Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRalpha) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRalpha protein level in the cell is low and the LXRalpha protein itself is very hard to detect. We have previously reported that the mRNA for LXRalpha is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRalpha protein in the human macrophage, we have established a monoclonal antibody against LXRalpha, K-8607. The binding of mAb K-8607 to the human LXRalpha protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRalpha antibody should prove to be a useful tool in the analysis of the human LXRalpha protein.     

alpha-Selective glycosylation with 5-thioglucopyranosyl donors; synthesis of an isomaltotetraoside mimic composed of 5-thioglucopyranose units

A general method for alpha-selective glycosylation with 5-thioglucopyranosyl donors followed by efficient deprotection of the resulting products was developed. This methodology was utilized in the synthesis of an isomaltotetraoside analogue.     

Use of an activated carbon column for the synthesis of disaccharides by use of a reversed hydrolysis activity of β-galactosidase

Summary An activated carbon column was utilized for the synthesis of disaccharides by use of a reversed hydrolysis activity of an immobilized -galactosidase column in order to shift the equilibrium to the direction of condensation. The yields of lactulose and allo-lactulose from galactose and fructose, and N-acetyl lactosamine and N-acetyl allolactosamine from galactose and N-acetyl glucosamine, were 11.3% and 10.0%, respectively.     

Identification of a key amino acid residue of Streptomyces phospholipase D for thermostability by in vivo DNA shuffling

To isolate thermostability-related amino acid residues of Streptomyces phospholipase D (PLD), we constructed a chimeral genes library between two highly homologous plds, which exhibited different thermostabilities, by an in vivo DNA shuffling method using Escherichia coli that has a mutation of a single-stranded DNA-binding protein gene. To confirm the location of the recombination site, we carried out the restriction mapping of 68 chimeral pld genes. The recombination sites were widely dispersed over the entire pld sequence. Moreover, we examined six chimeral PLDs by comparing their thermostabilities with those of parental PLDs. To identify a thermostability-related amino acid residue, we investigated the thermostability of chimera C that was the most thermolabile among the six chimeras. We identified the thermostability-related factor Gly-188, which is located in the alpha-7 helix of PLD from Streptomyces septatus TH-2 (TH-2PLD). TH-2PLD mutants, in which Gly-188 was substituted with Phe, Val or Trp, exhibited higher thermostabilities than that of the parental PLD. Gly-188 substituted with the Phe mutant, which was the most stable among the mutants, showed an enzyme activity almost the same as that of TH-2PLD as determine by kinetic analysis.     

Conformational restriction approach to BACE1 inhibitors II: SAR study of the isocytosine derivatives fixed with a cis-cyclopropane ring

To improve the efficacy of the conformationally restricted BACE1 inhibitors, structural modifications were investigated using two strategies: (a) modification of the terminal aromatic ring and (b) insertion of a spacer between the aromatic rings. In the latter approach, another type of inhibitor 17 bearing an ethylene spacer between two aromatic rings was found to exhibit good BACE1 inhibitory activity, while the corresponding conformationally unrestricted compound 25 showed no activity. This result revealed an interesting effect of a conformational restriction with a cyclopropane ring.     

c-ABL tyrosine kinase stabilizes RAD51 chromatin association

The assembly of RAD51 recombinase on DNA substrates at sites of breakage is essential for their repair by homologous recombination repair (HRR). The signaling pathway that triggers RAD51 assembly at damage sites to form subnuclear foci is unclear. Here, we provide evidence that c-ABL, a tyrosine kinase activated by DNA damage which phosphorylates RAD51 on Tyr-315, works at a previously unrecognized, proximal step to initiate RAD51 assembly. We first show that c-ABL associates with chromatin after DNA damage in a manner dependent on its kinase activity. Using RAD51 mutants that are unable to oligomerize to form a nucleoprotein filament, we separate RAD51 assembly on DNA to form foci into two steps: stable chromatin association followed by oligomerization. We show that phosphorylation on Tyr-315 by c-ABL is required for chromatin association of oligomerization-defective RAD51 mutants, but is insufficient to restore oligomerization. Our findings suggest a new model for the regulation of early steps of HRR.     

Characterization of a variety of neutralizing anti-heparin-binding epidermal growth factor-like growth factor monoclonal antibodies by different immunization methods

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family. The accumulated evidence on the tumor-progressing roles of HB-EGF has suggested that HB-EGF-targeted cancer therapy is expected to be promising. However, the generation of neutralizing anti-HB-EGF monoclonal antibodies (mAbs) has proved difficult. To overcome this difficulty, we performed a hybridoma approach using mice from different genetic backgrounds, as well as different types of HB-EGF immunogens. To increase the number of hybridoma clones to screen, we used an electrofusion system to generate hybridomas and a fluorometric microvolume assay technology to screen anti-HB-EGF mAbs. We succeeded in obtaining neutralizing anti-HB-EGF mAbs, primarily from BALB/c and CD1 mice, and these were classified into 7 epitope bins based on their competitive binding to the soluble form of HB-EGF (sHB-EGF). The mAbs showed several epitope bin-dependent characteristics, including neutralizing and binding activity to human sHB-EGF, cross-reactivity to mouse/rat sHB-EGF and binding activity to the precursor form of HB-EGF. The neutralizing activity was also validated in colony formation assays. Interestingly, we found that the populations of mAb bins and the production rates of the neutralizing mAbs were strikingly different by mouse strain and by immunogen type. We succeeded in generating a variety of neutralizing anti-HB-EGF mAbs, including potent sHB-EGF neutralizers that may have potential as therapeutic agents for treating HB-EGF-dependent cancers. Our results also suggest that immunization approaches using different mouse strains and immunogen types affect the biological activity of individual neutralizing antibodies.     

Differential regulation of intracellular redox state by extracellular matrix proteins in glomerular mesangial cells: potential role in diabetic nephropathy

Advanced diabetic nephropathy is characterized by abnormal synthesis of extracellular matrix (ECM) proteins, such as collagen I (COL I). The present experiments were designed to test the hypothesis that the presence of abnormal ECM proteins may be responsible for increased generation of reactive oxygen species (ROS) that are thought to have an important role in the pathogenesis of diabetic nephropathy. SV40 MES 13 murine mesangial cells were plated on COL I or collagen IV (COL IV) for 3 h at 5.5 or 25 mM D-glucose concentration. Increased intracellular ROS generation and reduced intracellular nitric oxide (NO) production was measured in cells attached to COL I compared with cells attached to COL IV. Treatment with N(omega)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of NO synthase, reduced this difference in ROS generation between cells attached to either COL I or IV. The results using antibodies against integrins also indicated that an alpha(2) integrin-mediated pathway was involved in the different response in ROS generation caused by ECM proteins. These results suggest that contact between altered ECM proteins that are present in advanced diabetic nephropathy and mesangial cells has the potential to increase intracellular oxidative stress, leading to progressive glomerular damage.