Enhanced antiviral activity of soybean β-conglycinin-derived peptides by acylation with saturated fatty acids

Abstract: Peptide mixtures prepared from soybean β‐conglycinin (7S‐peptides) were acylated with saturated fatty acids of different chain length (6C‐18C) in order to improve their antiviral activity against Feline calicivirus (FCV) strain F9 which is a typical norovirus surrogate. Among the fatty acids varieties, it was revealed that 7S‐peptides acylated with myristic and palmitic acids potently inhibited FCV replication. Myristorylation and palmitoylation of 7S‐peptides kept host cells viability at 91.51% and 98.90%, respectively. The infectivity of FCV on Crandell–Reese feline kidney cells was further determined after exposure of initial titer of 10^6.47 TCID₅₀/mL. Myristoylated and palmitoylated 7S‐peptides significantly (P < 0.006) reduced FCV infectivity as compared to native 7S‐peptides. Native 7S‐peptides showed 25% FCV inhibitory activity while myristoylated and palmitoylated 7S‐peptides exhibited 98.59% and 99.98% reduction in FCV infectivity, respectively. Myristoylated and palmitoylated 7S‐peptides demonstrated higher anti‐FCV activity in a wide range of concentration with complete reduction at 25 μg/mL. Surface hydrophobicity was significantly (P < 0.05) increased after attachment of long hydrocarbon fatty acids to 7S‐peptides as supported by changes in fluorescence intensity. Enzymatic hydrolysis together with acylation will give an insight into surface and physiological functional lipopeptides derived from soy β‐conglycinin.     

Purification, bacteriolytic activity, and specificity of beta-lytic protease from Lysobacter sp. IB-9374

Lysobacter sp. IB-9374, which was isolated from soil as a high lysyl endopeptidase-producing strain (Chohnanet al., FEMS Microbiol. Lett., 213, 13-20, 2002), was found to produce a beta-lytic protease capable of lysing gram-positive bacteria such as Staphylococcus aureus, Microccocuseus, and Bacillus subtilis. The Lysobacter strain secreted the beta-lytic protease into the culture medium at a 2.4-fold higher level than Achromobacter lyticus. The enzyme was highly purified through a series of six steps with a high yield. The enzyme was strongly inhibited by tetraethylene-pentamine and 1,10-phenanthroline. The purified enzyme lysed more efficiently almost all the gram-positive bacteria tested than lysozyme, lysostaphin, and mutanolysin. The enzyme was very similar to Achromobacter beta-lytic protease containing one zinc atom in terms of amino acid composition and N-terminal sequence. The nucleotide sequence revealed that the mature enzyme was composed of 179 amino acid residues with additional 198 amino acids at the amino-terminal end of the enzyme. The deduced amino acid sequence of the mature enzyme coincided with that of the Achromobacter enzyme, although the prepro-region showed a 41% sequence identity with the counterpart. These results indicate that Lysobacter sp. is a useful strain for an efficient large-scale preparation of beta-lytic protease capable of lysing bacteria.     

Introduction of sulfhydryl groups and disulfide linkage to mungbean 8Sα globulin and effects on physicochemical and functional properties

Sulfhydryl groups and disulfide bond were introduced in mungbean's major storage protein, 8Sα globulin, by protein engineering to improve structural stability and functional properties. Five modified proteins or mutants (F59C, I99C, A213C, one free sulfhydryl group; I99C/A213, one disulfide bridge; F59C/I99C/A213C, one free sulfhydryl group and one disulfide linkage) were expressed in Escherichia coli at a yield similar to that of the unmodified protein or wild type (WT) in soluble form (38%). The number of introduced groups in the mutants was confirmed by Ellman analysis. Mutant and WT proteins exhibited similar elution patterns on gel filtration indicating their trimeric native conformation. Mutants had 2 to 3.8 °C higher T_m values than WT and were digested by chymotrypsin at 52–58% in 60 min but exhibited different digestion patterns. All mutants showed greater hardness of heat-induced gels than WT, especially I99C/A213C and F59C/I99C/A213C. Results indicate the improved structural stability of the modified 8Sα globulin.     

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.     

NMR study on sulfonation of internal olefins

Terminal olefins can be converted into the corresponding sulfonates by sulfonation with SO₃ followed by hydrolysis, the products are used as detergent ingredients. Although internal olefins were also expected to be good feed stocks, it was difficult to convert them into the corresponding sulfonates with as good of a yield as that of terminal olefins under the same synthesis conditions. Our studies have been carried out in order to clarify the reason for poorer conversion of the internal olefins. It was found by a nuclear magnetic resonance spectroscopic study that the major components of the sulfonated intermediate of internal olefins wereβ-sultones, the amounts of which were usually very small in sulfonated terminal olefins. A portion of theseβ-sultones was desulfonated, depending on temperature, to the original olefins and the corresponding sulfate salts during alkaline hydrolysis. The prevention of desulfonation of theβ-sultones in the hydrolysis process was one of the most important aspects for the production of internal olefin sulfonates. Finally, by keeping the hydrolysis temperature below 35°C, the conversion of internal olefins into sulfonates was achieved with the same yield as that of conventional terminal olefins.     

