Abstract. Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H+-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60-and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatmerit of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K+-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through vacuolar H+-ATPase.STEOCLASTS are multinucleated giant cells that are responsible for bone resorption. Like secreting epithelial cells, they are polarized when active in bone resorption, showing three distinct specialized cell membrane areas (for review see Vaes et al., 1988). In addition to the basolateral membrane, which is rich in Na+,K+-ATPase (Baron et al., 1986), resorbing osteoclast exhibits a clear zone that mediates the attachment of resorbing cells to the bone matrix. The cytoplasm in the vicinity of this clear zone contains specialized cytoskeletal structures (Holtrop et al., 1974;Lakkakorpi et al., 1989). The third specialized membrane area, the ruffled border, faces the actual bone resorption site on the bone surface. The resorption lacuna underneath the ruffled border membrane is acidic. This has been shown by acridine orange accumulation experiments (Anderson et al., 1986; Bar6n et al., 1985) and direct micropuncture measurements (Fallon, 1984). The acidic pH favors dissolution of the bone mineral. In addition, proteinases active at acid pH and capable of collagen degradation are present in osteoclasts (Vaes et al., 1988; Blair et al., 1986). Enzyme histochemistry suggests the presenc$ of ATPase activity in the plasma membrane of the osteoclast- (Akisaka et al., 1986). Baron et al. (1985) found at the ruffled:border of the osteoclast a 100-kD lysosomal membrane polypeptide that showed immunological similarity to gastric H+,K+-ATPase. Now we report that osteoclasts contain an ATP-dependent proton pump that is clearly different from the gastric proton pump and from the mitochondrial proton pump but shows considerable immunological similarities to the vacuolar type H+-ATPase of Neurospora crassa. Materials and Methods Preparation of Bone MicrosomesBone microsomes were prepared from medullary bone of regularly laying hens. Medullary bone from the tibia and femur was dissected out and immediately homogenized in a medium containing 5 mM Tris pH 7.4, 250 mM sucrose, 1 mM K2CO3, 1 mM DTT, and 1 mM EGTA in a glass-Teflun ...

Abstract. During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycinearginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts . When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed in-0 TEOC1.ASTs are multinucleated cells responsible for bone resorption . When actively resorbing they are polarized and have three specialized cell membrane areas. Resorption of bone takes place in the ruffled border area, where protons and proteases are secreted into the resorption lacuna between the ruffled border membrane of the osteoclast and the bone surface (Baron et al., 1988 ;Blair et al ., 1989;Bekker and Gay, 1990;Sundquist et al ., 1990; . The ruffled border area is surrounded by a sealing zone, which forms a tight attachment ofthe cell membrane to the underlying bone surface, thereby isolating the resorption lacuna from the extracellular fluid and permitting the maintenance of a cell-generated pH gradient. Previously we have described the formation of a specific microfilament organization around resorption lacunae in resorbing osteoclasts (Lakkakorpi et al., 1989). Recently we have further characterized the kinetics of this microfilament structure and demonstrated its stepwise formation into the sealing zone area (Lakkakorpi and Vaananen, 1991) . The third specialized membrane domain of the active osteoclast, the basolateral membrane, faces the bone marrow and is not in contact with the mineralized bone matrix .The molecular mechanisms by which osteoclasts attach to the bone surface are not well understood. The vitronectin receptor (a,a3), one member of the integrin superfamily (Hynes, 1987), has been shown to be expressed in os- subunits of the vitronectin receptor were similarly localized . These results strongly suggest that, although the vitronectin receptor is important in the function of osteoclasts, it is not mediating the final sealing zone attachment of the osteoclasts to the mineralized bone surface .

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Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Tularemia is a zoonotic disease which, in Scandinavia, is usually acquired through a mosquito bite. As the infecting organism, Francisella tularensis, is highly virulent the culturing of F. tularensis has generally been avoided. PCR offers a safe way to rapidly confirm diagnosis of tularemia. The case of a 9-y-old boy with ulceroglandular tularemia is presented. The diagnosis was made rapidly with DNA amplification from a pus specimen. The efficacy of ciprofloxacin treatment of tularemia in children is also discussed.