Screening of Neutrophil Activating Factors from a Metagenome Library of Sponge-Associated Bacteria

Marine sponge-associated bacteria are known as bio-active compound produce. We have constructed metagenome libraries of the bacteria and developed a metagenomic screening approach. Activity-based screening successfully identified novel genes and novel enzymes; however, the efficiency was only in 1 out of 10⁴ clones. Therefore, in this study, we thought that bioinformatics could help to reduce screening efforts, and combined activity-based screening with database search. Neutrophils play an important role for the immune system to recognize excreted bacterial by-products as chemotactic factors and are recruited to infection sites to kill pathogens via phagocytosis. These excreted by-products are considered critical triggers that engage the immune system to mount a defense against infection, and identifying these factors may guide developments in medicine and diagnostics. We focused on genes encoding amino acid ligase and peptide synthetase and selected from an in-house sponge metagenome database. Cell-free culture medium of each was used in a neutrophil chemiluminescence assay in luminol reaction. The clone showing maximum activity had a genomic sequence expected to produce a molecule like a phospho-N-acetylmuramyl pentapeptide by the metagenome fragment analysis.     

Determination of antioxidant activity and characterization of antioxidant phenolics in the plum vinegar extract of cherry blossom (Prunus lannesiana)

Sakura-cha (salted cherry blossom tea) is a Japanese tea that is traditionally served at celebrations such as wedding ceremonies. The production of Sakura-cha includes the immersion of cherry blossom flowers in Japanese plum vinegar, and through this process, the byproduct (plum vinegar extract of cherry blossom) is obtained. In this study, the antioxidant activity of the plum vinegar extract of cherry blossom was examined. The plum vinegar extract of cherry blossom had a greater superoxide anion scavenging activity compared with red wine, which is a well-known strong antioxidant. Liquid chromatography-mass spectrometry analysis showed that cyanidin-3-glucoside, cyanidin-3-rutinoside, and caffeic acid were the major components in the phenolic extract prepared from plum vinegar extract of cherry blossom, and they possessed superoxide anion scavenging activity. Caffeic acid is mainly responsible for the scavenging activity of phenolic extract; the contributions of cyanidin-3-glucoside and cyanidin-3-rutinoside were minor.     

Environmental controls on photosynthetic production and ecosystem respiration in semi-arid grasslands of Mongolia

The Mongolian steppe zone comprises a major part of East Asian grasslands. The objective of this study was to separately evaluate the quantitative dependencies of gross primary production (GPP) and ecosystem respiration (R_eco) on the environmental variables of temperature, moisture, radiation, and plant biomass in a semi-arid grassland ecosystem. We determined GPP and R_eco using transparent and opaque closed chambers in a grassland dominated by Poaceae species in central Mongolia during five periods: July 2004, May 2005, July 2005, September 2005, and June 2006. Values of GPP were linearly related to live aboveground biomass (AGB) enclosed by the chamber. The amount of GPP per unit ground area differed among the study periods, whereas GPP normalized by the amount of AGB did not differ significantly among the periods, suggesting that plant production per unit green biomass did not depend on the phenological stage. GPP/AGB fit well a rectangular hyperbolic light–response curve for all the study periods. When the air and soil were dry, considerable reduction in GPP was observed. The GPP/AGB ratio was also expressed as individual functions of air temperature, vapor pressure deficit, and volumetric soil water content. R_eco was exponentially related to the soil temperature and the relationship was modified by soil moisture. The amount of R_eco and its temperature sensitivity (Q₁₀) declined with decreasing soil moisture. Sharp increases of R_eco after rainfall events were observed. The values of R_eco, even including the rain-induced pulses, were expressed well as a bivariate function of soil temperature and soil moisture near the soil surface.     

Loop-mediated isothermal amplification for the rapid, sensitive, and specific detection of the O9 group of Salmonella in chickens

In the present study, the loop-mediated isothermal amplification (LAMP) assay was developed to amplify the fragments of the O9 Salmonella-specific insertion element and evaluated in the laboratory for its potential use in a field situation, such as poultry farms. Among the bacteria tested, a positive reaction was observed only for 128 strains of 6 serovars of the O9 group Salmonella, such as Enteritidis (SE) and Pullorum. The detection limit of the LAMP assay was 10(3)CFU/ml, which was more sensitive than that of the polymerase chain reaction (PCR) assay with the same target gene (10(6)CFU/ml). The final results were obtained within 30 min for the LAMP assay, while the PCR assay needed a total of 120 min. When the LAMP assay was applied to the enrichment broth mixed with cecal dropping samples either spiked with SE in vitro or excreted by SE-inoculated hens, the results were comparable to those of the conventional plating method including 2 separate enrichments. In conclusion, the LAMP assay developed in the present study is an effective method for the specific detection of the O9 group Salmonella serovars, including SE.     

