Analytical method for keratan sulfates by high-performance liquid chromatography/turbo-ionspray tandem mass spectrometry

We established a highly sensitive LC/MS/MS method for the analysis of the disaccharides produced from keratan sulfates (KS). It was revealed that the disaccharides produced by keratanase II enzymatic digestion of KS could be determined with high sensitivity by the negative-ion mode of multiple reaction monitoring. Furthermore, monosulfated and disulfated disaccharides can be separated using a short column of Capcell Pak NH2 UG80 (35 mm x 2 mm i.d.). The complete analysis of one sample can be performed within 5 min. The assay method was validated and showed satisfactory sensitivity, precision, and accuracy, which enabled quantitation at subpicomole levels. From the results of analyses of KS obtained from cornea, nasal cartilage, and brain, it was found that the degree of sulfation at the C-6 position of the galactose residues differed among those samples in the following order: nasal cartilage > cornea > brain. Our analytical method is very useful for the analyses of KS in various biological materials and for comparison of the degree of sulfation of KS from various biological samples.     

Comparison of oligosaccharides derived from salivary mucin of Japanese secretor and non-secretor individuals of blood group type-A

Comparison of oligosaccharide components derived from salivary mucin was performed between secretor and non-secretor individuals. Salivary mucin was collected from four secretors and three non-secretors having blood group type-A. Compositional analysis showed that the contents of galactose and N-acetylglucosamine in the non-secretor were higher than those in the secretor. The O-linked oligosaccharides obtained by treatment with alkaline borohydride were separated by gel filtration using Sephadex G-50. The results indicated that the size of the type-A active oligosaccharides from the secretor was similar to or smaller than that of the non-secretor. Ion-exchange chromatography showed that the secretors had strong type-A activities in both the neutral and acidic fractions but the non-secretors showed type-A activity mainly in the neutral fraction. These results suggest that compositional differences in blood group substances exist between secretors and non-secretors.     

Analysis of glucose tolerance in twin gestations using an oral glucose load.

The effect of twin gestation on carbohydrate metabolism was evaluated using a 75 g oral glucose tolerance test (75 g OGTT). A 75 g OGTT was performed in 63 twin gestations and 3 791 singleton gestations during the third trimester. Plasma glucose concentrations were measured in the pregnant women under fasting conditions as well as 30 min, 1 h, and 2 h after ingestion of glucose (75 g oral load), and serum insulin concentrations were measured in fasting and 30 min post-ingestion samples. Women with twin gestations showed significantly lower plasma glucose concentrations during fasting and 30 min after the glucose load in the samples taken than those with singleton gestations. No significant difference in serum glucose concentrations was found in the other specimens. There were no cases of gestational diabetes mellitus in our study. Although women with twin gestations demonstrated lower plasma glucose concentrations than women with singleton gestations, the difference observed was subtle. We could not find any significant differences in these plasma glucose values as used to define a pathologic OGTT between twin and singleton pregnancies, with the exception of the fasting value.     

Granulocyte-macrophage colony-stimulating factor is a keratinocyte-derived factor involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanocytes in culture

Mouse epidermal melanoblasts and melanocytes preferentially proliferated from disaggregated epidermal cell suspensions derived from newborn mouse skin in a serum-free melanocyte-proliferation medium (MDMD) and a melanoblast-proliferation medium (MDMDF) supplemented with dibutyryl adenosine 3':5'-cyclic monophosphate (DBcAMP) and/or basic fibroblast growth factor (bFGF). Pure cultured primary melanoblasts and melanocytes were further cultured with MDMD/MDMDF supplemented with granulocyte-macrophage colony-stimulating factor (GMCSF) from 14 days (keratinocyte depletion). GMCSF stimulated the number of melanoblasts/melanocytes as well as the percentage of differentiated melanocytes in keratinocyte-depleted cultures. Flow cytometry analysis showed that melanoblasts and melanocytes in the S and G(2)/M phases of the cell cycle were increased by the treatment with GMCSF. Moreover, anti-GMCSF antibody added to MDMD/MDMDF from the initiation of the primary culture (in the presence of keratinocytes) inhibited the proliferation of melanoblasts/melanocytes as well as the differentiation of melanocytes. Enzyme-linked immunosorbent assay of culture media revealed that GMCSF was secreted from keratinocytes, but not from melanocytes. These results suggest that GMCSF is one of the keratinocyte-derived factors involved in regulating the proliferation and differentiation of neonatal mouse epidermal melanoblasts/melanocytes in culture in cooperation with cAMP elevator and bFGF.     

