Application of several types of substrates to ficin-catalyzed peptide synthesis

Summary. The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction. The products were obtained in 72–96% yield using 10 different substrates, within a few minutes of reaction time. Authors’ address: Prof. Haruo Sekizaki, Faculty of Pharmaceutical Sciences, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Hokkaido 061-0293, Japan     

Phenolic and other constituents of fresh water fern Salvinia molesta

Two glycosides, 6'-O-(3,4-dihydroxy benzoyl)-beta-D-glucopyranosyl ester (1), and 4-O-beta-d-glucopyranoside-3-hydroxy methyl benzoate (2), along with five known compounds methyl benzoate (3), hypogallic acid (4), caffeic acid (5), paeoniflorin (6) and pikuroside (7) were isolated for the first time from a fresh water fern Salvinia molesta D.S. Mitch. These compounds showed a potent antioxidant radical scavenging activity in a non-physiological assay. Their structures were determined by NMR spectroscopic and CID mass spectrometry techniques.     

Dynamics of C, N, P and microbial community composition in particulate soil organic matter during residue decomposition

Decomposing residues can be an important source of nutrients for plants, especially of N and P, but the relationship between N and P release and microbial community dynamics have rarely been studied. Two pea (Pisum sativum L.) residues with contrasting chemical composition, shoots from flowering pea (Pea-Y) with 2.9 mg P and 36 mg N kg⁻¹ and from mature pea (Pea-M) with 0.3 mg P and 13 mg N kg⁻¹, were added at a rate of 20 g kg soil⁻¹ to a sandy soil low in nutrients. Particulate organic matter (POM) was isolated on days (d) 0, 5, 15, 28, 42 and 61 after residue addition and analysed for C, N, P and microbial community structure (fatty acid methyl ester analysis). The recovery of POM from residue-amended soils decreased over time to 30–40% of added amounts for both residues. Apart from d 0, the N concentration in POM was lower in residue-amended soil than in the control. Due to a rapid decrease in P concentration during the first 5 days in Pea-Y and a slow increase over the whole experiment in Pea-M, P concentrations in POM on d 61 were similar in all treatments. In Pea-Y, the dynamics of C, N and P were coupled, with amounts of C, N and P decreasing during the first 15 days and remaining stable thereafter. In Pea-M, a steady loss of C from POM was contrasted by a slight increase in P. As a result, the C/P ratio decreased from 1,330 on d 0 to 390 on d 61. The C/N ratio of Pea-M decreased only during the second phase of decomposition. The different nutrient dynamics in Pea-Y and Pea-M led to similar amounts of N and P in POM towards the end of the incubation. Microbial community composition in the POM in Pea-Y and Pea-M remained distinct from the control, even though it changed over time. POM was shown to be an important source of potentially available nutrients after addition of plant residues. In the unamended soil, stable nutrient amounts in POM suggested very low net nutrient release from native POM compared to POM after residue addition.     

Preparation and characterization of the graft copolymer of chitosan with poly[rosin-(2-acryloyloxy)ethyl ester]

Graft copolymerization of rosin-(2-acryloyloxy)ethyl ester (RAEE) onto chitosan (Cts) was carried out under microwave irradiation using potassium persulfate as an initiator. The structures, morphology, and thermal properties of the Cts graft copolymer (Cts-g-PRAEE) were characterized by means of FT-IR, XRD, SEM, and TG. Also, Cts and Cts-g-PRAEE copolymer were used as carriers of fenoprofen calcium (FC), and their controlled release behavior in artificial intestinal juice were studied. The results show that the rate of release of fenoprofen calcium from the carrier of Cts-g-PRAEE copolymer becomes very slower than that of Cts in artificial intestinal juice.     

