The role of glyceraldehyde 3-phosphate dehydrogenase (GapA-1) in <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> adherence to human cells

Background  

Glyceraldehyde 3-phosphate dehydrogenases (GAPDHs) are cytoplasmic glycolytic enzymes, which although lacking identifiable secretion signals, have also been found localized to the surface of several bacteria (and some eukaryotic organisms); where in some cases they have been shown to contribute to the colonization and invasion of host tissues. Neisseria meningitidis is an obligate human nasopharyngeal commensal which can cause life-threatening infections including septicaemia and meningitis. N. meningitidis has two genes, gapA-1 and gapA-2, encoding GAPDH enzymes. GapA-1 has previously been shown to be up-regulated on bacterial contact with host epithelial cells and is accessible to antibodies on the surface of capsule-permeabilized meningococcal cells. The aims of this study were: 1) to determine whether GapA-1 was expressed across different strains of N. meningitidis; 2) to determine whether GapA-1 surface accessibility to antibodies was dependant on the presence of capsule; 3) to determine whether GapA-1 can influence the interaction of meningococci and host cells, particularly in the key stages of adhesion and invasion.     

Multiple reaction monitoring assay for pre-eclampsia related calcyclin peptides in formalin fixed paraffin embedded placenta

Although the cause of pre-eclampsia during pregnancy has not been elucidated yet, it is evident that placental and maternal endothelial dysfunction is involved. We previously demonstrated that in early onset pre-eclampsia placental calcyclin (S100A6) expression is significantly higher compared to controls ( De Groot , C. J. ; Clin. Proteomics 2007 , 1 , 325 ). In the current study, the results were confirmed and relatively quantified by using multiple reaction monitoring (MRM) on two peptide fragments of calcyclin. Cells were obtained from control (n = 5) and pre-eclamptic placental (n = 5) tissue collected by laser capture microdissection from formalin-fixed paraffin-embedded (FFPE) material treated with a solution to reverse formalin fixation. Two calcyclin peptides with an extra glycine inserted in the middle of the amino acid sequence were synthesized and used as an internal reference. Data presented show that MRM on laser microdissected material from FFPE tissue material is possible. The developed MRM assay to study quantitative levels of proteins in FFPE laser microdissected cells using nonisotopic-labeled chemical analogs of mass tagged internal references showed that in pre-eclamptic patients elevated levels of calcyclin is observed in placental trophoblast cells compared to normal trophoblast cells. By immunohistochemistry, we were able to confirm this observation in a qualitative manner.     

The complete mitochondrial DNA sequence of the shark Mustelus manazo: evaluating rooting contradictions to living bony vertebrates

A remarkable example of a misleading mitochondrial protein tree is presented, involving ray-finned fishes, coelacanths, lungfishes, and tetrapods, with sea lampreys as an outgroup. In previous molecular phylogenetic studies on the origin of tetrapods, ray-finned fishes have been assumed as an outgroup to the tetrapod/lungfish/coelacanth clade, an assumption supported by morphological evidence. Standard methods of molecular phylogenetics applied to the protein-encoding genes of mitochondria, however, give a bizarre tree in which lamprey groups with lungfish and, therefore, ray-finned fishes are not the outgroup to a tetrapod/lungfish/coelacanth clade. All of the dozens of published phylogenetic methods, including every possible modification to maximum likelihood known to us (such as inclusion of site heterogeneity and exclusion of potentially misleading hydrophobic amino acids), fail to place the ray-finned fishes in a biologically acceptable position. A likely cause of this failure may be the use of an inappropriate outgroup. Accordingly, we have determined the complete mitochondrial DNA sequence from the shark, Mustelus manazo, which we have used as an alternative and more proximal outgroup than the lamprey. Using sharks as the outgroup, lungfish appear to be the closest living relative of tetrapods, although the possibility of a lungfish/coelacanth clade being the sister group of tetrapods cannot be excluded.