Synthetic glycopeptides reveal specific binding pattern and conformational change at O-mannosylated position of α-dystroglycan by POMGnT1 catalyzed GlcNAc modification.

Structural and functional effects of core M1 type glycan modification catalyzed by protein O-linked mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) were investigated using a core M1 glycoform focused library of an α-dystroglycan fragment, 372TRGAIIQTPTLGPIQPTRV390. Evanescent-field fluorescence-assisted microarray system illuminated the specific binding pattern of plant lectins that can discriminate the glycan structure of core M1 glycan of the library. The comparative NMR analysis of synthetic glycopeptide having different length of the O-mannosylated glycans revealed a conformational change of the peptide backbone along with core M1 disaccharide formation. No long-range NOE signals of glycan-amino acid nor inter amino acid indicate the conformational change is induced by steric hindrance of core M1, the sole 1,2-O-modified form among protein binding sugar residue found in mammals.

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides.

BACKGROUND: Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear. METHODS: A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies. RESULTS: Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galß1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats. CONCLUSION: We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption. GENERAL SIGNIFICANCE: The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.

Inhibition of CUTIN DEFICIENT 2 Causes Defects in Cuticle Function and Structure and Metabolite Changes in Tomato Fruit.

Tomato (Solanum lycopersicum) fruit cuticle has been extensively studied due to its effect on the biochemical and physiological properties of the fruit. To date, several tomato mutants defective in proper cuticle formation have been identified. To gain insight into tomato cuticle formation, we investigated one such mutant, sticky peel/light green (pe lg). We verified the responsible gene by fine mapping and obtained the same conclusion as a previous report. To elucidate the pleiotropic effects of cuticle deficiency caused by the cd2 mutation, CD2 suppression lines were constructed. As found in the pe lg mutant, the suppression lines showed enhanced water permeability and aberrant leaf and fruit cuticles. Water use efficiency of the suppression line was lower than that of the wild type. However, photosynthetic ability was not affected in the suppression line. Since these phenotypes are related to altered deposition of wax and cutin, other lipidic metabolites might be changed, too. To confirm this hypothesis, we conducted metabolite profiling. The metabolite profiling revealed that not only lipid but also sugar, flavonoid and glycoalkaloid metabolites in fruit were changed in the cd2 mutant. These results indicate that CD2 is essential both for normal cutin and wax deposition and for proper accumulation of specific metabolites in tomato fruit.

A sulfated glycosaminoglycan array for molecular interactions between glycosaminoglycans and growth factors or anti-glycosaminoglycan antibodies.

Glycosaminoglycans (GAGs) take part in numerous biological processes by binding to protein molecules and functionally regulating protein-ligand interactions; therefore, molecular interactions of GAGs have been studied by several methods, including surface plasmon resonance, enzyme-linked immunosorbent assays (ELISAs), and GAG microarrays. To achieve rapid, sensitive, and high-throughput screening of GAG interactions, we have developed a novel microarray in which GAGs, including chondroitin sulfate, heparan sulfate, and heparin, were immobilized. The microarray is made from cyclic polyolefin substrate coated with metacrylate polymers, which have phospholipid groups as side chains. The polymer also has aminooxy groups that react specifically with aldehyde groups at the reducing termini of GAG chains, whereas the phospholipid groups prevent nonspecific adsorption of proteins. Thus, minute amounts of GAGs can be chemically immobilized on the surface with low nonspecific binding of proteins. Using this array, interactions between GAGs and antibodies against chondroitin or heparan sulfate and heparin-binding growth factors were examined. The results were in agreement with previously reported specificities, suggesting that the GAG array is useful for high-throughput interaction analyses between GAGs and functional proteins in miniscule amounts and can be applied to both basic studies of GAGs and the development of diagnostic methods for metabolic diseases involving GAGs.

Production of (Z)-Lycopene-Rich Tomato Concentrate: A Natural Catalyst-Utilized and Oil-Based Study for Practical Applications.

Since lycopene Z-isomers exhibit greater bioavailability and biological activity than the naturally occurring all-E-isomer, efficient manufacturing methods for (Z)-lycopene-rich materials are urgently needed. Herein, a method was developed for Z-isomerization of (all-E)-lycopene in tomato oleoresin using heat treatment and a natural catalyst, viz. allyl isothiocyanate (AITC). For practical application of this isomerization technology, no organic solvents were used, and instead, oils and fats were used as the reaction medium. The Z-isomerization of (all-E)-lycopene was promoted by heating (>120 °C) even when oil and fat media were used. Allyl isothiocyanate enhanced thermal Z-isomerization and improved the (5Z)-lycopene content, which shows higher biological activity compared to the other Z-isomers. The thermal isomerization efficiency with AITC was further improved by using certain vegetable oils such as argan and olive oils. In addition, the storage stability of (Z)-lycopene-rich tomato concentrates dispersed in olive oil was evaluated. The total Z-isomer ratio and lycopene concentration decreased with longer storage periods, and it was revealed that (5Z)-lycopene showed excellent storage stability among the mono-Z-isomers.

Synergistic Effects of Food Ingredients and Vegetable Oils on Thermal Isomerization of Lycopene.

