RESUMEN
Color variation is a well-known feature of sea cucumbers (Apostichopus japonicus), which are classified into three groups based on their colors of red, green and black. It is also one of the most important traits related to how they taste, and it thereby affects their market price. Attempts were made to identify single-nucleotide polymorphisms (SNPs) and to analyze differences associated with SNP genotypes between green and red color variants using HSP70 as the target gene. The HSP70 gene, which is found universally in organisms from bacteria to humans, is one of the most evolutionarily conserved genes and the most widely studied biomarker of stress response. DNA fragments of 1074 bp covering a partial sequence of the sea cucumber HSP70 gene, were amplified from both red and green variants, and subsequently analyzed for the presence of SNPs. Twenty-seven polymorphic sites in total, including heterozygous sites, were observed. Of these, six sites were found to be significantly different SNP genotypes between green and red variants. Furthermore, PCR with an internal primer designed to include an allele-specific SNP at the 3' end (site 443) showed differentiation between the two variants, 100% and 4.2% amplification in green and red variants, respectively. The validated SNPs may serve as informative genetic markers that can be used to distinguish variants at the early developmental stage, prior to color differentiation.
Asunto(s)
Proteínas HSP70 de Choque Térmico/genética , Pigmentación/genética , Pepinos de Mar/genética , Alelos , Animales , Secuencia de Bases , Marcadores Genéticos , Técnicas de Genotipaje , Heterocigoto , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Nucleótido Simple , Sensibilidad y EspecificidadRESUMEN
Random mutagenesis was performed on beta-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K-T307I were approx. 140, 190 and 200%, respectively, of wild type beta-agarase (661 U/mg) at 40 degrees C. All three mutant enzymes were stable up to 50 degrees C and E99K-T307I had the highest thermostability. The melting temperature (Tm) of E99K-T307I, determined by CD spectra, was increased by 5.2 degrees C over that of the wild-type enzyme (54.6 degrees C). Activities of both the wild-type and E99K-T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl2. The E99K-T307I enzyme was stable at 55 degrees C with 1 mM CaCl2, reaching 260% of the activity the wild-type enzyme held at 40 degrees C without CaCl2.
Asunto(s)
Proteínas Bacterianas/química , Flavobacteriaceae/enzimología , Glicósido Hidrolasas/química , Calor , Mutagénesis , Sustitución de Aminoácidos/genética , Proteínas Bacterianas/genética , Cloruro de Calcio/metabolismo , Dicroismo Circular , Análisis Mutacional de ADN , Estabilidad de Enzimas , Flavobacteriaceae/genética , Glicósido Hidrolasas/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Mutación Missense , Estabilidad Proteica , Temperatura de TransiciónRESUMEN
Flavonoids are known to be effective scavengers of free radicals. In particular, proanthocyanidins are flavonoids that possess cardiovascular protection, antioxidative activities, and immunomodulatory activities. Here, we evaluated proanthocyanidin contents in the total polyphenolic compounds of pine needle extracts prepared by hot water, ethanol, hexane, hot water-hexane (HWH), and hot water-ethanol (HWE). Analysis of each extract indicated that the ethanol extract contained the highest proanthocyanidin concentration. The HWH and hexane extracts also contained relatively high concentrations of proanthocyanidin. On the other hand, proanthocyanidin content analyses out of the total polyphenolic compounds indicated that the HWH extract contained the highest content. These results suggest that HWH extraction is a suitable method to obtain an extract with a high level of pure proanthocyanidins and a relatively high yield. The HWH extract possessed superior activity in diverse antioxidative analyses such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferrous ion chelating (FIC), and ferric-ion reducing power (FRAP) assays. In addition, upon assessing the effects of the pine needle extracts on macrophages (Raw 264.7 cell), the HWH extract exhibited the highest activity. In this study, we discerned an efficient extraction method to achieve relatively pure proanthocyanidins from pine needles and evaluated the biological functions of the resulting extract, which could potentially be used for its efficacious components in functional food products.
RESUMEN
Whitening effect, tyrosinase inhibitions, and cytotoxicity of neoagarotetraose were measured after purification from hydrolyzed agar by gel filtration chromatography. In melanoma B16F10 cells, melanin content of neoagarotetraose-treated cells was as same as that treated by kojic acid or arbutin. In addition, tyrosinase of melanoma cells was strongly inhibited by neoagarotetraose at a concentration of 1 micron/ml and similarly inhibited at 10 and 100 microg/ml compared to those by arbutin or kojic acid. The activity of mushroom tyrosinase showed a 38% inhibition by neoagarotetraose at 1 microg/ml, and this inhibitory effect was more efficient than that by kojic acid. Neoagarotetraose revealed similar IC(50) (50% inhibition concentration) value for mushroom tyrosinase as that by kojic acid. These data suggest that neoagarotetraose generated from agar by recombinant beta-agarase might be a good candidate as a cosmetic additive for whitening effect.