Molecular characterisation of the recovery process in the entomopathogenic nematode Heterorhabditis bacteriophora.

In Heterorhabditis bacteriophora, an insect-parasitic nematode, the third juvenile is the infective, developmentally arrested form. When it infects a suitable host, the infective juvenile recovers from developmental arrest and resumes growth and development. This process is called recovery and it is the first outcome of the host-parasite interaction. Recovery is also very important from a commercial point of view. To characterise the recovery in H. bacteriophora, we sought to identify genes involved in this process. A large-scale bioassay for recovery was established and subtraction libraries of recovering infective juvenile from arrested infective juvenile transcripts were constructed at different time points. Most of the genes identified as differentially expressed between recovering and developmentally arrested infective juveniles belonged to metabolic pathways. Elevated expression levels of 23 selected genes during recovery were confirmed by quantitative PCR. For eight of these genes, transcription silencing in H. bacteriophora resulted in a significant decline in infective juvenile recovery rates, suggesting that these genes are critical to the recovery process. Two of the genes were associated with the insulin-like growth factor-1 (insulin/IGF-1) pathway, known to regulate dauer formation in the free-living nematode Caenorhabditis elegans, whereas the other six genes were associated with pathways not previously associated with recovery in nematodes. These results suggest that although little is known about parasitism-unique genes, the pathways regulating recovery in H. bacteriophora include those activated in C. elegans and those that might be unique to parasitic nematodes; the latter may be activated in response to host signals and enable the parasite to recognise its host.

CMF608-a novel mechanical strain-induced bone-specific protein expressed in early osteochondroprogenitor cells.

Microarray gene expression analysis was utilized to identify genes upregulated in primary rat calvaria cultures in response to mechanical force. One of the identified genes designated CMF608 appeared to be novel. The corresponding full-length cDNA was cloned and characterized in more details. It encodes a putative 2597 amino acid protein containing N-terminal signal peptide, six leucine-rich repeats (LRRs), and 12 immunoglobulin-like repeats, 10 of which are clustered within the C-terminus. Expression of CMF608 is bone-specific and the main type of CMF608-positive cells is mesenchymal osteochondroprogenitors with fibroblast-like morphology. These cells reside in the perichondral fibrous ring of La Croix, periosteum, endosteum of normal bone as well as in the activated periosteum and early fibrous callus generated postfracture. Expression of CMF608 is notably absent from the regions of endochondral ossification. Mature bone cell types do not produce CMF608 with the exception of chondrocytes of the tangential layer of the articular cartilage, which are thought to be under constant mechanical loading. Ectopic expression of CMF608 in HEK293T cells shows that the protein is subjected to post-translational processing and its N-terminal approximately 90 kDa polypeptide can be found in the conditioned medium. Ectopic expression of either the full-length cDNA of CMF608 or of its N-terminal region in CMF608-negative ROS17/2.8 rat osteosarcoma cells results in transfected clones displaying increased proliferation rate and the characteristics of less-differentiated osteoblasts compared to the control cells. Our data indicate that CMF608 is a unique marker of early osteochondroprogenitor cells. We propose that it could be functionally involved in maintenance of the osteochondroprogenitor cells pool and its down-regulation precedes terminal differentiation.