In order to reduce vehicle emitted greenhouse gases (GHGs) on a global scale, the scope of consideration should be expanded to include the manufacturing, fuel extraction, refinement, power generation, and end-of-life phases of a vehicle, in addition to the actual operational phase. In this paper, the CO2 emissions of conventional gasoline and diesel internal combustion engine vehicles (ICV) were compared with mainstream alternative powertrain technologies, namely battery electric vehicles (BEV), using life-cycle assessment (LCA). In most of the current studies, CO2 emissions were calculated assuming that the region where the vehicles were used, the lifetime driving distance in that region and the CO2 emission from the battery production were fixed. However, in this paper, the life cycle CO2 emissions in each region were calculated taking into consideration the vehicle’s lifetime driving distance in each region and the deviations in CO2 emissions for battery production. For this paper, the US, European Union (EU), Japan, China, and Australia were selected as the reference regions for vehicle operation. The calculated results showed that CO2 emission from the assembly of BEV was larger than that of ICV due to the added CO2 emissions from battery production. However, in regions where renewable energy sources and low CO2 emitting forms of electric power generation are widely used, as vehicle lifetime driving distance increase, the total operating CO2 emissions of BEV become less than that of ICV. But for BEV, the CO2 emissions for replacing the battery with a new one should be added when the lifetime driving distance is over 160,000 km. Moreover, it was shown that the life cycle CO2 emission of ICV was apt to be smaller than that of BEV when the CO2 emissions for battery production were very large.

We found a novel human gene (GenBank TM accession number AB037823, Kazusa DNA Research Institute KIAA1402) that possesses homology with chondroitin synthase. The full-length open reading frame consists of 772 amino acids and encodes a typical type II membrane protein. This enzyme had a domain containing ␤3-glycosyltransferase motifs, which might be a ␤3-glucuronyltransferase domain, but no domain with ␤4-glycosyltransferase motifs, although both are found in chondroitin synthase. The putative catalytic domain was expressed in COS-7 cells as a soluble enzyme. Its glucuronyltransferase activity was observed when chondroitin and chondroitin sulfate polysaccharides and oligosaccharides were used as acceptor substrates. However, it was not detected when dermatan sulfate, hyaluronan, heparan sulfate, heparin, N-acetylheparosan, lactosamine tetrasaccharide, and linkage tri-and tetrasaccharide acceptors were employed. The reaction product, which was speculated to exhibit a GlcA␤1-3GalNAc linkage structure at its non-reducing terminus, showed the following characteristics. 1) It was catabolized by ␤-glucuronidase. 2) It was an acceptor for Escherichia coli K4 chondroitin polymerase (K4 chondroitin polymerase). 3) The product of K4 chondroitin polymerase was cleaved by chondroitinase ACII. On the other hand, no N-acetylgalactosaminyltransferase activity was detected toward any acceptors. Quantitative real time PCR analysis revealed that its transcripts were highly expressed in the placenta, small intestine, and pancreas, although they were ubiquitously expressed in various tissues and cell lines. This enzyme could play a role in the synthesis of chondroitin sulfate as a glucuronyltransferase.

By a tblastn search with ␤1,4-galactosyltransferases as query sequences, we found an expressed sequence tag that showed similarity in ␤1,4-glycosyltransferase motifs. The full-length complementary DNA was obtained by a method of 5-rapid amplification of complementary DNA ends. The predicted open reading frame encodes a typical type II membrane protein comprising 543 amino acids, the sequence of which was highly homologous to chondroitin sulfate N-acetylgalactosaminyltransferase (CSGalNAcT-1), and we designated this novel enzyme CSGalNAcT-2. CSGalNAcT-2 showed much stronger Nacetylgalactosaminyltransferase activity toward glucuronic acid of chondroitin poly-and oligosaccharides, and chondroitin sulfate poly-and oligosaccharides with a ␤1-4 linkage, i.e. elongation activity for chondroitin and chondroitin sulfate, but showed much weaker activity toward a tetrasaccharide of the glycosaminoglycan linkage structure (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), i.e. initiation activity, than CSGalNAcT-1. Transfection of the CSGalNAcT-1 gene into Chinese hamster ovary cells yielded a change of glycosaminoglycan composition, i.e. the replacement of heparan sulfate on a syndecan-4/fibroblast growth factor-1 chimera protein by chondroitin sulfate, however, transfection of the CSGalNAcT-2 gene did not. The above results indicated that CSGalNAcT-1 is involved in the initiation of chondroitin sulfate synthesis, whereas CSGalNAcT-2 participates mainly in the elongation, not initiation. Quantitative real-time PCR analysis revealed that CSGalNAcT-2 tran-

