We had previously identified the cDNA for a novel protein called osteoblast-specific factor 2 (OSF-2) from an MC3T3-E1 cDNA library using subtraction hybridization and differential screening techniques. Here we describe the localization, regulation, and potential function of this protein. Immunohistochemistry using specific antiserum revealed that in adult mice, the protein is preferentially expressed in periosteum and periodontal ligament, indicating its tissue specificity and a potential role in bone and tooth formation and maintenance of structure. Based on this observation and the fact that other proteins have been called OSF-2, the protein was renamed "periostin." Western blot analysis showed that periostin is a disulfide linked 90 kDa protein secreted by osteoblasts and osteoblast-like cell lines. Nucleotide sequence revealed four periostin transcripts that differ in the length of the C-terminal domain, possibly caused by alternative splicing events. Reverse transcription-polymerase chain reaction analysis revealed that these isoforms are not expressed uniformly but are differentially expressed in various cell lines. Both purified periostin protein and the periostin-Fc recombinant protein supported attachment and spreading of MC3T3-E1 cells, and this effect was impaired by antiperiostin antiserum, suggesting that periostin is involved in cell adhesion. The protein is highly homologous to ␤ig-h3, a molecule induced by transforming growth factor ␤ (TGF-␤) that promotes the adhesion and spreading of fibroblasts. Because TGF-␤ has dramatic effects on periosteal expansion and the recruitment of osteoblast precursors, this factor was tested for its effects on periostin expression. By Western blot analysis, TGF-␤ increased periostin expression in primary osteoblast cells. Together, these data suggest that periostin may play a role in the recruitment and attachment of osteoblast precursors in the

Annexin V can be used to detect apoptotic cells in vitro and in vivo, based on its ability to identify extracellular phosphatidylserine, which arises during apoptosis. In the present study, we examined the synthesis of fluorine-18 labelled annexin V as a positron emission tomography tracer for apoptosis imaging. The distribution of [18F]annexin V and technetium-99m labelled annexin V, a well-characterised SPET tracer for apoptosis imaging, was compared. [18F]annexin V was synthesised using N-succinimidyl 4-[18F]fluorobenzoate as an 18F labelling reagent. Synthesised and purified [18F]annexin V was confirmed by SDS-PAGE. In an ex vivo imaging experiment, [18F]annexin V was intravenously injected into rats 24 h after the induction of myocardial ischaemia, and accumulation in the left ventricle was examined. [18F]annexin V accumulated in the infarct area of the left ventricle, where apoptotic cells were observed. In separate experiments, [18F]annexin V or [(99m)Tc]annexin V was intravenously injected into ischaemic or normal animals, and the distribution of the tracers was compared. In ischaemic animals, accumulation of [18F]annexin V and [(99m)Tc]annexin V in the infarct area was about threefold higher than in the non-infarct area. Furthermore, the ratio of accumulation in the normal heart to the blood radioactivity was not significantly different between the tracers. In normal animals, however, the uptake of [18F]annexin V in the liver, spleen and kidney was much lower than that of [(99m)Tc]annexin V. The low uptake of [18F]annexin V in these organs might represent an advantage over [(99m)Tc]annexin V.

