miRConnect: identifying effector genes of miRNAs and miRNA families in cancer cells

micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.     

Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening

Salmonella enterica serovar Typhi and Typhimurium are closely related serovars. However, S. Typhi, a human-specific pathogen, has 5% of genes as pseudogenes, far more than S. Typhimurium, which only has 1%. One of these pseudogenes corresponds to sopD2, which in S. Typhimurium encodes an effector protein involved in Salmonella-containing vacuole biogenesis in human epithelial cell lines, which is needed for full virulence of the pathogen. We investigated whether S. Typhi trans-complemented with the functional sopD2 gene from S. Typhimurium (sopD2(STM) ) would reduce the invasion of human epithelial cell lines. Our results showed that the presence of sopD2(STM) in S. Typhi significantly modified the bacterial ability to alter cellular permeability and decrease the CFUs recovered after cell invasion of human epithelial cell line. These results add to mounting evidence that pseudogenes contribute to S. Typhi adaptation to humans.     

Structure of a ganglioside with Cad blood group antigen activity

The Cad antigen is a rare erythrocyte blood group antigen expressed on both sialoglycoprotein and ganglioside structures. It is related both serologically and biochemically to the Sda blood group antigen expressed on over 90% of Caucasian erythrocytes. We reported previously that Cad erythrocytes contain a novel ganglioside that binds Helix pomatia lectin and inhibits human anti-Sda antibody. We have now purified the Cad ganglioside and determined its structure. The ganglioside contained Glc-Gal-GlcNAc-GalNAc-NeuAc in a molar ratio of 1.00:1.94:0.95:0.93:1.05. Its chromatographic mobility was between that of GM1 and GD3. After treatment with beta-hexosaminidase (human placenta Hex A), the product migrated with 2-3-sialosylparagloboside (IV3NeuAcnLc4OseCer), it no longer bound H. pomatia lectin, and it acquired the ability to bind an antibody to sialosylparagloboside. Treatment of this material with neuraminidase (Vibrio cholerae) yielded a product with the mobility of paragloboside (nLc4OseCer) that bound monoclonal antibody 1B2, which is specific for terminal N-acetyllactosaminyl structures. Treatment of the Cad ganglioside with Arthrobacter ureafaciens neuraminidase yielded a product reactive with monoclonal antibody 2D4, which is specific for terminal GalNAc beta (1-4)Gal structures. These data provide strong evidence that the Cad ganglioside structure is GalNAc beta (1-4)[NeuAc alpha (2-3)]Gal beta (1-3)Gal beta (1-4)GlcCer. 1H NMR analysis also supports the conclusion that the terminal GalNAc is linked beta (1-4) to Gal. High-performance thin-layer chromatographic ganglioside patterns from three blood group Cad individuals showed a direct correlation between the quantity of Cad ganglioside and the strength of Cad antigen expression on the erythrocytes, as measured by hemagglutination.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Microclimatic buffering by logging residues and forest edges reduces clear‐cutting impacts on forest bryophytes

Question: The practice of extracting logging residues after clear‐cutting for bioenergy purposes is spreading. Logging residues constitute a shelter in clear‐cut areas and therefore concerns have been expressed that their removal could make the ground and its vegetation more exposed to extreme microclimatic conditions. We asked whether logging residues and forest edges can protect ground‐dwelling forest bryophytes from fatal microclimate events following clear‐cutting. Location: Boreal forests of central Sweden. Methods: Using transplants of eight forest floor bryophyte species we experimentally analysed the sheltering effect (less solar radiation and less wind) of logging residues and forest edges in seven clear‐cut areas. Transplants were placed in two contrasting positions in each area; near a north‐facing forest edge and in the centre of the clear‐cut area. In each position, half of the transplants were covered by a layer of spruce branches and the other half was left uncovered. We estimated proportion of apparently living shoots (apparent vitality) and measured radial growth of transplants during one growing season. Results: Position in the clear‐cut area, but not cover of spruce branches, clearly influenced radial growth. Vitality scores were higher among transplants covered with branches and the lowest apparent vitality was observed in uncovered transplants in the middle of clear‐cut areas. The change in area of apparently living shoots during the course of the experiment (growth minus mortality) was unaffected by branch cover close to the edge but positively affected in the centre of the clear‐cut area. In general, the effect of branch cover on bryophytes was higher in the centre of clear‐cut areas. Here, climatic measurements showed that branch cover buffers during periods of extreme microclimates. Conclusions: Extraction of logging residues after clear‐felling may reduce the survival of some ground‐dwelling forest organisms. The additional sheltering provided by branches was unimportant close to forest edges. We suggest smaller clear‐cut areas, green‐tree retention and other ways to make logged areas shadier and less windy to mitigate the reduced shelter caused by harvest of logging residues.     

