Therapy using recombinant human bone morphogenetic protein-2 (rhBMP-2) is expected to promote bone healing and regeneration. Previous studies using protein or virus vectors for direct clinical application had problems, including a lack of efficiency, safety, and simplicity of the delivery system, and required an expensive protein, carrier matrix, or antigenic viral vector. In vivo gene transfer by electroporation is a simple and inexpensive method that only requires a plasmid and an electroporation device. Here, we created a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of rats after transferring different doses of this plasmid (25 microg, 100 microg, and 400 microg) by transcutaneous electroporation (8 electrical pulses of 100 V and 50 msec, in 1 to 5 sessions). First, we verified the gene transfer by transcutaneous electroporation using pCAGGS-lacZ. Next, the BMP-2 gene transfer and the production and localization of BMP-2 were identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blots, and immunohistochemistry. Ectopic bone formation was verified by radiography, histologic and immunohistochemical analyses, and quantitative examination. Ectopic bone formation, consisting of active osteoblasts and osteoclasts, was observed in all rats treated with electroporation. Thus, transcutaneous electroporation with pCAGGS-BMP-2 induced ectopic bone formation in the skeletal muscle of rats. This supports the possibility of applying human BMP-2 gene transfer using transcutaneous electroporation clinically.

Asymmetric synthesis of diarylmethyl amines has attracted growing attention owing to their importance in biological activity.[1] Among several methods for performing the asymmetric synthesis, [2,3] catalytic asymmetric addition of aryl metal reagents to imine derivatives seems to be most promising, provided that both high enantioselectivity and high catalytic activity are realized.[4] After our publication on the rhodium-catalyzed asymmetric addition of aryl stannanes to N-sulfonylimines, [5] two reports appeared on catalytic asymmetric arylation: 1) Bräse, Bolm, and co-workers described the addition of a phenylzinc reagent to masked Nformylimines in the presence of a chiral ketimine catalyst, [6] and 2) Tomioka illustrated the rhodium-catalyzed addition of aryl boroxines to N-tosylimines in which high enantioselectivity was observed for sterically tuned aryl imines.[7] Herein we report another rhodium-catalyzed asymmetric arylation in which the addition of aryl titanium reagents to sulfonylimines proceeds with high enantioselectivity under mild conditions (20 8C, 1 h) to give diarylmethyl amines with up to 96 % ee.During our studies on rhodium-catalyzed asymmetric 1,4-additions to a,b-unsaturated ketones, [8] we found that the phenyltitanium reagent PhTi(OiPr) 3 is highly reactive toward transmetalation and forms a phenyl-rhodium bond. In the presence of a rhodium/(S)-binap catalyst in THF at 20 8C, the catalytic 1,4-addition gives titanium enolates as 1,4-addition products with high enantioselectivity.[9] Under similar reaction conditions (Scheme 1), the addition of PhTi(OiPr) 3 [10] (4 a) to N-tosylarylimine 1 a, which was prepared from 4-trifluoromethylbenzaldehyde and 4-toluenesulfonamide, [11] took place rapidly to give the tosylamide of diarylmethyl amine 5 aa after aqueous workup, unfortunately with only 28 % ee (Table 1, entry 1). The use of (S)-H 8 -binap [12] and (S)-segphos [13] in place of (S)-binap [14] improved the enantioselectivity up to 43 % and 76 % ee, respectively (Table 1, entries 4 and 6). The relatively narrow dihedral angle of the biaryl bisphosphine ligand segphos [14] is considered to exert a positive influence on the enantioselectivity in the present

Background: Transcutaneous in vivo electroporation is expected to be an effective gene-transfer method for promoting bone regeneration using the BMP-2 plasmid vector. To promote enhanced osteoinduction using this method, we simultaneously transferred cDNAs for BMP-2 and BMP-7, as inserts in the non-viral vector pCAGGS.

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Histochemical, immunohistochemical and electron energy-loss spectroscopic studies were performed to examine the relationship between sulphated glycosaminoglycans and medullary bone calcification using oestrogen-injected male Japanese quail. Sulphated glycosaminoglycans, detected by high iron diamine (HID) or HID-thiocarbohydrazide-silver protein (HID-TCH-SP) methods, were distributed throughout the matrix of medullary bone, some periphery and extending tips of the trabeculae stained weakly, and the globular structures at osteoid areas were exclusively positive for HID-TCH-SP stain. Immunohistochemistry identified keratan sulphate located in the globular structures at osteoid areas and calcified matrix, but chondroitin-4 sulphate and chondroitin-6 sulphate were not detected in the matrix. Using electron spectroscopic imaging, sulphur was determined to be localized in the globular structures. These results demonstrate that medullary bone matrix accumulates keratan sulphate in the globular structures, which are the foci for calcification, and eventually in the calcified areas. This suggests that keratan sulphate containing sulphur is maintained in the calcified matrix. These results indicate a unique process of calcification exists in medullary bone.