An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3‐N‐Acetyl‐D‐glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose‐Binding Protein

A fusion protein composed of β1,3‐N‐acetyl‐D ‐glucosaminyltransferase (β1,3‐GlcNAcT) from Streptococcus agalactiae type Ia and maltose‐binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP‐β1,3‐GlcNAcT) did not show any sugar‐elongation activity to some simple low‐molecular weight acceptor substrates such as galactose, Galβ(1→4)Glc (lactose), Galβ(1→4)GlcNAc (N‐acetyllactosamine), Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)Glc (lacto‐N‐tetraose), and Galβ(1→4)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer‐mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β‐GlcNAc residue at the O‐3 position of the non‐reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(1→3)Galβ(1→4)Glc was subjected to further enzymatic modifications by using recombinant β1,4‐D ‐galactosyltransferase (β1,4‐GalT), α2,3‐sialyltransferase (α2,3‐SiaT), α1,3‐L ‐fucosyltransferase (α1,3‐FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→3)Galβ(1→4)GlcCerα(IV3Neu5Acα,III3Fucα‐nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP‐β1,3‐GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N‐acetyl‐D ‐galactosamine served from UDP‐GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(1→3)Galβ(1→4)Glc. The versatility of the MBP‐β1,3‐GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding‐affinity with MBP.     

Catalytic Friedel–Crafts Acylation of Heteroaromatics

Catalytic Friedel–Crafts acylation of heteroaromatics has been achieved using metal triflates as catalysts. While conventional Friedel–Crafts acylation often requires the use of more than stoichiometric amounts of aluminum chloride, metal triflates such as tin(II) triflate, scandium triflate, and gallium triflate, etc. have enabled efficient acylation reactions by catalytic use.     

Increased Lipid Synthesis and Decreased β-Oxidation in the Liver of SHR/NDmcr-cp (cp/cp) Rats, an Animal Model of Metabolic Syndrome

SHR/NDmcr-cp (cp/cp) rats (SHR/NDcp) are an animal model of metabolic syndrome. A previous study of ours revealed drastic increases in the mass of palmitic (16:0), oleic (18:1n-9), palmitoleic (16:1n-7), cis-vaccenic (18:1n-7) and 5,8,11-eicosatrienoic acids in the liver of SHR/NDcp. However, detailed information on the class of lipid accumulated and the mechanism responsible for the overproduction of the accumulated lipid in the liver was not obtained. This study aimed to characterize the class of lipid accumulated and to explore the mechanism underlying the lipid accumulation in the liver of SHR/NDcp, in comparison with SHR/NDmcr-cp (+/+) (lean hypertensive littermates of SHR/NDcp) and Wistar Kyoto rats. In the liver of SHR/NDcp, de novo synthesis of fatty acids (16:0, 18:1n-9 and 16:1n-7) and triacylglycerol (TAG) synthesis were up-regulated and fatty acid β-oxidation was down-regulated. These perturbations of lipid metabolism caused fat accumulation in hepatocytes and accumulation of TAG, which were enriched with 16:0, 18:1n-9 and 16:1n-7, in the liver of SHR/NDcp. On the other hand, no changes were found in hepatic contents of diacylglycerol and unesterified fatty acid (FFA); among FFA, there were no differences in the hepatic concentrations of unesterified 16:0 and stearic acid between SHR/NDcp and two other groups of rats. Moreover, little change was brought about in the expression of genes responsive to endoplasmic reticulum stress in the liver of SHR/NDcp. These results may reinforce the pathophysiological role of stearoyl-CoA desaturase 1 and fatty acid elongase 6 in the liver of SHR/NDcp.     

Energy value and digestibility of dietary oil containing mainly 1,3-diacylglycerol are similar to those of triacylglycerol

Diacylglycerol (DAG) is a component of various vegetable oils. Approximately 70% of the DAG in edible oils are in the configuration of 1,3-DAG. We recently showed that long-term ingestion of dietary oil containing mainly 1,3-DAG reduces body fat accumulation in humans as compared to triacylglycerol (TAG) oil with a similar fatty acid composition. As the first step to elucidate the mechanism for this result, we examined the difference in the bioavailabilities of both oils by measuring food energy values and digestibilities in rats. Energy values of the DAG oil and the TAG oil, measured by bomb calorimeter, were 38.9 and 39.6 kJ/g, respectively. Apparent digestibility expressed according to the formula: (absorbed) x (ingested)⁻¹x100=(ingested—excreted in feces)x(ingested)⁻¹x100 for the DAG oil and the TAG oil were 96.3±0.4 and 96.3±0.3% (mean±SEM), respectively. The similarity in the bioavailabilities of both oils supports the hypothesis that the reduced fat accumulation by dietary DAG is caused by the different metabolic fates after the absorption into the gastrointestinal epithelial cells.     

Effect of the Helical Tether of a Resin‐Supported Peptide Catalyst for Friedel–Crafts‐Type Alkylation in Water

A detailed investigation of the helical part of the resin‐supported peptide catalyst possessing a turn motif and helical unit was conducted to clarify the structure‐activity relationship. The peptide catalyst with an α‐ or 3₁₀‐helical tether was effective for an enantioselective Friedel–Crafts‐type alkylation in water. From the spectral analysis of the peptide with an optimum sequence, it was demonstrated that the helical moiety of the peptide catalyst played a role for stabilizing a terminal turn structure.     

