Masao Akagi

Synthesis and properties of mirror-image DNA.

We have investigated the conformations of the hexadeoxyribonucleotide, L-d(CGCGCG) composed of L-deoxyribose, the mirror image molecule of natural D-deoxyribose. In this paper, we report the synthesis of four L-deoxynucleosides and the L-oligonucleotide-ethidium bromide interactions. The L-deoxyribose synthon 9 was synthesized from L-arabinose with an over all yield of 28.5% via the Barton-McCombie reaction. The L-deoxynucleosides were obtained by a glycosylation of appropriate nucleobase derivatives with the 1-chloro sugar 9. After derivatization to nucleoside phosphoramidites, L-deoxycytidine and L-deoxyguanosine were incorporated into a hexadeoxynucleotide, L-d(CGCGCG) by a solid-phase beta-cyanoethylphosphoramidite method. This L-hexanucleotide was resistant to digestion with nuclease P1. The conformations of L-d(CGCGCG) were an exact mirror image of that of the corresponding natural one as described previously, and the conformations of the L-d(CGCGCG)-ethidium bromide complex were also the mirror images of those of the D-d(CGCGCG)-ethidium bromide complex under both low and high salt conditions. These results suggest that ethidium bromide prefers not a right-handed helical sense, but the base-base stacking geometry of the B-form rather than that of the Z-form. Thus, L-DNA would be a useful tool for studying DNA-drug interactions.

30 mm regeneration of rat sciatic nerve along collagen filaments.

This paper describes 30 mm regeneration of peripheral nerve axons along collagen filaments; 31-mm-long collagen filaments or collagen tube were grafted to bridge a 30-mm defect of rat sciatic nerve. The mean number and the diameter of regenerated myelinated axons were 330+/-227 and 2.7+/-0.9 microm at the distal end of the collagen-filaments 12 weeks postoperatively; while at the distal end of the tube no axon was found.

Reactive oxygen species depolymerize hyaluronan: involvement of the hydroxyl radical

We have previously demonstrated that reactive oxygen species (ROS) are involved in cartilage degradation. A decrease in the size of hyaluronan (HA), which is the major macromolecule in synovial fluid and is responsible for imparting viscosity to it, is reported in arthritis patients. The purpose of this study is to determine the ROS that depolymerize HA. The luminol derivative, L-012, was used to determine the generation of ROS. To generate hydroxyl radicals, a mixture of hydrogen peroxide (H(2)O(2)) and ferrous ions (Fe(2+)) was added to HA. The antioxidants and the depolymerization of HA were studied in this system. The hydroxyl radical is one of the ROS, causing the depolymerization of HA, which reacts with L-01. These data suggest that hydroxyl radicals play an important role at the site of inflammation.

Functional restoration of rabbit spinal cord using collagen‐filament scaffold

We report the first success of functional restoration of transected rabbit spinal cord using collagen‐filament nerve scaffold. We grafted 5 mm‐long 6000 collagen filaments parallel to the axis of the spinal cord to bridge 3 mm defects of 21 adult rabbit spinal cords; 18 rabbits were used as controls. Of the 39 rabbits, 22 survived the experimental period. At 12 weeks postoperatively, regenerated axons crossed the proximal spinal cord–implant interfaces in four out of six rabbits. At 24 weeks postoperatively, regenerated axons crossed the proximal and distal spinal cord–implant interfaces in four out of six rabbits. At 24 weeks postoperatively, the Basso–Beattie–Bresnahan (BBB) locomotor rating scale scores of the rabbits in the collagen‐filament grafted group were 4.7 ± 2.3, while the score in the control group was 2.8 ± 0.5. The BBB scale scores of the grafted group were significantly better than the control group. The results suggest that the collagen‐filament nerve scaffold supports the axonal regeneration of the transected spinal cord and the restoration of function when grafted parallel to the axis of the spinal cord. The functional restoration appeared to be permanent, raising the possibility of therapeutic application in humans. Copyright

Triterpenoids from Euphorbia maculata

Abstract Two new triterpenoids were isolated together with β-amyrin acetate, taraxeryl acetate, lupenyl acetate, 3β-acetoxy-30-norlupan-20-one, α-amyrenonol and sitosterol from the whole herb of Euphorbia maculata. The structures of the new compounds were characterized as gult-5-en-3β-yl acetate and ursa-9(11):12-dien-3β-ol on the basis of chemical and spectral evidence.

Induction of hypertrophic chondrocyte-like phenotypes by oxidized LDL in cultured bovine articular chondrocytes through increase in oxidative stress

OBJECTIVE It has been reported that the lectin-like oxidized low-density lipoprotein (Ox-LDL) receptor 1 (LOX-1) is expressed by chondrocytes in osteoarthritis (OA) cartilage and that Ox-LDL binding to LOX-1 increases intracellular oxidative stress in cultured bovine articular chondrocytes (BACs). It was recently demonstrated that reactive oxygen species (ROS) induce hypertrophic differentiation of chondrocytes in the growth plate. It has also been shown that activated chondrocytes in OA have hypertrophic chondrocyte-like phenotypes. The purpose of this study was to determine whether Ox-LDL induces hypertrophic chondrocyte-like phenotypes in BACs. DESIGN Changes in type X collagen (COL10) and runt-related transcription factor 2 (Runx2) mRNA expression in BACs after Ox-LDL stimulation were investigated using real-time polymerase chain reaction (PCR). Western blotting and immunofluorescent cell staining were used to investigate changes in protein level. The antioxidant N-acetyl cysteine (NAC) was used to ascertain whether oxidative stress is involved in COL10 and Runx2 expression. We induced LOX-1 knockdown cells using small interfering RNA (siRNA) to examine the receptor specificity of Ox-LDL. RESULTS COL10 expression was upregulated by Ox-LDL in a time- and dose-dependent manner. Immunofluorescent staining showed that Ox-LDL increased COL10 production in the extracellular matrix. Ox-LDL-induced upregulation of COL10 was suppressed by pretreatment with NAC and siRNA. Expression of Runx2 was upregulated by Ox-LDL and H(2)O(2), and these effects were suppressed by NAC pretreatment. CONCLUSION Ox-LDL binding to LOX-1 induces a hypertrophic chondrocyte-like phenotype through oxidative stress, indicating that Ox-LDL plays a role in the degeneration of cartilage.