Correlations of Hepatic Hemodynamics,Liver Function,and Fibrosis Markers in Nonalcoholic Fatty Liver Disease: Comparison with Chronic Hepatitis Related to Hepatitis C Virus

The progression of chronic liver disease differs by etiology. The aim of this study was to elucidate the difference in disease progression between chronic hepatitis C (CHC) and nonalcoholic fatty liver disease (NAFLD) by means of fibrosis markers, liver function, and hepatic tissue blood flow (TBF). Xenon computed tomography (Xe-CT) was performed in 139 patients with NAFLD and 152 patients with CHC (including liver cirrhosis (LC)). The cutoff values for fibrosis markers were compared between NAFLD and CHC, and correlations between hepatic TBF and liver function tests were examined at each fibrosis stage. The cutoff values for detection of the advanced fibrosis stage were lower in NAFLD than in CHC. Although portal venous TBF (PVTBF) correlated with liver function tests, PVTBF in initial LC caused by nonalcoholic steatohepatitis (NASH-LC) was significantly lower than that in hepatitis C virus (C-LC) (p = 0.014). Conversely, the liver function tests in NASH-LC were higher than those in C-LC (p < 0.05). It is important to recognize the difference between NAFLD and CHC. We concluded that changes in hepatic blood flow occurred during the earliest stage of hepatic fibrosis in patients with NAFLD; therefore, patients with NAFLD need to be followed carefully.     

Single-Component Pheromone Consisting of Bombykal in a Diurnal Hawk Moth, <Emphasis Type="Italic">Neogurelca himachala sangaica</Emphasis>

Recent work has suggested that hawk moths share pheromone components but are sexually separated by qualitative and quantitative differences in their pheromone blends. During field assays on the sex pheromones of other species, a diurnal hawk moth, Neogurelca himachala sangaica (Lepidoptera: Sphingidae), was frequently captured, but the composition of the sex pheromone of this species was not known. Analysis of hexane extracts of the pheromone glands of calling female by gas chromatography (GC) using an electroantennographic detector (EAD) revealed two components that elicited EAD responses from male moth antennae. These components were identified by their mass spectra and retention indices on two GC columns as (10E,12Z)-10,12-hexadecadienal (E10,Z12–16:Ald) and a trace of its (10E,12E)-isomer (E10,E12–16:Ald) in 98:2 ratio. In field experiments, E10,Z12–16:Ald alone attracted male moths, and addition of E10,E12–16:Ald significantly reduced the attractiveness, even at the naturally-occurring ratio. Analysis of the data using a generalized linear mixed model showed that E10,Z12–16:Ald positively contributed to attractiveness, whereas E10,E12–16:Ald did so negatively, and it was concluded that the sex pheromone of N. himachala sangaica consists solely of E10,Z12–16:Ald, bombykal. The negative effect of E10,E12–16:Ald on attractiveness could promote the species-specificity of this single-component pheromone system.     

Highly hydrogenated dietary soybean oil modifies the responses to polychlorinated biphenyls in rats

The effects of dietary highly hydrogenated soybean oil (HSO) upon the changes caused by dietary polychlorinated biphenyls (PCBs) were examined in rats. Six groups of rats were fed the following diets for 30 d: a 20% soybean oil-containing diet (control diet), a diet in which a half of soybean oil was substituted with HSO (HSO-A diet), a diet in which cellulose powder was replaced with HSO (HSO-B diet) and these diets supplemented with 100 ppm PCBs (control+PCBs, HSO-A+PCBs and HSO-B+PCBs diets). Hepatic concentration of PCBs and relative liver weight were markedly decreased in rats fed with the HSO-A+PCBs diet compared with those fed with the other diets containing PCBs. Liver lipids and liver cholesterol were considerably decreased with a reciprocal increase in fecal sterol excretion by rats fed the HSO-A+PCBs and the HSO-B+PCBs diets compared with those fed with the control+PCBs diet. The fatty acid composition in hepatic phospholipids showed an independent increase of the saturated fatty acid content induced by dietary HSO and PCBs. Dietary PCBs also caused decreases in the amounts of monounsaturated and n-3 polyunsaturated fatty acids. These results suggest that dietary HSO prevents accumulation of PCBs in the liver and promotes the excretion of lipids stimulated by PCBs, accompanied by a change in fatty acid metabolism.     

Electrochemical DNA methylation detection for enzymatically digested CpG oligonucleotides

We describe the electrochemical detection of DNA methylation through the direct oxidation of both 5-methylcytosine (mC) and cytosine (C) in 5'-CG-3' sequence (CpG) oligonucleotides using a sputtered nanocarbon film electrode after digesting a longer CpG oligonucleotide with endonuclease P1. Direct electrochemistry of the longer CpG oligonucleotides was insufficient for obtaining the oxidation currents of these bases because the CG rich sequence inhibited the direct oxidation of each base in the longer CpG oligonucleotides, owing to the conformational structure and its very low diffusion coefficient. To detect C methylation with better quantitativity and sensitivity in the relatively long CpG oligonucleotides, we successfully used an endonuclease P1 to digest the target CpG oligonucleotide and yield an identical mononucleotide 2'-deoxyribonucleoside 5'-monophosphate (5'-dNMP). Compared with results obtained without P1 treatment, we achieved 4.4 times higher sensitivity and a wider concentration range for mC detection with a resolution capable of detecting a subtle methylated cytosine difference in the CpG oligonucleotides (60mer).