We found a novel glycosyltransferase gene having a hypothetical ␤1,4-galactosyltransferase motif (GenBank TM accession number AB081516) by a BLAST search and cloned its full-length open reading frame using the 5-rapid amplification of cDNA ends method. The truncated form was expressed in insect cells as a soluble enzyme. It transferred N-acetylgalactosamine, not galactose, to para-nitrophenyl-␤-glucuronic acid. The N-acetylgalactosamine-glucuronic acid linkage has been identified only in chondroitin sulfate; therefore, we examined its chondroitin elongation and initiation activities. N-Acetylgalactosaminyltransferase activity was observed toward chondroitin poly-and oligosaccharides, chondroitin sulfate oligosaccharides, and linkage tetrasaccharide (GlcA-Gal-Gal-Xyl-O-methoxyphenyl), and the chondroitin polysaccharide and linkage tetrasaccharide were better acceptor substrates than the others. Northern blot analysis and quantitative realtime PCR analysis revealed that its 4-kb transcripts were highly expressed in thyroid and placenta, although they were ubiquitously expressed in various tissues and cells. These results suggest that this enzyme has N-acetylgalactosaminyltransferase activity in both the elongation and initiation of chondroitin sulfate synthesis. Furthermore, we performed enzymatic synthesis of chondroitin pentasaccharide in vitro. In one tube reaction with four enzymes, ␤1,4-galactosyltransferase-VII, ␤1,3-galactosyltransferase-VI, glucuronyltransferase-I, and this enzyme, and a synthetic xylose-peptide acceptor, the structure GalNAc-GlcA-Gal-Gal-Xyl-peptide was constructed. This is the first report of a chondroitin pentasaccharide constructed with recombinant glycosyltransferases in vitro.

35 S]PAPS, the highest incorporation of 35 S was observed, and digestion of the product with a mixture of heparin lyases yielded two major 35 S-labeled disaccharides, which were determined as ⌬HexA-GlcN(NS,3S,6S) and ⌬HexA(2S)-GlcN(NS,3S) by further digestion with 2-sulfatase and degradation with mercuric acetate. However, when used heparin as acceptor, we identified a highly sulfated disaccharide unit as a major product. This had a structure of ⌬HexA(2S)-GlcN(NS,3S,6S). Quantitative real-time PCR analysis revealed that 3-OST-5 was highly expressed in fetal brain, followed by adult brain and spinal cord, and at very low or undetectable levels in the other tissues. Finally, we detected a tetrasulfated disaccharide unit in bovine intestinal heparan sulfate. To our knowledge, this is the first report to describe not only the natural occurrence of tetrasulfated disaccharide unit but also the enzymatic formation of this novel structure.

Crude enzyme obtained from chondroitin sulfate-induced Proteus vulgaris NCTC 4636 has been fractionated into 1) an endoeliminase capable of depolymerizing chondroitin sulfate and related polysaccharides to produce, as end products, a mixture of ⌬ 4 -unsaturated tetra-and disaccharides and 2) an exoeliminase preferentially acting on chondroitin sulfate tetra-and hexasaccharides to yield the respective disaccharides. Isolation of the two enzymes was achieved by a simple two-step procedure: extracting the enzymes from intact P. vulgaris cells with a buffer solution of nonionic surfactant and then treating the extract by cation-exchange chromatography. Each of the enzymes thus prepared was apparently homogeneous as assessed by SDS-polyacrylamide gel electrophoresis and readily crystallized from polyethylene glycol solutions. Both enzymes acted on various substrates such as chondroitin sulfate, chondroitin sulfate proteoglycan, and dermatan sulfate at high, but significantly different, initial rates. They also attacked hyaluronan but at far lower rates and were inactive to keratan sulfate, heparan sulfate, and heparin. Our results show that the known ability of the conventional enzyme called "chondroitinase ABC" to catalyze the complete depolymerization of chondroitin sulfates to unsaturated disaccharides may actually result from the combination reactions by endoeliminase (chondroitin sulfate ABC endolyase) and exoeliminase (chondroitin sulfate ABC exolyase).Chondroitin sulfate ABC lyase (EC 4.2.2.4) was first purified from extracts of Proteus vulgaris NCTC 4636 adapted to chondroitin 6-sulfate (1). It is believed to be an endoeliminase that splits ␤1,4-galactosaminidic bonds between N-acetylgalactosamine and either D-glucuronic acid or L-iduronic acid and degrades, therefore, a variety of glycosaminoglycans of the chondroitin sulfate and dermatan sulfate type to the respective unsaturated disaccharides. Using this enzyme, both chondroitin sulfates and dermatan sulfates were shown to contain, in addition to predominant 4-or 6-sulfated disaccharide residues, a smaller proportion of nonsulfated or disulfated disaccharide residues, or both (2). A variety of studies have since shown that the proportion of these disaccharide residues varies greatly with species and anatomical sites, during development and aging, and in pathology (3-11 among others). Many of these data suggest that variation in the carbohydrate sequence and sulfation pattern may be used to specify the functional properties of chondroitin sulfate and dermatan sulfate proteoglycans.There is a commercially available chondroitin sulfate ABC lyase ("chondroitinase ABC" from P. vulgaris, the product of Seikagaku Corp.) which has found wide applications including the quantification of chondroitin sulfate and dermatan sulfate (12), the structural analysis of the carbohydrate moiety of proteoglycans (13-16), the preparation of core proteins from proteoglycans (13, 17), the formation of antigenic epitopes to prepare anti-proteoglycan monoclonal antibodies (18), and ...

