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.     

Schizosaccharomyces pombe gmd3(+)/alg11(+) is a functional homologue of Saccharomyces cerevisiae ALG11 which is involved in N-linked oligosaccharide synthesis

The oligosaccharide of glycoproteins in the fission yeast Schizosaccharomyces pombe is unique in containing galactose. We isolated four mutants that had reduced amounts of galactose residues on their cell surface glycoproteins by fluorescence-activated cell sorter. The isolated four recessive mutants, gmd1 to gmd4, showed a defect in glycosylation of acid phosphatase, a cell surface glycoprotein. In gmd3 mutant cells, the amounts of both mannose and galactose residues were decreased on the cell surface galactomannoproteins, suggesting an underglycosylation of galactomannoproteins. The gmd3(+) gene encodes a protein that has significant similarity with Saccharomyces cerevisiae Alg11p and is likely to be involved in N-linked core oligosaccharide synthesis. ALG11 suppressed the gmd3 mutation, indicating that gmd3(+) gene is a functional homologue of the ALG11 gene. We therefore designated gmd3(+) as alg11(+).     

Simulation of migration from a multi-layer laminated film intended for retort foods

Migration from multi-layer laminated film pouches intended for retort foods was investigated through HPLC analysis with a fluorescence detector, and measurements of residue on evaporation, consumption of potassium permanganate and total organic carbon. HPLC analysis revealed that the levels of migrants in oil and the water which were heated in the pouches (121 degrees C, 30 min) were ten times of those in the corresponding official simulants under the official conditions; n-heptane (25 degrees C, 60 min), and water (95 degrees C, 30 min). Bisphenol A diglycidyl ether and related compounds were found in the oil and the water heated in the pouches, as well as in the simulants. These compounds were thought to have been present in the adhesive between the laminated films, and migrated through the food-contact film of the package. Consumption of potassium permanganate and residue on evaporation of the heated water were ten times of those of the water simulant, while the total organic carbon level of the heated water was several-hold greater than that of the water simulant. In addition, migrant levels per surface area of the pouch were one-fourth of the concentrations per content volume of the pouch. Since compliance with the legal limits is evaluated based on the concentration per surface area, real migration into foods would be underestimated by a factor of another four.     

Site-directed mutagenesis study of the antibody 2D7 which catalyzes a reaction for insertion of Cu2+ into mesoporphyrin

Monoclonal antibody 2D7 generated against a transition-state analog N-methyl mesoporphyrin catalyzes a reaction for insertion of a cupric ion into mesoporphyrin. To investigate amino acid residues responsible for the catalytic activity, site-directed mutagenesis of the amino acid residues in the third complementarity determining region of the heavy chain (CDRH3) was performed on the antigen-binding fragment (Fab) of the antibody. Recombinant Fab mutants, in which Arg95 is replaced with Ala (R95A), Asp96 with Asn (D96N) and Met97 with Gly (M97G), were examined in terms of the catalytic efficiency of the reaction (k/K(S)) and the dissociation constant for N-methyl mesoporphyrin binding (K(d)) and these values were compared with those of the wild type. The k/K(S) values of the R95A and D96N mutants were 0.96% and 1.0% of that of the wild type, respectively, whereas the M97G mutant had no detectable catalytic activity. The K(d) values of the R95A and D96N mutants were 165 and 69 times that of the wild type, respectively, while that of the M97G mutant was similar to that of the wild type. The relationship between the k/K(S) and 1/K(d) values in the wild type and the R95A and D96N mutants suggests that Arg95 and Asp96 are responsible for stabilizing the transition-state in the catalytic reaction. The results of the M97G mutant allow us to propose that Met97 plays an important role in the catalytic activity probably due to a subtle and specific conformation of the antibody.     

Improvements in the cell-free production of functional antibodies using cell extract from protease-deficient Escherichia coli mutant

Expression of a functional antibody fragment (Fab) using an Escherichia coli cell-free expression system has been reported previously [Jiang et al., FEBS Lett., 514, 290-294 (2002)]. The low yield of the synthesized antibody, however, limits the usefulness of the cell-free expression system, partly due to the degradation of product by endogenous proteases from the E. coli extract. To determine which proteases are responsible for the degradation, we compared the expression of a 6D9 Fab fragment under conditions whereby several protease inhibitors were added into the cell-free system. The addition of serine protease inhibitor increased the amount of the Fab fragment, indicating that serine proteases caused the antibody degradation. Therefore, several serine protease-deficient mutants of E. coli BW25113 were constructed by targeted homologous recombination. The use of extract from a double protease-deficient mutant (DeltadegP-ompT) significantly increased the amount and antigen-binding activities of an anti-HSA scFv and a 6D9 Fab fragment. These results suggest that the DegP- and OmpT-deleted mutant is a useful source of S30 extract for the production or screening of antibodies using the cell-free expression system.     

Physicochemical characteristics of rapidly dried onion powder and its anti-atherogenic effect on rats fed high-fat diet

Rapidly dried onion (Allium cepa L. cv. Momiji No. 3) powder (OP) prepared from the outer layers (from second to fourth scale leaves from the surface) of onion bulbs was analysed for its quercetin and polyuronide contents, the effects of enzymatic treatment and the anti-atherogenic effect on rats fed a high-fat diet. Quercetin 4′-glucoside (50%), free quercetin (30%) and quercetin 3,4′-diglucoside (20%) were identified as quercetin derivatives, and boiling-water extraction was effective in extracting these compounds. OP contained 12.9% of polyuronides, the basic skeleton of pectin. Enzymatic degradation (cellulase and pectinase, 50 °C for 12 h, pH 6.0) of OP was effective in obtaining a slurry of smaller particle sizes. The free quercetin increased and the glucosides decreased with enzyme treatment. In Wistar rats fed an OP-added high-fat diet, the total cholesterol, HDL-cholesterol and triglyceride concentrations were not significantly different from the rats fed a high-fat diet without OP. However, the atherogenic index (AI) of Wistar rats fed an OP-added high-fat diet was lower (AI = 3.3) than rats fed the diet without OP (AI = 4.1). The incremental elastic modulus (IEM) of the aorta from rats fed the OP-added diet was also significantly lower than that of the rats fed the diet without OP. The AI and IEM values of the rats fed the OP-added diet were quite similar to the values of rats fed the diet without OP but were allowed spontaneous exercise. These results suggest that OP intake is effective for decreasing the risk of arteriosclerosis.     

微量润滑油润滑和冷风冷却加工法对高硅铝合金切削面的影响

由于高硅铝合金中含有大量硅结晶粒子,硬度较大的硅结晶粒子在切削中会加速刀具的磨损,所以,其被切削性非常不好。为解决这一问题,以往是使用大量切削油进行切削加工。但是,随着环境意识的提高,大量切削油的使用所造成的环境污染的问题不得不被认识并进行改变。使用对切削点进行微量植物性润滑油润滑和冷风冷却的方法进行研究,探讨对高硅铝合金切削面的影响。并因此探讨半干式切削加工的可行性。     

Induction of platelet-activating factor in mice by intravenous administration of a neutral fraction of bakers' yeast mannan

A neutral subfraction of mannan of bakers' yeast (WNM) was found to show a lethal effect in mice when administered intravenously. Symptoms caused by intravenous (i.v.) administration of WNM resembled those resulting from the administration of platelet-activating factor (PAF). CV-3988 and ONO-6240, selective PAF antagonists, prevented hypotension and death caused by the administration of WNM or PAF. A β-adrenoceptor agonist was shown to prevent death caused by WNM, whereas propranolol increased the lethal activity of WNM. Intravenous administration of WNM into mice produced PAF in gall bladder fluid which was determined by platelet aggregation assay. The findings indicate that WNM is able to induce PAF in mice and that the resultant PAF may participate in the WNM-induced lethal activity observed in mice.     

Ozone treatment of water-soluble polymers. V. Ultraviolet irradiation effects on the ozonization of polyacrylamide

Polyacrylamide was ozonized in aqueous solution (at pH 2, 7, and 11) under UV irradiation with a low-pressure mercury lamp. The breakage of the polymer chains by ozone was strongly accelerated by UV irradiation under acidic and neutral conditions. Formaldehyde was produced characteristically in the UV ozonization. From the correlation between the amount of formaldehyde and the number of breaks of polymer chains, it was presumed that one molecule of formaldehyde resulted from the chain breakage, at least under acidic conditions. The intensity of the UV absorption peak at 266 nm (285 nm in the UV ozonization at pH 2), which was presumed to be due to the carbonyl groups, namely, ketone and terminal aldehyde produced by ozonization, was very much stronger than that in simple ozonization. Oxamic acid and oxamide as end products were observed in the solution which was ozonized exhaustively under UV irradiation.     

Verification of a model for estimating the void fraction in a three-component randomly packed bed

The void fraction in a three-component randomly packed bed was calculated from the authors' model, and the calculated values were compared with published experimental data for spherical and irregularly shaped particles and with results from computer simulations. Results from the model were in good agreement with simulated and published experimental data.