Extracellular proteasome in the human alveolar space: a new housekeeping enzyme?

We hypothesized that 20S proteasome is present and functional in the extracellular alveolar space in humans. Proteasomal activity was measured in bronchoalveolar lavage (BAL) supernatant from eight humans using specific proteasomal fluorogenic substrates and I(125)-albumin with and without specific proteasome inhibitors. Furthermore, gelfiltration, Western blot technique, and mass spectrometry were applied for proteasome characterization. All proteasomal fluorogenic substrates were hydrolyzed by BAL supernatant, with hydrolysis inhibited by epoxomicin (P = 0.024) and other proteasome inhibitors as well. E64, a lysosomal inhibitor, did not inhibit enzyme activity. The majority of proteolytic activity was detected in BAL supernatant rather than in the cell pellet. No correlation was found between proteasomal hydrolysis in BAL supernatant and lactate dehydrogenase activity, the total cell count in the cell pellet, and the fraction of avital cells in the cell pellet, ruling out cell lysis as a major source of proteasomal activity. Gelfiltration revealed hydrolyzing activity in the supernatant at 660 kDa and proteasome core proteins after analysis by ESI-QqTOF mass spectrometry. Furthermore, Western blots using a polyclonal antibody against proteasomal alpha-/beta-subunits detected proteasomal proteins in the typical 20- to 30-kDa range in BAL supernatant. Incubation of BAL supernatant with I(125)-albumin showed a high mean cleavage rate (101.8 microg/ml x h lavage +/- 46 SD) that was inhibited by epoxomicin (P = 0.013) and was ATP and ubiquitin independent. We identified for the first time extracellular, biologically active, ATP- and ubiquitin-independent 20S proteasome in the human alveolar space, with a high albumin cleavage rate. Possibly, the proteasome assists in maintenance of a low intra-alveolar oncotic pressure and/or alveolar protein degradation.

Bone apposition to titanium implants biocoated with recombinant human bone morphogenetic protein-2 (rhBMP-2). A pilot study in dogs.

The aim of the present study was to investigate bone formation to recombinant human bone morphogenetic protein-2 (rhBMP-2)-biocoated and rhBMP-2-nonbiocoated titanium implants after implantation in dogs. Implantation of sand-blasted and acid-etched (C), chromosulfuric acid surface-enhanced (CSA), and rhBMP-2-biocoated CSA [BMP-A: noncovalently immobilized rhBMP-2 (596 ng/cm(2)), BMP-B: covalently immobilized rhBMP-2 (819 ng/cm(2))] implants was performed in both the mandible and tibia of dogs. After 4 weeks of healing, the percentage of direct bone to implant contact (BIC) and the induced bone density (BD) at a distance of less than and greater than 1 mm adjacent to each implant was assessed. Histomorphometric analysis of implants inserted in the mandible and tibia revealed that BIC values appeared to be highest in the BMP-B group, followed by BMP-A, CSA, and C. BD as measured at a distance of <1 mm revealed obvious differences between groups: BMP-B>BMP-A>CSA>C. However, no differences between groups were observed at a distance of >1 mm. Within the limits of the present study, it may be concluded that rhBMP-2 immobilized by covalent and noncovalent methods on CSA-treated implant surfaces seemed to be stable and promoted direct bone apposition in a concentration-dependent manner.