Plasmodium falciparum serine-repeat antigen (SERA) forms a homodimer through disulfide bond

Plasmodium falciparum serine-repeat antigen (SERA) is one potential blood-stage vaccine candidate and is expressed as a protomer that is subsequently processed into four fragments (P47, P50, P6, and P17). Although recent evidence shows that P50 exhibits chymotrypsin-like protease activity, the function of SERA is still largely unknown. Here, we found that apart from cathepsin L-like cysteine protease, P50 showed significant homology to silicatein-α and testin which were shown to bind to cellular components, suggesting that SERA may have similar function. Immunoprecipitation of schizont lysate and molecular assignment of its precipitate by mass spectrometry provided evidence that SERA forms a homodimer through disulfide bond. Moreover, analysis of the fate of SERA using cell-free system revealed that the kinetics of conversion of SERA dimer into monomer is faster than that of processing of SERA monomer into various fragments. These findings may contribute to elucidate a possible function of SERA other than a protease.     

Hepatocyte adhesion on a poly[N-p-vinylbenzyl-4-O-beta-D-galactopyranosyl-D-glucoamide]-coated poly(L-lactic acid) surface

A surface of poly(l-lactic acid) (PLLA) was modified by coating with poly[N-p-vinylbenzyl-4-O-beta-d-galactopyranosyl-d-glucoamide] (PVLA), which was employed to improve the hepatocyte adhesion owing to its amphiphilic property and the presence of a hepatocyte recognition motif. We characterized the surface properties through water contact angle, electron spectroscopy for chemical analysis (ESCA), and scanning probe microscopy (SPM). The effect of PVLA coating on the efficiency of hepatocyte adhesion was evaluated by protein assay and optical microscopy. The surface morphology was under the influence of the concentration of PVLA coating solution and it played a critical role in hepatocyte adhesion. It was confirmed that galactose moieties in PVLA, which can bind to the asialoglycoprotein receptor (ASGPR) on hepatocytes, have a more dominant effect on hepatocyte adhesion than enhanced hydrophilicity. We suggest that the PVLA-PLLA system will be a useful method to improve hepatocyte cell seeding and adhesion onto scaffold matrices.     

Validation study of a method for assaying DE-310, a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, and the free drug in human plasma by HPLC and LC/MS/MS

DE-310 is a macromolecular carrier conjugate containing an anti-tumor camptothecin derivative, DX-8951, conjugated to a water-soluble polymer by means of a peptide spacer. New assay methods have been developed to determine the polymer-bonded DX-8951 conjugate, free DX-8951, and Glycyl-DX-8951 in human plasma. Solid-phase extraction was used to extract free DX-8951 and Glycyl-DX-8951 from plasma, and LC/MS/MS (Method I) was used to determine the amount of each analyte. Protein precipitation was used to extract Conjugated DX-8951, which was then digested with thermolysin. HPLC (Method II) was used to determine the productive compound (Phenylalanyl-Glycyl-DX-8951). The lower limit of quantitation of DX-8951 was 50 pg/ml, of Glycyl-DX-8951 was 80 pg/ml, and of Conjugated DX-8951 was 100 ng/ml (as DX-8951 equivalent). Both methods showed satisfactory sensitivity, precision, and accuracy.     

Measurement of homocysteine thiolactone hydrolase activity using high-performance liquid chromatography with fluorescence detection and polymorphisms of paraoxonase in normal human serum

We developed a non-radioactive and sensitive assay method for measurement of the HTL hydrolase (HTLase) activity in biological samples, using OPA as a fluorescent post-labeling agent, l-homocysteine thiolactone (L-HTL) as the substrate, and HPLC to achieve rapid and selective separation of the substrate and product. The method was applied to measure the activity of HTLase in human, rabbit, rat and mouse serum samples. In addition, the correlation between the serum HTLase activity and PON1 polymorphisms in Japanese subjects was also investigated. The serum HTLase activity in humans, as determined by measurement of the enzyme activity in 22 subjects, was found to be in the range of 0.89-2.06 nmol/min mg protein, with a mean activity of 1.44 nmol/min mg protein.     

Anti-tumor-initiating effects of monascin, an azaphilonoid pigment from the extract of Monascus pilosus fermented rice (red-mold rice)

Monascin (1) constitutes one of the azaphilonoid pigments in the extracts of Monascus pilosus-fermented rice (red-mold rice). Compound 1 was evaluated for its anti-tumor-initiating activity via oral administration on the two-stage carcinogenesis of mouse skin tumor induced by peroxynitrite (ONOO-; PN) or by ultraviolet light B (UVB) as an initiator and 12-O-tetradecanoylphorbol-13-acetate (TPA) as a promoter. Compound 1 exhibited marked inhibitory activity on both PN- and UVB-induced mouse skin carcinogenesis tests. These findings suggest that compound 1 may be valuable as potential cancer chemopreventive agent in chemical and environmental carcinogenesis.     

Formation of retinoylated proteins from retinoyl-CoA in rat tissues

Retinoylation (acylation of proteins by retinoic acid) is considered as one mechanism of retinoic acid (RA) action occurring in cells in vitro and in vivo. Previously, our studies showed that in rat tissues the formation of retinoyl-CoA from RA, the first step of retinoylation, required ATP, CoA and MgCl(2). In the current study, we examined whether the transfer of retinoyl-CoA into proteins, the second step of retinoylation, occurs in rat tissues. [(3)H]-Labeled-retinoyl-CoA bound covalently to proteins in rat liver, kidney, testis, and brain. The levels of incorporation of retinoyl-CoA into proteins were higher in vitamin A-deficient rats than in normal ones. The formation of retinoylated proteins depended on the incubation time, and the concentrations of retinoyl-CoA and homogenate. The reaction was suppressed by fatty acyl-CoAs and palmitic acid, but not by arachidonic acid. The Vmax and Km values for retinoyl-CoA in the formation of retinoylated proteins using a crude liver extract were estimated to be 2,597.3 pmol/min/mg protein and 9.5 x 10(-5) M, respectively. Retinoylated proteins formed from retinoyl-CoA, including a 17 kDa protein exhibiting high radioactivity, disappeared in the presence of 2-mercaptoethanol, indicating that RA was linked to the proteins through a thioester bond. These results demonstrate that retinoylation in rat tissues occurs via retinoyl-CoA formed from RA. This process may play a significant physiological role in cells.     

A novel eukaryotic selenoprotein in the haptophyte alga Emiliania huxleyi

The diversity of selenoproteins raises the question of why many life forms require selenium. Especially in photosynthetic organisms, the biochemical basis for the requirement for selenium is unclear because there is little information on selenoproteins. We found six selenium-containing proteins in a haptophyte alga, Emiliania huxleyi, which requires selenium for growth. The 27-kDa protein EhSEP2 was isolated, and its cDNA was cloned. The deduced amino acid sequence revealed that EhSEP2 is homologous to protein disulfide isomerase (PDI) and contains a highly conserved thioredoxin domain. The nucleotide sequence contains an in-frame TGA codon encoding selenocysteine at the position corresponding to the cysteine residue in the reaction center of known PDIs. However, no typical selenocysteine insertion sequence was found in the EhSEP2 cDNA. The EhSEP2 mRNA level was related to the abundance of selenium. E. huxleyi possesses a novel PDI-like selenoprotein and may have a novel type of selenocysteine insertion machinery.     

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy.