Superoxide production contributes to osteoclastic bone resorption. Evidence strongly indicates that NADPH oxidase is an enzyme system responsible for superoxide generation in osteoclasts. A membranebound subunit, p91, is the catalytic domain of NADPH oxidase. However, osteoclasts from p91 knockout mice still produce superoxide at a rate similar to that observed in wild type mice. This unexpected phenomenon prompted us to examine the osteoclasts for an alternative to the p91-containing oxidase. In this study, the cloning of a NADPH oxidase subunit (Nox 4) with 578 amino acids is reported. Nox 4 has 58% similarity in amino acids with the known p91 subunit of NADPH oxidase. Nox 4 is present and active in osteoclasts. Antisense oligonucleotides of Nox 4 reduced osteoclastic superoxide generation as well as resorption pit formation by osteoclasts. This new oxidase complex was present and functional in osteoclasts from p91 knockout mice, explaining the normal resorptive activity seen in the osteoclasts where no p91 is present.

Long-term therapy with interferon gamma in patients with osteopetrosis increases bone resorption and hematopoiesis and improves leukocyte function.

The S-layer of Bacillus stearothermophilus PV72/p2 shows oblique lattice symmetry and is composed of identical protein subunits with a molecular weight of 97,000. The isolated S-layer subunits could bind and recrystallize into the oblique lattice on native peptidoglycan-containing sacculi which consist of peptidoglycan of the A1␥ chemotype and a secondary cell wall polymer with an estimated molecular weight of 24,000. The secondary cell wall polymer could be completely extracted from peptidoglycan-containing sacculi with 48% HF, indicating the presence of phosphodiester linkages between the polymer chains and the peptidoglycan backbone. The cell wall polymer was composed mainly of GlcNAc and ManNAc in a molar ratio of 4:1, constituted about 20% of the peptidoglycan-containing sacculus dry weight, and was also detected in the fraction of the S-layer self-assembly products. Extraction experiments and recrystallization of the whole S-layer protein and proteolytic cleavage fragments confirmed that the secondary cell wall polymer is responsible for anchoring the S-layer subunits by the N-terminal part to the peptidoglycan-containing sacculi. In addition to this binding function, the cell wall polymer was found to influence the in vitro self-assembly of the guanidinium hydrochloride-extracted S-layer protein. Chemical modification studies further showed that the secondary cell wall polymer does not contribute significant free amino or carboxylate groups to the peptidoglycan-containing sacculi.

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