cell aggregates. As the integrin-associated protein/CD47 was shown to be a SHPS-1 ligand in neural cells, we investigated whether CD47 played a role in the aggregation of SHPS-1-expressing Ba/F3 cells. In support of this idea, aggregate formation was inhibited by an anti-CD47 Ab. Furthermore, erythrocytes from control, but not from CD47-deficient mice, were able to form rosettes on SHPS-1-expressing Ba/F3 cells. Because erythrocytes do not express integrins, this result suggested that SHPS-1-CD47 interactions can take place in the absence of a CD47-integrin association. We also present evidence that the amino-terminal Ig domain of SHPS-1 mediates the interaction with CD47. Although SHPS-1-CD47 binding likely triggers bidirectional intracellular signaling processes, these results demonstrate that this interaction can also mediate cell-cell adhesion.     

Tissue Selenium in Pigs with Dietetic Microangiopathy (MAP, “Mulberry Heart”)

Mean selenium contents of liver, heart muscle and skeletal muscle of pigs with dietetic microangiopathy (MAP), but without waxy muscle degeneration (MD) and hepatosis diaetetica (HD), were 1113, 850 and 265 ng/g d.s., respectively. These values were not lower than corresponding values of control pigs without MAP, MD and HD.     

Purification of homogeneous forms of fungal peroxygenase

Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient.     

Jekyll encodes a novel protein involved in the sexual reproduction of barley

Cereal seed development depends on the intimate interaction of filial and maternal tissues, ensuring nourishment of the new generation. The gene jekyll, which was identified in barley (Hordeum vulgare), is preferentially expressed in the nurse tissues. JEKYLL shares partial similarity with the scorpion Cn4 toxin and is toxic when ectopically expressed in Escherichia coli and tobacco (Nicotiana tabacum). In barley, jekyll is upregulated in cells destined for autolysis. The gene generates a gradient of expression in the nucellar projection, which mediates the maternal-filial interaction during seed filling. Downregulation of jekyll by the RNA interference technique in barley decelerates autolysis and cell differentiation within the nurse tissues. Flower development and seed filling are thereby extended, and the nucellar projection no longer functions as the main transport route for assimilates. A slowing down in the proliferation of endosperm nuclei and a severely impaired ability to accumulate starch in the endosperm leads to the formation of irregular and small-sized seeds at maturity. Overall, JEKYLL plays a decisive role in the differentiation of the nucellar projection and drives the programmed cell death necessary for its proper function. We further suggest that cell autolysis during the differentiation of the nucellar projection allows the optimal provision of basic nutrients for biosynthesis in endosperm and embryo.     

Tropomyosins are present in lamellipodia of motile cells

This paper shows that high-molecular-weight tropomyosins (TMs), as well as shorter isoforms of this protein, are present in significant amounts in lamellipodia and filopodia of spreading normal and transformed cells. The presence of TM in these locales was ascertained by staining of cells with antibodies reacting with endogenous TMs and through the expression of hemaglutinin- and green fluorescent protein-tagged TM isoforms. The observations are contrary to recent reports suggesting the absence of TMs in regions,where polymerization of actin takes place, and indicate that the view of the role of TM in the formation of actin filaments needs to be significantly revised.