A convenient synthesis of oligonucleotides with a 3′-phosphoglycolate and 3′-phosphoglycaldehyde terminus

Abstract A simple and rapid method for solid phase synthesis of oligonucleotides carrying a 3′-phosphoglycerol terminus was developed. The modified oligonucleotide was readily oxidized by NaIO4 to a 3′-phosphoglycaldehyde. The treatment of this aldehyde with NaClO2 afforded a 3′-phosphoglycolate.

Oxidized LDL binding to LOX-1 enhances MCP-1 expression in cultured human articular chondrocytes

OBJECTIVE It has been suggested that oxidized low-density lipoprotein (ox-LDL) has some roles in progression of osteoarthritis. The purpose of this study is to investigate whether ox-LDL binding to lectin-like ox-LDL receptor 1 (LOX-1) enhances monocyte chemoattractant protein 1 (MCP-1) expression in cultured human articular chondrocytes (HACs). METHOD The time course and dose response of MCP-1 mRNA expression and MCP-1 protein release into medium following ox-LDL stimulation were investigated using quantitative Real time PCR (delta-delta Ct method) and enzyme-linked immunosorbent assay (ELISA), respectively. To examine the receptor specificity of ox-LDL action, HACs were preincubated with anti-human LOX-1 monoclonal antibody (TS92). RESULTS A time-course study revealed that MCP-1 mRNA expression increased 5.09+/-0.86 fold 12h after ox-LDL stimulation compared to time-0. ox-LDL stimulation increased MCP-1 protein level in conditioned medium in a time-dependent manner. Increased MCP-1 level was evident 6h after stimulation, reaching 830+/-91 pg/ml at 24h (33+/-8 pg/ml at time-0). Dose responses of MCP-1 expression were also evident in mRNA and protein levels. Pretreatment with TS92 markedly suppressed these stimulating effects of ox-LDL, although that with non-specific IgG did not. Native LDL did not affect MCP-1 expression. CONCLUSION Our results suggest that ox-LDL enhances MCP-1 expression in HACs and supports the hypothesis that ox-LDL is involved in cartilage degeneration.

Induction of bovine articular chondrocyte senescence with oxidized low-density lipoprotein through lectin-like oxidized low-density lipoprotein receptor 1.

OBJECTIVE Findings of recent in vivo and in vitro studies suggest that oxidized low-density lipoprotein (ox-LDL) plays a role in the degeneration of cartilage. The purpose of this study was to determine whether ox-LDL induces chondrocyte senescence through binding to lectin-like ox-LDL receptor 1 (LOX-1). METHODS The effects of ox-LDL on senescence of cultured bovine articular chondrocytes (BACs) were investigated by observing senescence-associated (SA) beta-galactosidase (beta-gal) activity, cell proliferation activity, and telomerase activity. Telomerase activity was measured after adding LY294002 (a specific inhibitor of phosphatidylinositol 3-kinase [PI3K]) or after adding insulin-like growth factor 1 (IGF-1; an activator of PI3K) plus ox-LDL to the culture medium to elucidate the involvement of the PI3K/Akt pathway. Immunoblot analysis was used to investigate whether ox-LDL affects the phosphorylation of Akt. To ascertain whether these effects were attributable to ox-LDL binding to LOX-1, BACs were preincubated with TS-20, an anti-bovine LOX-1 blocking antibody. RESULTS The activity of SA beta-gal was increased and the incorporation of bromodeoxyuridine into BACs was decreased by ox-LDL in a dose-dependent manner. The telomerase activity of BACs was suppressed by the addition of ox-LDL in a time- and dose-dependent manner. LY294002 suppressed the telomerase activity of BACs, and IGF-1 reversed the ox-LDL-induced suppression of telomerase activity. In addition, ox-LDL rapidly decreased the amount of phosphorylated Akt in BACs. Pretreatment of cultured BACs with TS-20 recovered these effects. CONCLUSION These data show that ox-LDL binding to LOX-1 induces stress-induced premature senescence of chondrocytes and results in suppression of telomerase activity by inactivating the PI3K/Akt pathway. Oxidized LDL may play an important role in the pathogenesis of osteoarthritis by inducing chondrocyte senescence.

Thermodynamic study of hybridization properties of heterochiral nucleic acids.

Heterochiral DNA and RNA heptamers, which contained an unnatural L-nucleotide, were synthesized, and thermodynamic analyses of their hybridization properties with complementary DNA and RNA strands were systematically conducted by UV melting experiments. The results clearly demonstrated that the incorporation of an L-ribonucleotide into the RNA strand leads to more significant destabilization of the duplexes than that of an L-deoxyribonucleotide into the DNA strand, regardless of whether the complementary strand is DNA or RNA. The destabilization of the duplexes by the substitution of D-thymidine with L-thymidine in the DNA strand is entropically driven, whereas that by the substitution of D-uridine with L-uridine in the RNA strand is enthalpically driven. The thermodynamic characteristic that the stability of homochiral duplex is far superior to that of heterochiral duplex is much more remarkable in RNA than in DNA. Thus, RNA might have been a self-replicating system superior to DNA to exclude the chiral antipode.