Plasma transport and tissue distribution of β-carotene, vitamin A and retinol-binding protein in domestic cats

The objective of this study was to determine retinol, retinyl esters and retinol-binding protein (RBP) as well as carotenoids in plasma, urine, liver and kidneys of randomly selected domestic cats. Retinol (240±64 ng/ml, mean±S.D.) represented one-third of total retinyl esters (736±460 ng/ml) in plasma. Retinyl esters were stearate, palmitate and oleate representing 61±6, 36±13 and 5±3% of total retinyl esters, respectively. In half of the cats, retinyl esters (22±21 ng/ml) were found in the urine. Vitamin A in the livers (4317±1956 μg/g) was significantly higher than in the kidney cortex and medulla (14.16±8.92 and 7.59±4.52 μg/g, respectively, both P<0.001). RBP was detected in the plasma but not in the urine. Immunoreactive RBP was observed in hepatocytes and in the cells of the proximal tubules. β-Carotene was present in plasma but never in tissues. The results show that similar to canines differences in vitamin A metabolism in cats are related to the occurrence of retinyl esters in plasma. They differ, however, with regard to the tissue distribution of β-carotene and the excretion of vitamin A in the urine.     

化学发光蛋白芯片用于检测肝癌患者血清CA19-9水平的方法研究

目的:探讨一种简单、有效、低成本、低耗时的化学发光蛋白芯片方法用于检测血清中的糖链抗原19-9(Carbohydrate Antigen19-9,CA19-9),以有助于对原发性肝癌的早期辅助诊断。方法:预先在醛基芯片上包被鼠源CA19-9单克隆抗体,建立CA19-9抗体蛋白芯片,共筛选出46份肝癌血清和32份正常健康人血清,然后用蛋白芯片方法进行检测,并以化学发光成像对检测结果进行判定。结果:24份肝癌血清的CA19-9水平高于37 U/m L,22份肝癌血清的CA19-9水平低于37 U/m L;30份正常人血清CA19-9含量低于37 U/m L,2份正常人血清的CA19-9含量为37 U/m L;灵敏度为52.17%,特异性为93.75%,ROC曲线下面积0.688[95%CI:0.566,0.811]。结论:本研究成功的建立了血清CA19-9的化学发光蛋白芯片检测方法。     

Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis

In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an initial step prior to initiation of necrotic cell death. In addition, the dual addition led to black circular structures of 10–20 nm, interpreted as stain-filled cylindrical channels on transmission electron microscopy. The formation of palmitoyl-ceramide channels in the lysosome membrane causes the liberation of cathepsin B from lysosomes for necrotic cell death. On the other hand, necrotic cell death in the dual addition was not caused by oxidative stress or cathepsin B activity, and the cell death was free from the contribution of the translation of Bax protein to the lysosome membrane.     

Physiological functions of pyruvate:NADP+ oxidoreductase and 2-oxoglutarate decarboxylase in Euglena gracilis under aerobic and anaerobic conditions

In Euglena gracilis, pyruvate:NADP⁺ oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon and energy metabolisms in Euglena under aerobic and anaerobic conditions, physiological significances of these enzymes involved in 2-oxoacid metabolism were examined by gene silencing experiments. The pyruvate dehydrogenase complex was indispensable for aerobic cell growth in a glucose medium, although its activity was less than 1% of that of pyruvate:NADP⁺ oxidoreductase. In contrast, pyruvate:NADP⁺ oxidoreductase was only involved in the anaerobic energy metabolism (wax ester fermentation). Aerobic cell growth was almost completely suppressed when the 2-oxoglutarate decarboxylase gene was silenced, suggesting that the tricarboxylic acid cycle is modified in Euglena and 2-oxoglutarate decarboxylase takes the place of the 2-oxoglutarate dehydrogenase complex in the aerobic respiratory metabolism.     

Key compounds contributing to the fruity aroma characterization in Japanese raw soy sauce

In order to clarify the aroma characteristics of raw soy sauce (RS), the application of gas chromatography?olfactometry analysis to the aroma concentrate from a RS revealed 76 aroma peaks, of which 25 peaks showed fruit-like aromas. Furthermore, the head space aromatic compounds of RS were analyzed with 32 peaks detected. Ethyl 2-methylpropanoate, ethyl butanoate, ethyl 2-methylbutanoate, ethyl 3-methylbutanoate, and ethyl 4-methylpentanoate were detected with higher flavor dilution factor (FD factor) than other aroma compounds by aroma extract dilution analysis. Quantitative analysis suggested that these compounds were common in all RS samples tested in this study, and were present at higher concentrations than their perception thresholds. The concentrations and the FD factors of these compounds were significantly decreased during the heating of the RS. Fruitiness is one of the key aroma characteristics of RS and the ethyl esters identified in this study are the key components contributing to this distinct aroma.