Molecular Design and Synthesis of a Novel Substrate for Assaying Lysozyme Activity

A novel substrate {Galβ1,4GlcNAcβ1,4GlcNAc-β-pNP [Gal(GlcNAc)₂-β-pNP]} for assaying lysozyme activity has been designed using docking simulations and enzymatic synthesis via β-1,4-galactosyltransferase-mediated transglycosylation from UDP-Gal as the donor to (GlcNAc)₂-β-pNP as the acceptor. Hydrolysis of the synthesized Gal(GlcNAc)₂-β-pNP and related compounds using hen egg-white lysozyme (HEWL) demonstrated that the substrate was specifically cleaved to Gal(GlcNAc)₂ and p-nitrophenol (pNP). A combination of kinetic studies and docking simulation was further conducted to elucidate the mode of substrate binding. The results demonstrate that Gal(GlcNAc)₂-β-pNP selectively binds to a subsite of lysozyme to liberate the Gal(GlcNAc)₂ and pNP products. The work therefore describes a new colorimetric method for quantifying lysozyme on the basis of the determination of pNP liberated from the substrate.     

Debugging methods in recursive structured FORTRAN

A version of structured FORTRAN named STAR is introduced. STAR allows recursive subroutines. Implementation problems such as parameter passing by value and local variable stacking are discussed. The major difficulty in the preprocessor approach is debugging. We claim that execution profiles are strong debugging aids, and demonstrate how to debug a STAR program at source language level with the clue in the profiles plus assertion statements. We make use of a quicksort program as an example.     

PTCR Characteristics in Barium Titanate Ceramics with Curie Points Between 60° and 360°C

PTCR characteristics in porous semiconducting barium titanate ceramics with Curie points from 60° to 360°C were investigated. The magnitude of the PTCR effect in these cerumics decreases self-onsistently with increasing Curie point within this temperature range. A PTCR efSect of more than 4 orders of magnitude was ahserved, for a Ba_0.44Pb_0.6TiO₃ ceramic with a Curie point of 360°C .     

Novel poly-gamma-glutamate-processing enzyme catalyzing gamma-glutamyl DD-amidohydrolysis

The pgdS gene product of Bacillus subtilis, PgdS, cleaves poly-gamma-glutamate (PGA) in an endo-peptidase-like fashion. However, its catalytic property remains obscure. In this study, a simple assay for the PgdS enzyme using 1-fluoro-2,4-dinitrobenzene was developed, and some characteristics of PgdS, such as optimal pH, were examined. The enzyme was strongly inhibited by a thiol-modifying reagent, suggesting that it possesses essential cysteine residue(s) in catalysis. PgdS exhibited a high affinity to PGA that consisted mainly of D-glutamate residues, but no affinity to PGA composed only of L-glutamate residues (L-PGA). The enzyme processed DL-copolymer-type PGA (DL-PGA) with an average molecular mass of 1,000 kDa to a high-molecular-mass L-glutamate-rich fragment (average 200 kDa), the L-rich PGA fragment, and low-molecular-mass fragment composed mostly of D-glutamate residues (average 5 kDa), D-fragment. To deepen our understanding of the catalytic property of the PgdS enzyme, we analyzed the structures of the N- and C-terminal regions and found that D-glutamyl residues successively lie even at both ends of the L-rich PGA fragment. Our observations indicate that PgdS is a novel endo-peptidase that specifically cleaves the gamma-amide linkage between two D-glutamate residues in PGA, i.e., gamma-glutamyl DD-amidohydrolase. The enzyme is possibly useful in the biochemical processing of B. subtilis DL-PGA.     

Mumefural and related HMF derivatives from Japanese apricot fruit juice concentrate show multiple inhibitory effects on pandemic influenza A (H1N1) virus

Fruit-juice concentrate of Japanese apricot (Prunus mume Sieb. et Zucc.) has been shown to be effective against influenza A infection in MDCK cells. In this study, we isolated five components from the fruit-juice concentrate of Japanese apricot, 5-(hydroxymethyl)-2-formylfuran (HMF), 1-[5-(2-formylfuryl)methyl]dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (mumefural, MF), 2-[5-(2-formylfuryl)methyl]dihydrogen 2-hydroxypropane-1,2,3-tricarboxylate (MF‘), 1-[5-(2-formylfuryl)methyl]hydrogen 1-hydroxyethane-1,2-dicarboxylate (MA1) and 2-[5-(2-formylfuryl)methyl]hydrogen 1-hydroxyethane-1,2-dicarboxylate (MA2), and investigated their inhibitory activities against the novel influenza A/Narita/1/2009 (H1N1) pandemic virus hemagglutinin and neuraminidase functions, which are essential for viral attachment and budding, respectively. An hemagglutination inhibition assay indicated that MF and MF‘ were effective at minimum hemagglutination concentrations of 3.1 and 6.3 mM, respectively. An inhibition study for sialidase activity of the neuraminidase spike showed that MF was the most active anti-sialidase compound with an IC₅₀ value of 0.21 ± 0.01 mM, followed by MA2 (IC₅₀, 0.71 ± 0.09 mM), MA1 (IC₅₀, 1.64 ± 0.31 mM) and MF‘(IC₅₀, 1.62 ± 0.22 mM). Furthermore, MF was shown to inhibit the growth of the pandemic virus in a dose-dependent manner (62 ± 3% inhibition at 5 mM). The results suggest that MF, a citric acid ester linked to HMF at the 1-position of the propane backbone, might be a lead compound for the development of anti-influenza A inhibitors.     

