Many studies have attempted to elucidate the mechanism of the biostimulatory effects of low-level laser irradiation (LLLI), but the molecular basis of these effects remains obscure. We investigated the stimulatory effect of LLLI on bone formation during the early proliferation stage of cultured osteoblastic cells. A mouse calvaria-derived osteoblastic cell line, MC3T3-E1, was utilised to perform a cDNA microarray hybridisation to identify genes that induced expression by LLLI at the early stage. Among those genes that showed at least a twofold increased expression, the osteoglycin/mimecan gene was upregulated 2.3-fold at 2 h after LLLI. Osteoglycin is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix which was previously called the osteoinductive factor. SLRP are abundantly contained in the bone matrix, cartilage cells and connective tissues, and are thought to regulate cell proliferation, differentiation and adhesion in close association with collagen and many other growth factors. We investigated the time-related expression of this gene by LLLI using a reverse transcription polymerase chain reaction (RT-PCR) method, and more precisely with a real-time PCR method, and found increases of 1.5-2-fold at 2-4 h after LLLI compared with the non-irradiated controls. These results suggest that the increased expression of the osteoglycin gene by LLLI in the early proliferation stage of cultured osteoblastic cells may play an important role in the stimulation of bone formation in concert with matrix proteins and growth factors.
Yeast porin, the major outer mitochondrial membrane protein, is synthesized without a cleavable extension peptide and post-translationally inserted into the membrane. When inserted into the membrane, it acquires resistance to externally added trypsin. To locate the sequences responsible for membrane insertion and topogenesis in the primary structure of yeast porin, we constructed several deletion and chimeric mutants of the porin cDNA. These cDNAs were expressed in vitro and the products were assayed for capacity to be correctly inserted into isolated mitochondria. It was thus found that deletion of the segment spanning residues 37-98 did not appreciably impair the insertion competence and the inserted protein became resistant to trypsin. On the other hand, the porin mutant lacking the segment consisting of residues 17-98 did not acquire the trypsin resistance, though it could bind to mitochondria specifically. Deletion of the carboxy-terminal 62 amino acid residues also abolished the capacity to be correctly inserted into mitochondria. We conclude that information required for membrane insertion and intramembranous topogenesis of the porin molecule is stored not only in the amino-terminal region but also in the carboxy-terminal portion.
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