Purification and characterization of polyphenol oxidase from cauliflower (Brassica oleracea L.)

Polyphenol oxidase (PPO) of cauliflower was purified to 282-fold with a recovery rate of 8.1%, using phloroglucinol as a substrate. The enzyme appeared as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The estimated molecular weight of the enzyme was 60 and 54 kDa by SDS-PAGE and gel filtration, respectively. The purified enzyme, called phloroglucinol oxidase (PhO), oxidized phloroglucinol (K(m) = 3.3 mM) and phloroglucinolcarboxylic acid. The enzyme also had peroxidase (POD) activity. At the final step, the activity of purified cauliflower POD was 110-fold with a recovery rate of 3.2%. The PhO and POD showed the highest activity at pH 8.0 and 4.0 and were stable in the pH range of 3.0-11.0 and 5.0-8.0 at 5 °C for 20 h, respectively. The optimum temperature was 55 °C for PhO and 20 °C for POD. The most effective inhibitor for PhO was sodium diethyldithiocarbamate at 10 mM (IC(50) = 0.64 and K(i) = 0.15 mM), and the most effective inhibitor for POD was potassium cyanide at 1.0 mM (IC(50) = 0.03 and K(i) = 29 μM).     

Metabolomic Analyses of Blood Plasma after Oral Administration of D-Glucosamine Hydrochloride to Dogs

D-Glucosamine hydrochloride (GlcN∙HCl) is an endogenous amino monosaccharide synthesized from glucose that is useful in the treatment of joint diseases in both humans and animals. The aim of this study was to examine amino acid metabolism in dogs after oral administration of GlcN∙HCl. Accelerated fumarate respiration and elevated plasma levels of lactic acid and alanine were observed after administration. These results suggest that oral administration of GlcN∙HCl induces anaerobic respiration and starvation in cells, and we hypothesize that these conditions promote cartilage regeneration. Further studies are required to evaluate the expression of transforming growth factor-beta (TGF-β).     

Breakdown of a hybridization barrier between Solanum pinnatisectum Dunal and potato using the S locus inhibitor gene (Sli)

Several attempts have been undertaken to exploit the resistance to late blight [Phytophthora infestans (Mont.) de Bary] of the Mexican diploid Solanum pinnatisectum Dunal (2n = 2x = 24). However, sexual hybridization between S. pinnatisectum and cultivated potatoes is almost impossible because of strict reproductive barriers. In this study, we first induced chromosome doubling of S. pinnatisectum for matching of the species-specific endosperm balance number (EBN), which is a genetic phenomenon responsible for normal endosperm development. We pollinated 4x (2EBN) S. pinnatisectum flowers with pollen from 2x (2EBN) clones carrying the S locus inhibitor gene (Sli), which originally was identified as an inhibitor of S-locus-controlled self-incompatibility in pollen of diploid potatoes. Nine putative hybrid plants were obtained from 613 pollinations, of which two plants were characterized. Both individuals were triploid hybrids with extreme resistance to P. infestans derived from S. pinnatisectum and showed an acceptable level of female fertility for backcrossing. Thus, utilization of the Sli gene, combined with chromosome-doubling for matching of the EBN, is proposed as a technique to overcome barriers to interspecific hybridization between Mexican 2x (1EBN) Solanum species and cultivated diploid potatoes.     

Apple necrotic mosaic virus,a novel ilarvirus from mosaic-diseased apple trees in Japan and China

The causal agent of apple mosaic disease has been previously thought to be solely caused by apple mosaic virus (ApMV). In this study, we report that a novel ilarvirus is also associated with apple mosaic disease. Next-generation sequencing analysis of an apple tree showing mosaic symptoms revealed that the tree was infected with three apple latent viruses (apple stem pitting virus, apple stem grooving virus, and apple chlorotic leaf spot virus) and a novel ilarvirus (given the name apple necrotic mosaic virus (ApNMV)) that is closely related to Prunus necrotic ringspot virus (PNRSV) and ApMV. The genome of ApNMV consists of RNA1 (3378 nt), RNA2 (2767 nt), and RNA3 (1956 nt). A phylogenetic analysis based on the coat protein amino acid sequences indicated that the novel virus belongs to the same subgroup 3 of the genus Ilarvirus as PNRSV and ApMV. The presence of mosaic leaves, which tend to be unevenly distributed in diseased apple trees, was correlated with the internal distribution of ApNMV. RT-PCR detection of mosaic-diseased apple trees in Japan indicated that ApNMV was detected in apple trees introduced from China, whereas ApMV was detected from cultivated apple trees in domestic orchards. Consistent with these findings, a survey of mosaic-diseased apple trees in major apple-producing provinces in China revealed that the majority of apple trees showing mosaic symptoms in China are infected with ApNMV.     

Metabolomic Analysis of Blood Plasma after Oral Administration of N-acetyl-d-Glucosamine in Dogs

N-acetyl-d-glucosamine (GlcNAc) is a monosaccharide that polymerizes linearly through (1,4)-β-linkages. GlcNAc is the monomeric unit of the polymer chitin. GlcNAc is a basic component of hyaluronic acid and keratin sulfate found on the cell surface. The aim of this study was to examine amino acid metabolism after oral GlcNAc administration in dogs. Results showed that plasma levels of ectoine were significantly higher after oral administration of GlcNAc than prior to administration (p < 0.001). To our knowledge, there have been no reports of increased ectoine concentrations in the plasma. The mechanism by which GlcNAc administration leads to increased ectoine plasma concentration remains unclear; future studies are required to clarify this mechanism.     

Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation

Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm²), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation.     

Flavonoid content in fresh, home-processed, and light-exposed onions and in dehydrated commercial onion products

Onion plants synthesize flavonoids as protection against damage by UV radiation and by intracellular hydrogen peroxide. Because flavonoids also exhibit health-promoting effects in humans, a need exists to measure their content in onions and in processed onion products. To contribute to the knowledge about the levels of onion flavonoids, HPLC and LC-MS were used to measure levels of seven quercetin and isorhamnetin glucosides in four Korean commercial onion bulb varieties and their distribution within the onion, in scales of field-grown onions exposed to home processing or to fluorescent light and in 16 commercial dehydrated onion products sold in the United States. Small onions had higher flavonoid content per kilogram than large ones. There was a graduated decrease in the distribution of the flavonoids across an onion bulb from the first (outside) to the seventh (innermost) scale. Commercial, dehydrated onion products contained low amounts or no flavonoids. Losses of onion flavonoids subjected to "cooking" (in percent) ranged as follows: frying, 33; sauteing, 21; boiling, 14-20; steaming, 14; microwaving, 4; baking, 0. Exposure to fluorescent light for 24 and 48 h induced time-dependent increases in the flavonoid content. The results extend the knowledge about the distribution of flavonoids in fresh and processed onions.     

Analysis by HPLC and LC/MS of pungent piperamides in commercial black, white, green, and red whole and ground peppercorns

Pepper plants accumulate pungent bioactive alkaloids called piperamides. To facilitate studies in this area, high-performance liquid chromatography (HPLC) and liquid chromatography/mass spectrometry methods were developed and used to measure the following piperamides in 10 commercial whole (peppercorns) and in 10 ground, black, white, green, and red peppers: piperanine, piperdardine, piperine, piperlonguminine, and piperettine. Structural identification of individual compounds in extracts was performed by associating the HPLC peak of each compound with the corresponding mass spectrum. The piperanine content of the peppers (in mg/g piperine equivalents) ranged from 0.3 for the ground white pepper to 1.4 in black peppercorns. The corresponding range for piperdardine was from 0.0 for seven samples to 1.8 in black peppercorns; for four isomeric piperines, from 0.7 for red to 129 in green peppercorns; for piperlonguminine, from 0.0 in red peppercorns to 1.0 in black peppercorns; and for piperyline, from 0.9 in ground black pepper to 5.9 for red peppercorn. Four well-separated stereoisomeric forms of piperettine with the same molecular weight were present in 19 peppers. The sums of the piperamides ranged from 6.6 for red to 153 for green peppercorns. In contrast to large differences in absolute concentrations among the peppers, the ratios of piperines to total piperamide were quite narrow, ranging from 0.76 for black to 0.90 for white peppercorns, with an average value of 0.84 +/- 0.04 ( n = 19). Thus, on average, the total piperamide content of the peppers consists of 84% piperines and 16% other piperamides. These results demonstrate the utility of the described extraction and analytical methods used to determine the wide-ranging individual and total piperamide contents of widely consumed peppers.     

Heavy Metals in a Tropical Eel Anguilla marmorata from The Central Part of Vietnam

In order to understand the present status of heavy metal contamination and the risk associated with human consumption, commercial freshwater eels in Vietnam were examined. The concentration of eight elements (Cr, Mn, Co, Cu, Zn, Sr, Cd, and Pb) was determined in muscle tissue of tropical eel Anguilla marmorata collected in four provinces, Quang Tri, Quang Ngai, Binh Dinh, and Phu Yen, from the central part of Vietnam. Concentrations of Cu, Zn, Cd, and Sr were found to be highest, whereas Co concentration showed the lowest at Quang Tri. Cr was recorded to be lowest in Quang Ngai, while the amounts of Mn and Pb did not vary among locations. Although the metal concentrations in the muscle of A. marmorata did not represent a risk for human consumption, elevated Cd concentrations in Quang Tri related to agriculture activities suggest that further investigations should be undertaken to clarify the potential risk to the eel population.