Osteogenic protein-1 (OP-1) is a member of the transforming growth factor beta superfamily and is a potent modulator of osteogenesis and bone cell differentiation. This preclinical study in dogs sought to assess the effects of OP-1 on periodontal wound healing in surgically created critical size Class III furcation defects. Eighteen male beagle dogs were subjected to the creation of bilateral mandibular 5 mm osseous defects. A split-mouth design was utilized which randomly assigned opposing quadrants to control therapy (surgery alone or collagen vehicle) or 1 of 3 ascending concentrations of OP-1 in a collagen vehicle (0.75 mg OP-1/g collagen, 2.5 mg/g, or 7.5 mg/g). Thus, 9 quadrants per test group received OP-1, 9 quadrants per control group received surgery alone, and 9 quadrants received collagen vehicle alone. Test articles were delivered by a surgeon masked to the treatment, and fluorogenic bone labels were injected at specified intervals post-treatment. Eight weeks after defect creation and OP-1 delivery, tissue blocks of the mandibulae were taken for masked histomorphometric analysis to assess parameters of periodontal regeneration (e.g., bone height, bone area, new attachment formation, and percent of defect filled with new bone). Histomorphometry revealed limited evidence of osteogenesis, cementogenesis, and new attachment formation in either vehicle or surgery-alone sites. In contrast, sites treated with all 3 concentrations of OP-1 showed pronounced stimulation of osteogenesis, regenerative cementum, and new attachment formation. Lesions treated with 7.5 mg/g of OP-1 in collagen regenerated 3.9+/-1.7 mm and 6.1+/-3.4 mm2 (mean +/-S.D.) of linear bone height and bone area, respectively. Furthermore, these differences were statistically different from both control therapies for all wound healing parameters (P < 0.0001). No significant increase in tooth root ankylosis was found among the treatment groups when compared to the surgery-alone group. We conclude that OP-1 offers promise as an attractive candidate for treating severe periodontal lesions.
We report the results of a long-term follow-up of subjects in a phase 1 study of AAV2-hAADC (adeno-associated virus type 2-human aromatic L-amino acid decarboxylase) gene therapy for the treatment of Parkinson's disease (PD). Ten patients with moderately advanced PD received bilateral putaminal infusions of either a low or a high dose of AAV2-hAADC vector. An annual positron emission tomography (PET) imaging with [(18)F]fluoro-L-m-tyrosine tracer was used for evaluation of AADC expression, and a standard clinical rating scale [Unified Parkinson's Disease Rating Scale (UPDRS)] was used to assess effect. Our previous analysis of the 6-month data suggested that this treatment was acutely safe and well tolerated. We found that the elevated PET signal observed in the first 12 months persisted over 4 years in both dose groups. A significantly increased PET value compared with the presurgery baseline was maintained over the 4-year monitoring period. The UPDRS in all patients off medication for 12 hr improved in the first 12 months, but displayed a slow deterioration in subsequent years. This analysis demonstrates that apparent efficacy continues through later years with an acceptable safety profile. These data indicate stable transgene expression over 4 years after vector delivery and continued safety, but emphasize the need for a controlled efficacy trial and the use of a higher vector dose.
We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse failed to release TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.
We characterized the tyrosine phosphorylation sites of free pp6O-src and of pp60CSrc associated with the polyomavirus middle tumor antigen (mT) in transformed avian and rodent cells. The sites of tyrosine phosphorylation in the two populations of pp60`-src were different, both in vitro and in vivo. Free pp60csrc was phosphorylated in vitro at a single site, tyrosine 416. pp60Src associated with mT was phosphorylated in vitro on tyrosine 416 and on one or more additional tyrosine residues located in the amino-terminal region of the molecule. Free pp60CSrC in polyomavirus mT-transformed cells was phosphorylated in vivo on tyrosine 527. In contrast, pp60csrc associated with mT was phosphorylated in vivo on tyrosine 416 and not detectably on tyrosine 527. Thus, the in vivo phosphorylation sites of pp60csrc associated with mT in transformed cells are identical to those of pp60(-src, the Rous sarcoma virus transforming protein. The results suggest that altered phosphorylation of pp60Csrc associated with mT may play a role in the enhancement of the pp660csrc protein kinase activity and in cell transformation by polyomavirus.The Rous sarcoma virus transforming protein pp6ov-src and its cellular homolog pp6oc-src are membrane-associated phosphoproteins with protein-tyrosine kinase activity (9,10,14,21,23,31,33,38). The specific activity of pp6Ocsrc, when assayed in vitro with peptide substrates, is 10% or less of the specific activity of pp6Ov-src (17, 25). Phosphorylation may regulate the enzymatic activities of these two proteins: phosphorylation of amino-terminal tyrosine residues in pp6v-src is accompanied by increased protein kinase activity (3,7,11,37); phosphorylation of tyrosine in the carboxyterminal region of pp60csrc decreases its protein kinase activity (13). The major site of tyrosine phosphorylation in vivo is different in the viral and cellular protein; pp6-src is phosphorylated on tyrosine 416, whereas pp60c-src is phosphorylated on a different tyrosine residue in the carboxy-terminal region of the molecule (8,30,36,44).The middle tumor antigen (mT) of polyomavirus plays a central role in transformation (50). mT is associated with a protein-tyrosine kinase activity (20,39,45) which correlates with the transforming ability of the protein (4,20,39,45,49). mT lacks intrinsic protein kinase activity (40, 41), but associates with a known cellular protein kinase, pp6-src (2,9,15,16,33,38). pp60c-src complexed to mT shows enhanced protein kinase activity and novel sites of tyrosine phosphorylation in vitro (2,5,13,53). Because the protein kinase activity of pp6Oc-src appears to be negatively regulated by carboxy-terminal tyrosine phosphorylation (13), we examined the possibility that binding to mT might prevent phosphorylation at this site on pp6c-src, thereby increasing its protein kinase activity. We compared the phosphorylation sites of free pp60csrc with those of pp6Oc-src associated with mT.
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