Isolation and structural characterization of rhamnogalacturonan II-borate complex fromPinus densiflora

A boron-containing pectic polysaccharide was isolated from Driselase digests of akamatsu (Pinus densiflora) cell wall. The boron-containing polysaccharide was purified by successive column chromatography of Cosmosil C₁₈ OPN, DEAE Sepharose FF, and Superdex 75. The complex had 0.12% (w/w) boron (B) and¹¹B-NMR spectroscopy showed the complex to be the tetrahedral diester form of boron. The sugar composition and glycosyl linkage analyses showed that the sugar portion of the complex was rhamnogalacturonan-II (RG-11). The boron-attached glycosyl residue was identified to be 2-O-Me-xylose-containing apiosyl side chain in RG-II. These results established that the location of borate in the cell wall is conserved among dicots, gramineous monocots, and coniferous gymnosperm.Part of this study was presented at the 8th Cell Wall Symposium, Norwich, September 3, 1998     

Rheological Properties of White Salted Noodles with Different Amylose Content at Small and Large Deformation

The rheological properties of cooked white salted noodles made from eight wheat cultivars with varied amylose content were analyzed at small and large deformation. Their dynamic shear viscoelasticity was measured using a rheometer with parallel plate geometry. Compressive force and creep‐recovery curves were measured using various probes and sample shapes. Noodles with lower amylose content showed a lower storage shear modulus (G′) and a higher frequency dependence of G′. The G′ values of noodles were highly correlated with amylose content in wheat flour and with G′ values of 30 and 40% starch gels. Remarkable differences in the characteristics of creep‐recovery curves were observed between cultivars. The difference in amylose content in wheat flour reflected the creep‐recovery properties of noodles. A negative correlation was demonstrated between amylose content and both maximum creep and recovery compliance. The compressive force required for 20, 50, 80, and 95% strains was compared. At 20 and 50% strain, noodles made from lower amylose wheat flour showed lower compressive force. Noodles of waxy wheat had a higher compressive force than nonwaxy noodles when the strain was >80%, indicating the waxy wheat noodles are soft but difficult to completely cut through.     

Expression and location of IGF binding proteins-2, -4, and -5 in developing fetal tissues.

Insulin-like growth factors are associated with myogenesis in vivo, and their actions are mediated by IGF binding proteins (IGFBP). Sites of IGFBP production and their location during early development are not clear. The objective of this research was to examine the developmental expression and location of IGFBP-2, -4, and -5 mRNA and peptides in developing porcine skeletal muscle and liver. Pregnant pigs were euthanatized at various times postconception (pc). Developmental expression of IGFBP was evaluated using total RNA extracted from skeletal muscle and liver of 30-, 44-, 59-, 68-, 75-, 89-, and 109-d pc fetuses and from adult and neonatal pigs. Localization of IGFBP-2, -4, and -5 mRNA and peptides was examined by in situ hybridization and immunocytochemistry of muscle samples from contralateral pelvic limbs of each pig. Overall muscle IGFBP gene expression decreased (P < .05) with increasing age. Moreover, expression of liver IGFBP-2 and -5, but not of IGFBP-4, was greater (P < .05) during prenatal than during postnatal periods. The majority of immunoreactive IGFBP was located in developing muscle cells, with little localized to connective tissue, except at later stages of development. These data show that IGFBP-2, -4, and -5 expression is time- and tissue-dependent in fetal liver and muscle.     

Incorporating accessibility limitation into the surplus production model

Limited access to aquatic populations hinders estimation of their status and establishment of effective management measures. We propose a modified surplus production model to cope with this problem. The model provides population parameters and biological reference points from a time series of annual accessible abundance data. Simulation tests showed that the model provided biological reference point estimates with little bias when sufficiently long time series were available. Even for short time series, we could obtain nearly unbiased estimates by providing information on the exploitation rate at the maximum sustainable yield (F _MSY). As an application, we fit the modified surplus production model to 7-year accessible biomass estimates of a local population of Japanese spiky sea cucumber Apostichopus japonicus using a Bayesian approach. The results indicated that the stock in the area studied was likely to have experienced recent overfishing and had a high probability of being overfished in the future.     

Effect of Amylose Content on Gelatinization,Retrogradation, and Pasting Properties of Starches from Waxy and Nonwaxy Wheat and Their F1 Seeds

We studied the effect of amylose content on the gelatinization, retrogradation, and pasting properties of starch using wheat starches differing in amylose content. Starches were isolated from waxy and nonwaxy wheat and reciprocal F1 seeds by crossing waxy and nonwaxy wheat. Mixing waxy and nonwaxy wheat starch produced a mixed starch with the same amylose content as F1 seeds for comparison. The amylose content of F1 seeds ranged between waxy and nonwaxy wheat. Nonwaxy‐waxy wheat had a higher amylose content than waxy‐nonwaxy wheat. Endothermic enthalpy and final gelatinization temperature measured by differential scanning calorimetry correlated negatively with amylose content. Gelatinization onset and peak temperature clearly differed between F1 and mixed starches with the same amylose content as F1 starches. Enthalpy for melting recrystallized starches correlated negatively with amylose content. Rapid Visco Analyser measurement showed that F1 starches had a higher peak viscosity than waxy and nonwaxy wheat starches. Mixed starches showed characteristic profiles with two low peaks. Setback and final viscosity correlated highly with amylose content. Some of gelatinization and pasting properties differed between F1 starches and mixed starches.