Synthesis of azide derivative and discovery of glyoxalase pathway inhibitor against pathogenic bacteria

A glyoxalase inhibitor was synthesized and tested against Staphylococcus aureus for first time and showed MIC₉₀ of 20 μg/ml. Henceforth, we synthesized unnatural azide derivative of the same inhibitor to improve the biological activity. In that order, an azide carboxylate was synthesized from dimethyl tartrate by tosylation and azide substitution. The synthesized, azide compound was coupled with glutathione derivative in high yield and tested against S. aureus and showed improved MIC₉₀ of 5 μg/ml. In general, it can be also easily converted to unnatural β-amino acid in good yield. The shown methodology will be extended to study induced suicide in Burkholderia mallei, Francisella tularensis and Mycobacterium tuberculosis in future.     

Antioxidant Properties of 2-O-β-D-Glucopyranosyl-L-ascorbic Acid

The antioxidant activity of a provitamin C agent, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), was compared to that of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS^?+)-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2βG slowly and continuously scavenged DPPH radicals and ABTS^?+ in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2βG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2βG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2βG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2βG appeared to be comparable not only to that of AA-2G but also to that of AA.     

Recognition of Ribose Moiety by Adenosine (Phosphate) Deaminase from Squid Liver

An adenosine (phosphate) deaminase from the squid liver had much lower activity for 5′-deoxyadenosine than that for adenosine, 2′-, or 3′-deoxyadenosine. 3′-IMP and inosine as well as purine riboside and adenine competitively inhibited the deamination of adenosine 3′ phenylphosphonate by the enzyme, but 5′-AMP and 5′-IMP did not. The enzyme deaminated the 5′-hydroxyl terminal adenosine residue in dinucleotides and trinucleotide, but not the 3′-hydroxyl terminal one in dinucleotides. The 5′-hydroxyl group of the ribose moiety was necessary for the substrate binding and catalytic activity of the squid enzyme. These results indicated that the recognition of ribose moiety in the substrate by the squid enzyme might be intermediate between those by adenosine deaminase and adenosine (phosphate) deaminase from microorganisms.     

Induction of Non-diapause Eggs by Imidazole Derivative KK-42 in the Diapause Type of Bombyx mori Silkworm

The injection of an imidazole compound, KK-42, into fifth instar larvae of a silkworm (Bombyx mori, Daizo strain), which had been destined to produce diapause eggs, induced the moths to lay non-diapause eggs. The critical period for KK-42 injection in the induction of non-diapause eggs was 24 h to 72 h after the fourth ecdysis. Topical application of KK-42 to 48 h-old fifth instar larvae also induced non-diapause eggs.     

Assay of DNA Photolyase Activity in Spinach Leaves in Relation to Cell Compartmentation-Evidence for Lack of DNA Photolyase in Chloroplasts

Spinach cyclobutane pyrimidine dimer (CPD)-specific DNA photolyase was successfully detected in leaf extracts by an assay system for plant photolyase using an improved enzyme-linked immunosorbent assay (ELISA) which was newly introduced by novel horseradish peroxidase (HRP)-linked CPD specific monoclonal antibodies. The assay system includes two main steps: a photorepair reaction of CPD introduced in substrate DNA and measurement of CPD remained after the photorepair by the improved ELISA. When CPD- induced salmon sperm DNA was used as a substrate, high CPD-photolyase activities were observed in the enzyme fraction prepared from whole spinach leaf extracts, but not from chloroplast extracts. This strongly suggests that spinach CPD-specific photolyases are localized in cell compartments other than chloroplasts.     

Sedimentation Analysis of the Interacting Mixture of Dextran Sulfate and Low Density Lipoprotein

The reversible interaction between dextran sulfate (D) and the low density lipoprotein of human serum (P) was investigated by sedimentation velocity. Analysis of the velocity patterns of dextran sulfate—lipoprotein mixtures revealed that the maximum number of binding sites on dextran sulfate molecule is approximately 6. It was also shown that the species of the complex formed is affected by the mixing ratio of the two constituents: at the molar ratio (P/D) 0.69, the complex exists in average as DP_1.6 and at 0.98 as DP_2.2. The linear increment of sedimentation coefficient of the complex due to the binding of one lipoprotein molecule was 7.8S. Finally, the mechanism of precipitation of the complexes was discussed.     

Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.     

Generation of the antioxidant yellow pigment derived from 4-methylthio-3-butenyl isothiocyanate in salted radish roots (takuan-zuke)

2-[3-(2-Thioxopyrrolidin-3-ylidene)methyl]-tryptophan (TPMT) is a yellow pigment of salted radish roots (takuan-zuke) derived from 4-methylthio-3-butenyl isothiocyanate (MTBITC), the pungent component of radish roots. Here, we prepared salted radish and analyzed the behavior of the yellow pigment and related substances in the dehydration process and long-term salting process. All salted radish samples turned yellow, and their b^* values increased with time and temperature. The salted radish that was sun-dried and pickled at room temperature turned the brightest yellow, and the generation of TPMT was clearly confirmed. These results indicate that tissue shrinkage due to dehydration, salting temperature, and pH play important roles in the yellowing of takuan-zuke.     

Nematocidal activity of 6-O-octanoyl- and 6-O-octyl-d-allose against larvae of Caenorhabditis elegans

ABSTRACT

The nematocidal activities of the fatty acid esters of d-allose were examined using the larvae of C. elegans. Among the fatty acid esters, 6-O-octanoyl-d-allose (3) showed significant activity. 6-O-octanoyl-d-glucose (5) showed no activity, indicating that the D-allose moiety is essential for the nematocidal activity of 3. A nonhydrolyzable alkoxy analog 6-O-octyl-d-allose (6) also showed activity equivalent to that of 3.     

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.