Biologically meaningful expression profiling across species using heterologous hybridization to a cDNA microarray

Background  

Unravelling the path from genotype to phenotype, as it is influenced by an organism's environment, is one of the central goals in biology. Gene expression profiling by means of microarrays has become very prominent in this endeavour, although resources exist only for relatively few model systems. As genomics has matured into a comparative research program, expression profiling now also provides a powerful tool for non-traditional model systems to elucidate the molecular basis of complex traits.     

Clustering cancer gene expression data: a comparative study

Background  

The use of clustering methods for the discovery of cancer subtypes has drawn a great deal of attention in the scientific community. While bioinformaticians have proposed new clustering methods that take advantage of characteristics of the gene expression data, the medical community has a preference for using "classic" clustering methods. There have been no studies thus far performing a large-scale evaluation of different clustering methods in this context.     

Mycobacteria mobility shift assay: a method for the rapid identification of Mycobacterium tuberculosis and nontuberculous mycobacteria

The identification of mycobacteria is essential because tuberculosis (TB) and mycobacteriosis are clinically indistinguishable and require different therapeutic regimens. The traditional phenotypic method is time consuming and may last up to 60 days. Indeed, rapid, affordable, specific and easy-to-perform identification methods are needed. We have previously described a polymerase chain reaction-based method called a mycobacteria mobility shift assay (MMSA) that was designed for Mycobacterium tuberculosis complex (MTC) and nontuberculous mycobacteria (NTM) species identification. The aim of this study was to assess the MMSA for the identification of MTC and NTM clinical isolates and to compare its performance with that of the PRA-hsp65 method. A total of 204 clinical isolates (102 NTM and 102 MTC) were identified by the MMSA and PRA-hsp65. For isolates for which these methods gave discordant results, definitive species identification was obtained by sequencing fragments of the 16S rRNA and hsp65 genes. Both methods correctly identified all MTC isolates. Among the NTM isolates, the MMSA alone assigned 94 (92.2%) to a complex or species, whereas the PRA-hsp65 method assigned 100% to a species. A 91.5% agreement was observed for the 94 NTM isolates identified by both methods. The MMSA provided correct identification for 96.8% of the NTM isolates compared with 94.7% for PRA-hsp65. The MMSA is a suitable auxiliary method for routine use for the rapid identification of mycobacteria.     

Microsporidia, amitochondrial protists, possess a 70-kDa heat shock protein gene of mitochondrial evolutionary origin

An intronless gene encoding a protein of 592 amino acid residues with similarity to 70-kDa heat shock proteins (HSP70s) has been cloned and sequenced from the amitochondrial protist Encephalitozoon cuniculi (phylum Microsporidia). Southern blot analyses show the presence of a single gene copy located on chromosome XI. The encoded protein exhibits an N-terminal hydrophobic leader sequence and two motifs shared by proteobacterial and mitochondrially expressed HSP70 homologs. Phylogenetic analysis using maximum likelihood and evolutionary distances place the E. cuniculi sequence in the cluster of mitochondrially expressed HSP70s, with a higher evolutionary rate than those of homologous sequences. Similar results were obtained after cloning a fragment of the homologous gene in the closely related species E. hellem. The presence of a nuclear targeting signal-like sequence supports a role of the Encephalitozoon HSP70 as a molecular chaperone of nuclear proteins. No evidence for cytosolic or endoplasmic reticulum forms of HSP70 was obtained through PCR amplification. These data suggest that Encephalitozoon species have evolved from an ancestor bearing mitochondria, which is in disagreement with the postulated presymbiotic origin of Microsporidia. The specific role and intracellular localization of the mitochondrial HSP70-like protein remain to be elucidated.      

PGE2 induces the transition from non-adherent to adherent bone marrow mesenchymal precursor cells via a cAMP/EP2-mediated mechanism

When mesenchymal precursor cells from bone marrow are cultured in the presence of dexamethasone, the existence of distinct non-adherent and adherent populations can be demonstrated. The addition of PGE₂, forskolin, or dibutyryl-cAMP can induce a transition from the former to the latter and this may be an important mechanism in the bone anabolic effects of PGE₂. On the other hand, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, and sulprostone, an agonist for the PGE₂ receptor EP₁/EP₃ subtypes, had no effect. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX), had a synergistic effect in combination with PGE₂, whereas neomycin, an inhibitor of inositol phosphate activity, had no effect, and LiCl, an inhibitor of inositol triphosphate metabolism, had an inhibitory effect on the PGE₂-induced transition. Consistent with this, the addition of PGE₂ to non-adherent bone marrow cells caused a 100% increase in cAMP synthesis. These results suggest that the induction of the transition from non-adherent to adherent osteoblast precursor is mediated by the EP₂-PGE₂ receptor subtype via an increase in intracellular cAMP synthesis.     

Stimulation of bone resorption by lipoxygenase metabolites of arachidonic acid

We have studied the effect of leukotrienes, (LT): B4, C4, D4 and E4 and the hydroxyeicosatetraenoic acids (HETEs) 5-HETE and 12-HETE on bone resorption in vitro. Resorption was measured by colorimetric assay of calcium released from neonatal mouse calvaria maintained in organ culture for 72h. All the LTs and HETEs stimulated bone resorption, with optimum responses at picomolar or nanomolar concentrations. The responses were biphasic, with a decreasing effect at higher concentrations. In contrast, prostaglandin E2 (PGE2) stimulated resorption only at 10nM and above. Indomethacin partially inhibited resorption by LTB4, LTC4 and LTD4, but did not affect resorption stimulated by LTE4, 5-HETE and 12-HETE. These results indicate that lipoxygenase products of arachidonic acid are highly potent bone resorbing factors and may play an important role in the localised bone loss associated with inflammatory lesions.