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We previously Isolated from a rat regeneratbig islet cDNA library a gene named Reg, which Is expressed in regenerating Islets but is not expressed in normal islets. Here we examined the effect of rat Reg protein on pancreatic beta-cell replication using both 90% depancreatized rats and isolated Islets. The depancreatized rats that received i. Hydrated 5-pm sections of paraffin-embedded pancreatic tissues were stained for insulin by the labeled streptavidinbiotin method, using an LSAB kit (Dakopatts, Glostrup, Denmark). After being stained, the relative volumes of beta cells were measured by the point-counting method (1, 13).In Vitro Experiment. Pancreatic islets were isolated from male Wistar rats (body weight 200-250 g) by using density separation on a dextran gradient and cultured free-floating in RPMI 1640 medium/10%o fetal calf serum/penicillin G at 100 pg/ml/streptomycin at 100 g/ml for 48 hr to allow recovery from the isolation procedure (14). After this initial period, islets were transferred to 24-well culture dishes in groups of 50 islets. The islets were cultured in RPMI 1640 medium/2.7 mM D-glucose/2% fetal calf serum/penicillin G at 100 ;g/ ml/streptomycin at 100 ;g/ml in the presence of increased concentrations of Reg protein for 72 hr. During the last 24 hr, the islets were cultured in the above medium to which [methyl-3H]thymidine at 10 puCi/ml (Amersham; 1 Ci = 37 GBq) had been added. To estimate the amount of [3H]thymidine incorporated into newly synthesized DNA, the islets were washed as described (14) after the culture period and sonicated in 10 mM Tris.HCl/5 mM EDTA. DNA was precipitated by the addition of 7% ice-cold trichloroacetic acid and trapped by filtration on a glass-fiber disc (Whatman GF/C). The discs were dried, and radioactivity was counted after the addition of scintillation fluid (Packard Ultima Gold F). The DNA content of the islets was measured by a flurometric DNA assay using Hoechst 33258. To estimate labeling indices for insulin-positive cells, the [3H]thymidinelabeled islets were fixed in 4% paraformaldehyde for 20 min and embedded in OCT compound. Ten-micrometer sections were cut with a cryostat and immunostained with antiporcine insulin guinea pig antiserum (DAKO, 1:500) and Vectastain ABC-GO kit (Vector Laboratories). Autoradiography of the immunostained sections was performed by using lTo whom reprint requests should be addressed at:

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Ginseng saponins, ginsenosides Rg (1), Re and Rb (1), decomposed under mild acidic conditions to yield prosapogenins. The structures of the prosapogenins were investigated by (13)C-NMR spectroscopy and Rg (1)-prosapogenin II was shown to be a mixture of ginsenoside Rh (1), and its C-20 epimer, produced by hydrolysis followed by epimerization at C-20. Rg (1)-prosapogenin III, the other prosapogenin derived from ginsenoside Rg (1); was a C-25,26 hydrated derivative of Rg (1)-prosapogenin II. Re-prosapogenin II was identified as a mixture of ginsenoside Rg (2) and its C-20 epimer, and Re-prosapogenine III as a C-25,26 hydrated derivative of Re-prosapogenin II.

BackgroundMultiple myeloma (MM) expands almost exclusively in the bone marrow and generates devastating bone lesions, in which bone formation is impaired and osteoclastic bone resorption is enhanced. TGF-β, a potent inhibitor of terminal osteoblast (OB) differentiation, is abundantly deposited in the bone matrix, and released and activated by the enhanced bone resorption in MM. The present study was therefore undertaken to clarify the role of TGF-β and its inhibition in bone formation and tumor growth in MM.Methodology/Principal FindingsTGF-β suppressed OB differentiation from bone marrow stromal cells and MC3T3-E1 preosteoblastic cells, and also inhibited adipogenesis from C3H10T1/2 immature mesenchymal cells, suggesting differentiation arrest by TGF-β. Inhibitors for a TGF-β type I receptor kinase, SB431542 and Ki26894, potently enhanced OB differentiation from bone marrow stromal cells as well as MC3T3-E1 cells. The TGF-β inhibition was able to restore OB differentiation suppressed by MM cell conditioned medium as well as bone marrow plasma from MM patients. Interestingly, TGF-β inhibition expedited OB differentiation in parallel with suppression of MM cell growth. The anti-MM activity was elaborated exclusively by terminally differentiated OBs, which potentiated the cytotoxic effects of melphalan and dexamethasone on MM cells. Furthermore, TGF-β inhibition was able to suppress MM cell growth within the bone marrow while preventing bone destruction in MM-bearing animal models.Conclusions/SignificanceThe present study demonstrates that TGF-β inhibition releases stromal cells from their differentiation arrest by MM and facilitates the formation of terminally differentiated OBs, and that terminally differentiated OBs inhibit MM cell growth and survival and enhance the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. Therefore, TGF-β appears to be an important therapeutic target in MM bone lesions.

The combined use of fine-needle aspiration and core biopsy improves the diagnostic ability of CT fluoroscopy-guided lung biopsy, even in small lesions.