To identify HLA alleles closely involved in the autoantigen presentation in acquired aplastic anemia (AA), we studied the HLA allelic loss frequencies of 312 AA patients, including 43 patients with loss of heterozygosity of 6p chromosome (6pLOH). An analysis of the HLA alleles contained in the lost haplotype revealed to be the most frequently lost allele. When we examined 28 AA (12 6pLOH[+] and 16 6pLOH[-]) patients with for the presence of leukocytes lacking HLA-B4002 (B4002) using a new monoclonal antibody specific to this allele, B4002 granulocytes were detected not only in all 6pLOH(+) patients but also in 9 (56%) of the 16 6pLOH(-) patients. Furthermore, 10 (83%) of the 12 6pLOH(+) patients possessed 1.0% to 78% B4002 granulocytes that retained the HLA-A allele on the same haplotype (B4002A), suggesting the frequent coexistence of granulocytes that underwent mutations restricted to with 6pLOH(+) (B4002A) granulocytes. Deep sequencing of the of sorted B4002A granulocytes revealed various somatic mutations, such as frameshift, nonsense, and splice site mutations, in all 15 patients studied. Surprisingly, missense mutations in the α-3 domain of that are not involved in the antigen presentation were detected exclusively in the B4002 granulocytes of 3 patients possessing B4002 granulocytes. The markedly high prevalence of leukocytes lacking HLA-B4002 as a result of either 6pLOH or structural gene mutations, or both, suggests that antigen presentation by hematopoietic stem/progenitor cells to cytotoxic T cells via the HLA-B allele plays a critical role in the pathogenesis of AA.

Arginine vasopressin- (AVP) and oxytocin- (OXT) secreting magnocellular neurons undergo gross structural changes with chronic physiological stimulation. Here, we investigated subcellular aspects of plasticity in rat neurohypophysial terminals during dehydration. Ultrastructural analyses demonstrated that chronic dehydration by 2% NaCl drinking for 7 days significantly decreased the numbers of neurosecretory granules and microvesicles but not the numbers of mitochondria. Moreover, in dehydrated rats, terminals making neurovascular contacts enlarged, whereas terminals in apposition to astrocytes, i.e., neuroglial contacts, became smaller. Western blot analyses demonstrated significant decreases in the levels of F3 and Thy-1 together with those of AVP- and OXT-neurophysin, but the levels of synaptophysin, SNAP-25, and GAP-43 were unchanged. Both F3 and Thy-1 were recovered in the buffer-insoluble pellet, and phosphatidyl inositol-specific phospholipase C treatment released both molecules from the crude membrane fraction, indicating that they are attached to terminal membranes by glycosylphosphatidyl inositol anchors. Confocal microscopic observations demonstrated that F3 colocalized with Thy-1 in the same terminals of magnocellular neurons. In contrast, the level of calretinin, a Ca(2+) binding protein was significantly increased with chronic dehydration. Thus, the present results suggest that enhancement of neurovascular contacts results from rearrangement of terminal-astrocyte and terminal-vessel contacts rather than enlargement or sprouting of magnocellular terminals themselves. The down-regulation of F3 and Thy-1 may contribute to enhancement of neurovascular contacts that accompany increased peptide release during dehydration.

SMTP-7 (Stachybotrys microspora triprenyl phenol-7), a small molecule that promotes plasminogen activation through the modulation of plasminogen conformation, has excellent therapeutic activity against cerebral infarction in several rodent models. Detailed evaluations of SMTP-7 in a primate stroke model are needed for effective, safe drug development. Here we evaluated SMTP-7 in a monkey photochemical-induced thrombotic middle cerebral artery (MCA) occlusion model (n=6), in which MCA occlusion was followed by recanalization/reocclusion. SMTP-7 (10 mg/kg, intravenous infusion) significantly increased the postinfusion MCA recanalization rate (32.5-fold, P=0.043) and ameliorated the post-24-h neurologic deficit (by 29%, P=0.02), cerebral infarct (by 46%, P=0.033), and cerebral hemorrhage (by 51%, P=0.013) compared with the vehicle control animals. In normal monkeys, SMTP-7 did not affect general physiologic or hemostatic variables, including coagulation and platelet parameters. Investigations in rodent models of transient and permanent focal cerebral ischemia, as well as arterial thrombosis and bleeding tests, suggest a role for SMTP-7's regulated profibrinolytic action and neuroprotective properties in the monkey MCA occlusion model. In conclusion, SMTP-7 is effective in treating thrombotic stroke in monkeys. SMTP-7 is thus a promising candidate for the development of alternative therapy for ischemic stroke.