Relationship between Expression of Onco-Related miRNAs and the Endoscopic Appearance of Colorectal Tumors

Accumulating data indicates that certain microRNAs (miRNAs or miRs) are differently expressed in samples of tumors and paired non-tumorous samples taken from the same patients with colorectal tumors. We examined the expression of onco-related miRNAs in 131 sporadic exophytic adenomas or early cancers and in 52 sporadic flat elevated adenomas or early cancers to clarify the relationship between the expression of the miRNAs and the endoscopic morphological appearance of the colorectal tumors. The expression levels of miR-143, -145, and -34a were significantly reduced in most of the exophytic tumors compared with those in the flat elevated ones. In type 2 cancers, the miRNA expression profile was very similar to that of the exophytic tumors. The expression levels of miR-7 and -21 were significantly up-regulated in some flat elevated adenomas compared with those in exophytic adenomas. In contrast, in most of the miR-143 and -145 down-regulated cases of the adenoma-carcinoma sequence and in some of the de novo types of carcinoma, the up-regulation of oncogenic miR-7 and/or -21 contributed to the triggering mechanism leading to the carcinogenetic process. These findings indicated that the expression of onco-related miRNA was associated with the morphological appearance of colorectal tumors.     

Investigation of controllable self-organized honeycomb micro-structure

Patterned micro- and nanostructure are of great significance in industrial applications such as electronics, optics and sensors. Microporous honeycomb film of polyhpenylene oxide (PPO) was fabricated as the template via evaporation of solution in carbon bisulfide under humid ambience. The effect of fabrication conditions on patterned microstructure was investigated by self-organization experiments to build quantitative relationship between ambient conditions such as humidity, concentration and honeycomb microstructure (diameter and height), through which the honeycomb film formation can be controlled to self-organize desirable PPO patterned microstructure. Especially, the height of honeycomb was derived from the diameter of honeycomb, and its validity was clarified by morphological comparison between PPO template and PDMS molded structure. Moreover, soft mold experiments were conducted to demonstrate its high efficiency and excellence as an alternative to construct regular micro-pattern.     

NAD+‐Dependent Dehydrogenase PctP and Pyridoxal 5′‐Phosphate Dependent Aminotransferase PctC Catalyze the First Postglycosylation Modification of the Sugar Intermediate in Pactamycin Biosynthesis

The unique five‐membered aminocyclitol core of the antitumor antibiotic pactamycin originates from d ‐glucose, so unprecedented enzymatic modifications of the sugar intermediate are involved in the biosynthesis. However, the order of the modification reactions remains elusive. Herein, we examined the timing of introduction of an amino group into certain sugar‐derived intermediates by using recombinant enzymes that were encoded in the pactamycin biosynthesis gene cluster. We found that the NAD⁺‐dependent alcohol dehydrogenase PctP and pyridoxal 5′‐phosphate dependent aminotransferase PctC converted N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophonone into 3′‐amino‐3′‐deoxy‐N‐acetyl‐d ‐glucosaminyl‐3‐aminoacetophenone. Further, N‐acetyl‐d ‐glucosaminyl‐3‐aminophenyl‐β‐oxopropanoic acid ethyl ester was converted into the corresponding 3′‐amino derivative. However, PctP did not oxidize most of the tested d ‐glucose derivatives, including UDP‐GlcNAc. Thus, modification of the GlcNAc moiety in pactamycin biosynthesis appears to occur after the glycosylation of aniline derivatives.     

Fabrication and piezoelectric properties of BaTiO3/BaTiO3-Bi(Mg1/2Ti1/2)O3-BiFeO3 composites

We fabricated xBaTiO₃ (BT)/(1-x)[BaTiO₃-Bi(Mg_1/2Ti_1/2)O₃-BiFeO₃] (BT-BMT-BF)?+?0.1?wt%MnCO₃ composites by spark plasma sintering and investigated the effect of BT content x, BT powder size, and BT-BMT-BF composition on piezoelectric properties. For xBT/(1-x)(0.3BT-0.1BMT-0.6BF) +?0.1?wt%MnCO₃ (x?=?0–0.75) composites with a 0.5-µm BT powder, the dielectric constant was increased with x, and the relative density was decreased at x?=?0.67 and 0.75, creating optimum BT content of x?=?0.50 with a piezoelectric constant d₃₃ of 107?pC/N. When a larger 1.5-µm BT powder was utilized for the composite with x?=?0.50, the d₃₃ value increased to 150?pC/N due to the grain size effect of the BT grains. To compensate for a compositional change from the optimum 0.3BT-0.1BMT-0.6BF due to partial diffusion between the BT and 0.3BT-0.1BMT-0.6BF grains, a 0.5BT/0.5(0.275BT-0.1BMT-0.625BF)?+?0.1?wt%MnCO₃ composite with the 1.5-µm BT powder was fabricated. We obtained an increased d₃₃ value of 166?pC/N. These results provided a useful composite design to enhance the piezoelectric properties.