The dissociation of the fluid and particle phase in the forestomach as a physiological characteristic of large grazing ruminants: an evaluation of available, comparable ruminant passage data

Whether differences in digestive physiology exist between different ruminant feeding types has been an ongoing debate. In this regard, potential differences in ingesta retention have been understood to be of particular importance. We analyzed a data pool in which only mean retention time (MRT) data for the ruminoreticulum (RR) were collated that were obtained using comparable techniques with either chromium or cobalt EDTA as a fluid marker and/or with chromium-mordanted fiber of less than 2 mm in size as a particle marker. Data were compared using one averaged value per species. In general, the paucity of species in such a collection is striking and does not allow—in contrast to earlier statements—any final conclusions regarding the influence of body weight (BW) or feeding type on ruminant MRTs. In particular, there was no significant correlation between MRT_particlesRR or MRT_fluidRR and BW, neither in the interspecific nor in the intraspecific comparisons, and no difference between the feeding types. The trend that indicates longer MRT_particlesRR in grazers is based on too few species to be conclusive. Small browsers seemed to have shorter MRT_fluidRR than similar-sized grazers. In contrast, there was a trend for large grazers to have shorter MRT_fluidRR than large browsers. In direct pair-wise comparisons between cattle and the browsers giraffe, moose, and okapi, the latter difference was significant. Cattle also had the highest relative RR fluid outflow rates among the species investigated. This is in accord with the observation that grazers have larger omasa, a major function of which is water-reabsorption distal to the RR. Grazers seem to have longer MRT_particlesRR per unit MRT_fluidRR, and cattle are particular outliers in this respect. It is hypothesized that potentially shorter MRT_fluidRR in large grazers and higher relative outflow rates are linked to a higher saliva production and a lesser viscosity of both saliva and RR fluids. A constant supply of a fluid phase of low viscosity is proposed to be the prerogative for the physical mechanisms of flotation and sedimentation that result in the stratification of RR contents and its selective particle retention typical for large grazing species.     

A cross-strand Trp Trp pair stabilizes the hPin1 WW domain at the expense of function

Using the human Pin1 WW domain (hPin1 WW), we show that replacement of two nearest neighbor non-hydrogen-bonded residues on adjacent beta-strands with tryptophan (Trp) residues increases beta-sheet thermodynamic stability by 4.8 kJ mol(-1) at physiological temperature. One-dimensional NMR studies confirmed that introduction of the Trp-Trp pair does not globally perturb the structure of the triple-stranded beta-sheet, while circular dichroism studies suggest that the engineered cross-strand Trp-Trp pair adopts a side-chain conformation similar to that first reported for a designed "Trp-zipper" beta-hairpin peptide, wherein the indole side chains stack perpendicular to each other. Even though the mutated side chains in wild-type hPin1 WW are not conserved among WW domains and compose the beta-sheet surface opposite to that responsible for ligand binding, introduction of the cross-strand Trp-Trp pair effectively eliminates hPin1 WW function as assessed by the loss of binding affinity toward a natural peptide ligand. Maximizing both thermodynamic stability and the domain function of hPin1 WW by the above mentioned approach appears to be difficult, analogous to the situation with loop 1 optimization explored previously. That introduction of a non-hydrogen-bonded cross-strand Trp-Trp pair within the hPin1 WW domain eliminates function may provide a rationale for why this energetically favorable pairwise interaction has not yet been identified in WW domains or any other biologically evolved protein with known three-dimensional structure.