Poly(phenyl methacrylate) (PPhMA), poly(methyl methacrylate) (PMMA), poly(tert-butyl methacrylate), polystyrene (PS), poly(4-methylstyrene), poly(p-tert-butylstyrene), and polycarbonate (PC) were doped with the photochromic dye cis-1,2-dicyano-1,2-bis (2,4,5-trimethyl-3-thienyl)ethene (CMTE) using a simple vacuum process termed the "vapor transportation method." The CMTE-doped polymer molds and powder were examined by optical microscopy, SEM/energy-dispersive X-ray (EDX) elemental analysis, Fourier transform infrared absorption (FT-IR), and transmission electron microscopy (TEM). CMTE-doped PS, PC, and PMMA molds showed photochromism. Optical microscopy and sulfur (S) elemental analysis confirmed formation of CMTE-doped regions, with rapid CMTE concentration decreases at the CMTE-doped region/polymer substrate interface. CMTE doping rates into PS, PC, and PMMA were estimated by measuring the depths of the doped regions. CMTE showed more efficient penetration into PS than into PC or PMMA. FT-IR measurements showed saturated concentrations of CMTE in the CMTE-doped polymer powder. PS and PPhMA, with phenyl groups in their side chains, showed the highest concentrations of CMTE among PS derivatives and PMMA derivatives, respectively. Our results indicated that the affinity between CMTE and aromatic groups in these polymers, based on "π-π interactions," enhances CMTE doping. Ultrathin sectioned TEM images of CMTE-doped diblock copolymers, "PS-rich PS-block-PMMA (PS-b-PMMA)," and CMTE-doped "PMMA-rich PS-b-PMMA" films confirmed that CMTE was dispersed selectively into PS nanodomains in "PMMA-rich PS-b-PMMA" and the PS matrix in "PS-rich PS-b-PMMA." We demonstrated selective doping of CMTE into the PS regions of PS-b-PMMA.

We previously reported that the heparan sulfate 3-O-sulfotransferase (3OST)-5 produces a novel component of heparan sulfate, i.e. the tetrasulfated disaccharide (Di-tetraS) unit (Mochizuki, H., Yoshida, K., Gotoh, M., Sugioka, S., Kikuchi, N., Kwon, Y.-D., Tawada, A., Maeyama, K., Inaba, N., Hiruma, T., Kimata, K., and Narimatsu, H. (2003) J. Biol. Chem. 278, 26780 -26787). In the present study, we investigated the potential of other 3OST isoforms to produce Di-tetraS with heparan sulfate and heparin as acceptor substrates. 3OST-2, 3OST-3, and 3OST-4 produce Di-tetraS units as a major product from both substrates. 3OST-5 showed the same specificity for heparin, but the production from heparan sulfate was very low. Di-tetraS production by 3OST-1 was negligible. We then investigated the presence of Di-tetraS units in heparan sulfates from various rat tissues. Di-tetraS was detected in all of the tissues analyzed. Liver and spleen contain relatively high levels of Di-tetraS, 1.6 and 0.95%, respectively. However, the content of this unit in heart, large intestine, ileum, and lung is low, less than 0.2%. We further determined the expression levels of 3OST transcripts by quantitative real time PCR. The 3OST-3 transcripts are highly expressed in spleen and liver. The 3OST-2 and -4 are specifically expressed in brain. These results indicate that the Di-tetraS unit is widely distributed throughout the body as a rare and unique component of heparan sulfate and is synthesized by tissue-specific 3OST isoforms specific for Di-tetraS production.