Ribozyme Ablation Demonstrates That the Cardiac Subtype of the Voltage-sensitive Calcium Channel Is the Molecular Transducer of 1,25-Dihydroxyvitamin D3-stimulated Calcium Influx in Osteoblastic Cells

Ribozyme knockdown functionally links a 1,25(OH) 2 D 3 membrane binding protein (1,25D 3 -MARRS) and phosphate uptake in intestinal cells

“…After demonstration of in vitro activity (data not shown), complementary sense and antisense strands of single stranded ribozyme DNA were manufactured (Sigma Genosys) with 5Ј EcoRI and 3Ј SpeI overhangs and without an SP6 promoter. The complementary single strands were annealed into a double-stranded oligonucleotide insert that was then ligated into the pU1ZeoEcoSpe vector plasmid by using EcoRI and SpeI directional ligation (19). Insert ligation was confirmed by using restriction enzyme mapping, PCR, and sequencing.…”

“…The following morning, media were aspirated and each culture to be transfected was treated with 1 ml of medium containing 6 l of Lipofectamine (Invitrogen) and 1 g of the appropriate plasmid containing ribozyme cDNA. A complete description of ribozyme construction is given in Liu et al (19). Five hours after transfection, an additional 4 ml of RPMI with 10% FBS, antibiotics, and 1.5 mM EDTA/25 mM Hepes (final concentration; ref.…”

“…Sense and antisense single-stranded DNA coding for the active and scrambled ribozymes were manufactured (Sigma Genosys, The Woodlands, TX) with a 5Ј SP6 promoter. The complementary single strands were annealed and transcribed in vitro, and ribozyme activity was analyzed against a synthetic 1,25D 3 -MARRS target as described (19). After demonstration of in vitro activity (data not shown), complementary sense and antisense strands of single stranded ribozyme DNA were manufactured (Sigma Genosys) with 5Ј EcoRI and 3Ј SpeI overhangs and without an SP6 promoter.…”

Increased Ca2+ signaling through CaV1.2 promotes bone formation and prevents estrogen deficiency–induced bone loss

“…Indeed, while plasma membranes of bone cells (e.g., osteoblasts) have not been shown to prominently display the voltage-dependent changes that control Ca V 1.2 activity in excitable tissue, earlier reports identified functional L-type Ca 2+ channels, mainly Ca V 1.2, in human mesenchymal stem cells from BM cultured in osteogenic medium (5), in osteosarcoma cell lines (6), and in a rat osteoblast-like cell line (7). Further, Ca V 1.2 was shown in an osteosarcoma cell line to be the primary site for Ca 2+ influx, which promoted osteoblast differentiation (8,9). In addition, Ca V 1.2 expression and channel activity were regulated by calciotropic hormones such as 1,25(OH) 2 D 3 (8, 10) and parathyroid hormone (11), suggesting a functional contribution of Ca V 1.2 L-type Ca 2+ channel to bone development.…”

Ribozyme-mediated reduction of wild-type and mutant cartilage oligomeric matrix protein (COMP) mRNA and protein

“…The magnitude of the reduction suggests that this ribozyme may be more effective against MT-COMP than WT-COMP in native chondrocytes. The difference in efficiency of mRNA cleavage may be related to the difference in folding patterns of the WT-COMP mRNA compared with the MT-COMP (Fedor and Uhlenbeck 1990;Liu et al 2000;Liu et al 2002). Even though the site of cleavage of Ribo56 is near the 59 end of the COMP transcript, the changes in the secondary structures of MT-COMP compared with WT-COMP assessed by S-fold extend to the region complementary to the annealing arms of the ribozyme.…”