Recent investigations have demonstrated that some food ingredients and vegetable oils, such as onion, garlic, and sesame oil, enhanced thermal Z-isomerization of (all-E)-lycopene in tomatoes. However, the synergistic effects of these ingredients and oils have not yet been investigated. This study aims at clarifying how the combined use of lycopene Z-isomerization-promoting food ingredients and vegetable oils impacts thermal Z-isomerization of (all-E)-lycopene in tomato puree. Apart from a few exceptions, when olive oil was used as a reaction medium, the combined use of garlic, cabbage, broccoli, shiitake mushroom, and makonbu improved the total Z-isomer ratio of lycopene after heating compared to the separate use of the tested ingredients. However, when onion was used together with the other ingredients, the Z-isomer ratio significantly decreased compared to its individual use. Moreover, when garlic, cabbage, broccoli, shiitake mushroom, and makonbu were used with sesame and mustard oils, that exhibit higher Z-isomerizationpromoting effect than that of olive oil, the lycopene Z-isomerization reaction was further enhanced. However, when onion was combined with these oils, the Z-isomer ratio decreased compared to that measured upon the combined use of onion with olive oil. Our results on these synergistic effects are not only important for the food and drink manufacturing industries but also for daily home cooking.

Isomerization of Commercially Important Carotenoids (Lycopene, ß-Carotene, and Astaxanthin) by Natural Catalysts: Isothiocyanates and Polysulfides.

Effects of natural catalysts, isothiocyanates and polysulfides, on Z-isomerization and decomposition of (all-E)-carotenoids (lycopene, ß-carotene, and astaxanthin) after heat treatment were investigated. When isothiocyanates were added to (all-E)-carotenoid solutions and heated, Z-isomerization and decomposition of carotenoids were enhanced and the degree differed depending on the isothiocyanate type. Interestingly, when polysulfides were applied in the same manner, in addition to promoting the Z-isomerization reaction, they markedly improved the thermal stability of carotenoids. Successively, we investigated the reaction characteristics of allyl isothiocyanate (AITC) and diallyl disulfide (DADS) using (all-E)-lycopene; that is, effects of the amount added, solvent used, and reaction temperature and time, as well as the combination use on Z-isomerization and decomposition of lycopene, were investigated. With increases in the amount added and reaction temperature and time, Z-isomerization of lycopene was promoted for both catalysts. The high-temperature treatment tests clearly showed that AITC induced thermal decomposition of lycopene, whereas DADS improved the lycopene stability. Moreover, the simultaneous use of AITC and DADS resulted in a synergetic effect on the Z-isomerization efficiency.

Multiple primer extension by DNA polymerase on a novel plastic DNA array coated with a biocompatible polymer.

DNA microarrays are routinely used to monitor gene expression profiling and single nucleotide polymorphisms (SNPs). However, for practically useful high performance, the detection sensitivity is still not adequate, leaving low expression genes undetected. To resolve this issue, we have developed a new plastic S-BIO PrimeSurface with a biocompatible polymer; its surface chemistry offers an extraordinarily stable thermal property for a lack of pre-activated glass slide surface. The oligonucleotides immobilized on this substrate are robust in boiling water and show no significant loss of hybridization activity during dissociation treatment. This allowed us to hybridize the templates, extend the 3' end of the immobilized DNA primers on the S-Bio by DNA polymerase using deoxynucleotidyl triphosphates (dNTP) as extender units, release the templates by denaturalization and use the same templates for a second round of reactions similar to that of the PCR method. By repeating this cycle, the picomolar concentration range of the template oligonucleotide can be detected as stable signals via the incorporation of labeled dUTP into primers. This method of Multiple Primer EXtension (MPEX) could be further extended as an alternative route for producing DNA microarrays for SNP analyses via simple template preparation such as reverse transcript cDNA or restriction enzyme treatment of genome DNA.

Scheloribatid mites as the source of pumiliotoxins in dendrobatid frogs.

The strawberry poison frog Dendrobates pumilio (Anura: Dendrobatidae) and related poison frogs contain a variety of dendrobatid alkaloids that are considered to be sequestered through the consumption of alkaloid-containing arthropods microsympatrically distributed in the habitat. In addition to ants, beetles, and millipedes, we found that adults of two species of oribatid mites belonging to the cohort Brachypylina, trophically a lower level of animal than ants and beetles, contain dendrobatid alkaloids. Gas chromatography/mass spectrometry (GC/MS) of hexane extracts of adult Scheloribates azumaensis (Oribatida: Acari) revealed the presence of not only pumiliotoxin 251D (8-hydroxy-8-methyl-6-(2'-methylhexylidene)-1-azabicyclo[4.3.0]nonane), but also precoccinelline 193C and another coccinelline-type alkaloid. From the corresponding extracts of an unidentified Scheloribates sp., pumiliotoxin 237A (8-hydroxy-8-methyl-6-(2'-methylpentylidene)-1-azabicyclo[4.3.0]nonane) was detected as a minor component, and identified by synthesis. The presence of related alkaloids, namely deoxypumiliotoxin 193H, a 6,8-diethyl-5-propenylindolizidine, and tentatively, a 1-ethyl-4-pentenynylquinolizidine, were indicated by the GC/MS fragmentation patterns, along with at least another six unidentified alkaloid components. Thus, one possible origin of pumiliotoxins, coccinellid alkaloids, and certain izidines found in poison frogs may be mites of the genus Scheloribates and perhaps related genera in the suborder Oribatida.

Quantitative determination method for trace amount of penicillin contaminants in commercially available drug product by HPLC coupled with tandem mass spectrometry.

A quantitative determination method for trace amount of penicillin contaminants in an active pharmaceutical ingredient (API) has been developed. Selective extraction of penicillin contaminants from the matrix containing API and specific separation among penicillin contaminants were achieved through an on-line column switching technique with gradient elution, followed by tandem mass spectrometric determination. Validation was conducted on the developed method in terms of specificity, linearity, accuracy, precision, and detection limit, and appeared reasonable. The detection limit was estimated as 0.03 ng/ml or lower of the concentration of penicillin contaminants in the preparation, corresponding to 4 parts par billion (ppb) against the API. This fulfilled the regulatory requirement by the authorities.