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Warburg showed in 1929 that the photochemical action spectrum for CO dissociation from cytochrome c oxidase is that of a heme protein. Keilin had shown that cytochrome a does not react with oxygen, so he did not accept Warburg's view until 1939, when he discovered cytochrome a3. The dinuclear cytochrome a3-CuB unit was found by EPR in 1967, whereas the dinuclear nature of the CuA site was not universally accepted until oxidase crystal structures were published in 1995. There are negative redox interactions between cytochrome a and the other redox sites in the oxidase, so that the reduction potential of a particular site depends on the redox states of the other sites. Calculated electron-tunneling pathways for internal electron transfer in the oxidase indicate that the coupling-limited rates are 9 x 10(5) (CuA--a) and 7 x 10(6) s(-1) (a--a3); these calculations are in reasonable agreement with experimental rates, after corrections are made for driving force and reorganization energy. The best CuA-a pathway starts from the ligand His204 and not from the bridging sulfur of Cys196, and an efficient a-a3 path involves the heme ligands His378 and His376 as well as the intervening Phe377 residue. All direct paths from CuA to a3 are poor, indicating that direct CuA--a3 electron transfer is much slower than the CuA--a reaction. The pathways model suggests a means for gating the electron flow in redox-linked proton pumps.

The aim of this study was to evaluate the effect of deproteinized bovine bone graft material on new bone formation in a guided bone regeneration model system. In 20 rabbits, a periosteal skin flap was raised uncovering the calvaria. A form stable hemispherical dome made of poly-lactic acid (PLA) was placed onto the roughened calvaria. Prior to placement, the dome was either filled with peripheral blood alone (control group, 8 rabbits), or with blood and OsteoGraf/N-300 (test group, 12 rabbits). The wound was closed for primary healing. Morphometric assessment of 1- and 2-month undecalcified histologic specimens revealed better tissue fill in the test domes at 1 month (test 99%, control 55%) (P0.05) and 2 months (t, 100%; c, 82%). The fraction of the new bone within the regenerated tissue was higher in the test specimens at 1 month (t, 22%; c, 12%) (P0.05) and 2 months (t, 34%; c, 24%). The fraction of the entire space underneath the domes occupied by bone was higher in the test at 1 month, but higher in the controls at 2 months. The fraction of the bone substitute material in contact with bone increased from 1 month (34% +/- 14) to 2 months (45% +/- 5). The surface fraction of osteoblast layers was tendentially higher in the test at 1 month but higher in the control specimens at 2 months. In both test and control, initially woven bone was formed which underwent subsequent remodeling. Cellular degradation of the deproteinized bone graft was frequently detected. It is concluded that deproteinized bovine bone mineral has osteoconductive properties and can initially accelerate new bone formation during guided bone regeneration by increased recruitment of osteoblasts.

The aim of the present study was to evaluate the effect of natural deproteinized bone mineral on the temporal and spatial pattern of bone formation in a guided bone regeneration model system while using a bioresorbable membrane device. A periosteal skin flap was raised uncovering the calvaria of 20 rabbits. A stiff hemispherical dome made of polylactic acid was placed onto the roughened calvaria and anchored by screws. Prior to placement, the dome was either filled with peripheral blood (control group, 8 rabbits) or with blood and OsteoGraf/N-300 (test group, 12 rabbits). At 1 month, histologic sections revealed bone regeneration in both test and control domes to various degrees. In the test domes, bone height reached 78% (67-83) and bone volume was 11% (6-17), while in the control domes, bone height was 45% (14-67) and bone volume 6% (1-11). At 2 months, bone height was unchanged in the test group at 70% (67-83) and bone volume had only slightly increased to 16% (11-21). In the controls, height increased to 86% (60-100) and volume to 20% (9-27). Thus, in this model system, natural bone mineral fill contributed to accelerate initial bone neogenesis, while it did not contribute to increasing bone volume or bone height at later observation stages.

Rat liver lysosomes, isolated from metrizamide gradients by the method of Wattiaux et al. (Wattiaux, R., Wattiaux-de Coninck, S., Ronveaux-Dupol, M.F., and Dubois, F. (1978) J. Cell Biol. 78, 349-368) took up from the medium and degraded several marker protein preparations, viz. 125I-asialofetuin, [35S]methionine-labeled hemoglobin, and [3H]leucine-labeled rat liver cytosol proteins. Rates were indistinguishable for all the markers, indicating that uptake was by a nonspecific process analogous to fluid pinocytosis. No effect of added MgATP or K+ was observed. Lysosomal degradation of all the markers was inhibited by 10(-4) M chloroquine. Swainsonine, on the other hand, at 10(-5) M, inhibited the breakdown only of the glycoprotein, 125I-asialofetuin. In the presence of the inhibitors, there was an accumulation of markers in the lysosomes in amount corresponding to the decreased breakdown, indicating that uptake was unaffected. Degradation and inhibition were measured at pH 7.0, 6.0, and 5.0 with both intact lysosomes and with lysosomes disrupted by the addition of 0.2% Triton X-100. Degradation with intact lysosomes was relatively independent of pH. On the other hand, activity with disrupted lysosomes was negligible at pH 7.0 and rose rapidly with decreasing pH. Inhibition by 10(-4) M chloroquine and 10(-5) M swainsonine with intact lysosomes decreased sharply with decreasing pH and did not occur with disrupted lysosomes.

Swainsonine, an alpha-mannosidase inhibitor found in the Australian plant Swainsona canescens and in spotted locoweed, was tested for its effect on the degradation of endocytosed proteins in rat liver cells. The compound inhibited the release of proteolytically derived breakdown products from endocytosed glycoproteins but not from nonglycoproteins. Fifty per cent inhibition of the degradation of 125I-asialofetuin occurred at a concentration of swainsonine in the medium of 6 X 10(-7) M. In the presence of the inhibitor, there was an increased cellular accumulation of the glycoproteins corresponding quantitatively to the decreased degradation, so that total uptake was the same in the presence or absence of the inhibitor. The excess 125I-labeled material which accumulated within cells under these conditions was found entirely in the lysosomal peak on Percoll density gradient centrifugation and migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a mobility indistinguishable from that of the original material. Thus, the inhibition of breakdown occurs prior to any conversion of the protein to intermediates of detectably smaller size. In contrast to the results with swainsonine, degradation of both glycoproteins and nonglycoproteins was inhibited by chloroquine and by 3-methyladenine. The findings with 3-methyladenine are in conflict with earlier reports in the literature on the effect of this compound on degradation of endocytosed proteins.