Establishment and characterization of Roberts syndrome and SC phocomelia model medaka (Oryzias latipes)

Roberts syndrome and SC phocomelia (RBS/SC) are genetic autosomal recessive syndromes caused by establishment of cohesion 1 homolog 2 ( ESCO 2) mutation. RBS/SC appear to have a variety of clinical features, even with the same mutation of the ESCO2 gene. Here, we established and genetically characterized a medaka model of RBS/SC by reverse genetics. The RBS/SC model was screened from a mutant medaka library produced by the Targeting Induced Local Lesions in Genomes method. The medaka mutant carrying the homozygous mutation at R80S in the conserved region of ESCO2 exhibited clinical variety (i.e. developmental arrest with craniofacial and chromosomal abnormalities and embryonic lethality) as characterized in RBS/SC. Moreover, widespread apoptosis and downregulation of some gene expression, including notch1a, were detected in the R80S mutant. The R80S mutant is the animal model for RBS/SC and a valuable resource that provides the opportunity to extend knowledge of ESCO2. Downregulation of some gene expression in the R80S mutant is an important clue explaining non-correlation between genotype and phenotype in RBS/SC.     

Eutrophication of mountain lakes in Japan due to increasing deposition of anthropogenically produced dust

Atmospheric dust has wide-reaching effects, not only influencing climate conditions, but also ecosystems. The eastern region of the Asian continent is one of the largest emitters of dust in the world, and recent economic growth in the region has been accompanied by an increase in anthropogenic emissions. However, the effects of increased Asian dusts on aquatic ecosystems are not well understood. We examined fossil pigments and zooplankton remains from ²¹⁰Pb-dated sediments taken from high mountain lakes of Hourai-Numa and Hachiman-Numa, located in the Towada-Hachimantai National Park of Japan Islands, to uncover historical changes in the phyto- and zooplankton community over the past 100?years. Simultaneously, we measured the geochemical variables of TOC, TN, TP, ??¹³C, ??¹⁵N, and lead isotopes (²⁰⁷Pb/²⁰⁶Pb, ²⁰⁸Pb/²⁰⁶Pb) in the sediments to identify environmental factors causing such changes. As a result, despite few anthropogenic activities in the watersheds, alpine lakes in Japan had increased algal and herbivore plankton biomasses by 3?C6 fold for recent years depending on the surrounding terrestrial vegetation and landscape conditions. Biological and biogeochemical proxies recorded from the lake sediments showed that this eutrophication occurred after the 1990s when P deposition increased as a result of atmospheric loading of dust transported from the Asian continent. The continued increase of anthropogenically produced dust may therefore impart damaging impacts on mountain ecosystems even if they are protected from direct anthropogenic disturbances.     

Independent pathways leading to apoptotic cell death, oxidative burst and defense gene expression in response to elicitin in tobacco cell suspension culture.

We characterized pharmacologically the hypersensitive cell death of tobacco BY-2 cells that followed treatments with Escherichia coli preparations of INF1, the major secreted elicitin of the late blight pathogen Phytophthora infestans. INF1 elicitin treatments resulted in fragmentation and 180 bp laddering of tobacco DNA as early as 3 h post-treatment. INF1 elicitin also induced rapid accumulation of H2O2 typical of oxidative burst, and the expression of defense genes such as phenylalanine ammonia-lyase (PAL) gene at 1 h and 3 h after elicitin treatment, respectively. To investigate the involvement of the oxidative burst and/or the expression of defense genes in the signal transduction pathways leading to hypersensitive cell death, we analyzed the effect of several chemical inhibitors of signal transduction pathways on the various responses. The results indicated that (a) the cell death required serine proteases, Ca2+ and protein kinases, (b) the oxidative burst was involved in Ca2+ and protein kinase mediated pathways, but elicitin-induced AOS was neither necessary nor sufficient for cell death and PAL gene expression, and (c) the signaling pathway of PAL gene expression required protein kinases. These results suggest that the three signal transduction pathways leading to cell death, oxidative burst and expression of defense genes branch in the early stages that follow elicitin recognition by tobacco cells.     

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.