Potent antioxidative activity of non‐polyphenolic fraction of green tea (Camellia sinensis) – association with pheophytins a and b

The antioxidative activity of green tea‐derived polyphenols known as catechins has been extensively studied. However, the antioxidative activity of the non‐polyphenolic fraction of green tea has been poorly elucidated and is the subject of the present study. The non‐polyphenolic fraction of green tea showed a significant dose‐dependent suppressive effect against the autooxidation of linoleic acid. The chlorophyll‐related compounds pheophytins a and b showed similar antioxidative activities in the same assay, much higher than those of α‐tocopherol and the green tea catechin (−)‐epigallocatechin‐3‐gallate (EGCG). The non‐polyphenolic fraction of green tea and pheophytins a and b exhibited suppressive activities against superoxide anion (${\bf O}_{2}ˆ{‐}$ ) generation in mouse macrophages induced by 12‐O‐tetradecanoylphorbol‐13‐acetate, showing higher activities than that of EGCG. These results suggest that the non‐polyphenolic fraction of green tea leaves has potent antioxidative activity and that this activity is associated with pheophytins a and b. © 2000 Society of Chemical Industry     

口服透明质酸对小鼠佐剂性关节炎的作用

目的:探讨口服透明质酸(HA)对小鼠佐剂性关节炎(AA)的治疗作用。方法:将40只6周龄的雄性小鼠随机分成4个小组,正常组、模型组、HA组和双氯芬酸钠组。通过给模型组、HA组和双氯芬酸钠组小鼠右后足注射弗氏完全佐剂(FCA)建立佐剂性关节炎模型,经连续4周口服透明质酸进行治疗后,分别对小鼠的体质量、足跖厚度进行测量,观察小鼠膝关节肿胀程度并评分,同时采用酶联免疫吸附实验(ELISA)检测小鼠血清中白介素-1(IL-1)和HA的含量。结果:口服HA能显著降低小鼠足跖厚度和关节炎指数,同时血清中IL-1和HA含量亦显著降低;并且各项指标与双氯芬酸钠无显著性差异。结论:口服透明质酸能有效地缓解小鼠膝关节的肿胀,降低血清中IL-1的含量,增强活动能力,因而对小鼠佐剂性关节炎具有良好的治疗作用。     

食用沙拉酱对部分血脂指标的影响

目的:通过试食实验,评价沙拉酱对人体部分血脂指标影响的效果。方法:采用自身对照设计的方法。47例18～22岁青年健康男性参与为期70d的试食实验。在不影响试食者饮食习惯的前提下,每日提供15g沙拉酱。分别在实验前及试食过程中的30、60、70d后分别进行检测。并于实验结束后37d再进行一次检测,以观察试食者在食用沙拉酱后血脂指标的变化。结果:试食至30d,试食者低密度脂蛋白胆固醇(LDL-C)含量显著下降(P<0.01)。试食至60d,血清总胆固醇(TC)、甘油三酯(TG)的含量显著上升(P<0.01),LDL-C值显著下降(P<0.01)。试食至70d,TC、TG含量显著上升(P<0.01)、高密度脂蛋白胆固醇(HDL-C)含量显著上升(P<0.01)。结论:在实验所指定的剂量下,沙拉酱未对人体血脂造成不良影响。值得注意的是,试食至50d时,由于节日原因,试食者饮食结构发生突变,饮酒和动物性脂肪摄入较多,导致第60天及第70天检测所得数据中TC和TG等有异常变化,证明饮食结构的变化直接影响血脂指标的变化。本次试食实验的数据表明,沙拉酱对提高HDL-C含量、降低LDL-C含量具有一定的促进作用。     

Spatial distribution of submarine groundwater discharge and associated nutrients within a local coastal area

To understand the local-scale distribution of submarine groundwater discharge (SGD) and dissolved nutrients, a multiple-detector (222)Rn monitoring survey was undertaken along the Mt. Chokai volcanic coast in northern Japan. The surveys revealed that the highest SGD (calculated to be 6.2 × 10(4) m(3) d(-1), within an area of 2 × 10(4) m(2)) with the greatest nutrient fluxes (sum of NO(3)(-), NO(2)(-), and NH(4)(+) (DIN): 9.2 × 10(2) mol d(-1); PO(4)(3-) (DIP): 56 mol d(-1)) is present at the edge of the youngest volcanic lava flow in the area. Recharged groundwater transports nutrients through porous volcanic flows and discharges as SGD near shore. Our results demonstrate that the spatial distribution of SGD in the study area is closely regulated by the local geology and topography. Furthermore, we show that continuous (222)Rn monitoring with a multidetector system at boat speeds of 1-2 knots provides details at a scale one order of magnitude greater than has been reported previously. In addition, the results of our study suggest that SGD-borne DIP may play an important role in the important local oyster production.