A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate

Alaska pollack frame protein, which is normally discarded as an industrial byproduct in the processing of fish in plants, was hydrolyzed with pepsin. This was fractionated into five major types of Alaska pollack frame protein hydrolysates (APH-I, 10-30 kDa; APH-II, 5-10 kDa; APH-III, 3-5 kDa; APH-IV, 1-3 kDa; and APH-V, below 1 kDa) using an ultrafiltration membrane bioreactor system. Angiotensin I converting enzyme (ACE) inhibitory activities of the fractionated hydrolysates were investigated, and the fraction that exhibited the highest ACE inhibitory activity was further purified using consecutive chromatographic methods on SP-Sephadex C-25 column, Sephadex G-25 column, and high-performance liquid chromatography (HPLC) on an octadecylsilane column. Finally, we purified a novel ACE inhibitory peptide with an IC50 value of 14.7 microM, and the sequence of the peptide was Phe-Gly-Ala-Ser-Thr-Arg-Gly-Ala. In addition, the ACE inhibition pattern of the peptide was found to be noncompetitive.     

A comparison of two methods for the isolation of free and occluded particulate organic matter

Various methods exist for the isolation of particulate organic matter (POM), one of the soil‐organic‐matter (SOM) fractions reacting most sensitive on land‐use or soil‐management changes. A combination of density separation and ultrasonic treatment allows to isolate two types of POM: (1) free POM and (2) POM occluded in soil aggregates. POM fractions are closely linked to their biochemical function for the formation and stabilization of aggregates, therefore methods using different aggregate sizes may result in different POM fractions isolated. We evaluated two physical fractionation procedures to reveal whether they yield different POM fractions with respect to amount and composition, using grassland and arable soils with sandy‐loam to sandy–clay‐loam texture and thus low macroaggregate stability. Method I used air‐dried aggregates of <2.0 mm size and a low‐energy sonication for aggregate disruption, method II used field‐moist aggregates <6.3 mm and a high‐energy–sonication procedure for aggregate disruption. POM fractions were analyzed by elemental analysis (C, N) and CPMAS ¹³C‐NMR spectroscopy. With both methods, about similar proportions of the SOM are isolated as free or occluded POM, respectively. The free‐ and occluded‐POM fractions obtained with method I are also rather similar in C and N concentration and composition as shown by ¹³C‐NMR spectroscopy. Method II isolates a free‐ and occluded‐POM fraction with significantly different C and N concentrations. NMR spectra revealed significant differences in the chemical composition of both fractions from method II, with the occluded POM having lower amounts of O‐alkyl C and higher amounts of aryl C and alkyl C than the free POM. Due to the use of larger, field‐moist aggregates with minimized sample pretreatment, two distinctly different POM fractions are isolated with method II, likely to be more closely linked to their biochemical function for the formation and stabilization of aggregates. High‐energy sonication as in method II also disrupts small microaggregates <63 µm and releases fine intraaggregate POM. This fraction seems to be a significant component of occluded POM, that allows a differentiation between free and occluded POM in sandy soils with significant microaggregation. It can be concluded, that microaggregation in arable soils with sandy texture is responsible for the storage of a more degraded occluded POM, that conversely supports the stabilization of fine microaggregates.     

Modulations of the Bcl-2/Bax family were involved in the chemopreventive effects of licorice root (Glycyrrhiza uralensis Fisch) in MCF-7 human breast cancer cell

Recently, cancer chemoprevention with strategies using foods and medicinal herbs has been regarded as one of the most visible fields for cancer control. Genistein in soy, American ginseng, and resveratrol are well-known to have antiproliferative properties in human breast cancer. Licorice root is a botanical, a shrub native to southern Europe and Asia, which primarily has desirable qualities in sweetening and herbal medicine. In this study, licorice (Glycyrrhiza uralensis Fisch) root also inhibits cell proliferation in human breast cancer cell. The cell proliferation study demonstrated that licorice root reduced the proliferation of MCF-7 cells in a dose- and time-dependent manner. The extracts were fractionated in CHCl(3), EtOAc, C(6)H(14), and CH(3)OH-H(2)O (70:30), and these extracts of licorice root (50 microg/mL) induced DNA fragmentation demonstrated by Hoechst 33258 staining. Apoptosis also determined the sub-G1 accumulation by flow cytometry analysis. These results were consistent with specific cleavage of PARP and antiapoptotic protein Bcl-2 and up-regulation of proapoptotic protein Bax demonstrated by Western blotting. Our findings suggest that licorice root may have chemopreventive effects against human breast cancer through the modulation of the expression of the Bcl-2/Bax family of apoptotic regulatory factors.     

Spatial distribution of earthworms [Lumbricidae] in recultivated soils of the Rhenish lignite‐mining area,Germany

The spatial distribution of earthworms was studied by means of combined formalin expulsion and hand sorting in three arable fields of the Rhenish lignite‐mining area that differed in their recultivation age (6, 12, 24 yr). In addition, pH and the spatial distribution of penetration resistances were measured to see if they are corresponding with the distribution of earthworms. Already the 6 yr old field had a rich population of endogeic, anecic, and epigeic earthworms (119 ind. m^–2, 48 g m^–2, 6 species). This quantity was similar to the 24 yr old site. The 12 yr old field was only sparsely populated by earthworms (5 ind. m^–2, 5 g m^–2, 3 species). In the 6 yr old field, the spatial distribution pattern showed a center of maximal earthworm abundances, corresponding to the distributional pattern of penetration resistances. In the old field (24 yr), the species varied in their spatial distribution, and there was no correspondence with the distribution of penetration resistance. In general, the penetration resistance at the youngest site was clearly lower than at the two older sites. The earthworm population in the 6 yr old field can be explained by cocoons contained in the dumped material. A calculation using literature data on earthworm‐population dynamics shows that a founding population of 400–600 reproductive individuals per hectare and a continuity of favorable growth conditions during the time of soil management is necessary for the development of the situation found at the 6 yr old site in this study.     

Methylation methods for the quantitative analysis of conjugated linoleic acid (CLA) isomers in various lipid samples

Precise methylation methods for various chemical forms of conjugated linoleic acid (CLA), which minimize the formation of t,t isomers and allylmethoxy derivatives (AMD) with the completion of methylation, were developed using a 50 mg lipid sample, 3 mL of 1.0 N H(2)SO(4)/methanol, and/or 3 mL of 20% tetramethylguanidine (TMG)/methanol solution(s). Free CLA (FCLA) was methylated with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). CLA esterified in safflower oil (CLA-SO) was methylated with 20% TMG/methanol (100 degrees C, 5 min), whereas CLA esterified in phospholipid (CLA-PL) was methylated with 20% TMG/methanol (100 degrees C, 10 min), followed by an additional reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). Similarly, CLA esterified in egg yolk lipid (CLA-EYL) was methylated by base hydrolysis, followed by reaction with 1.0 N H(2)SO(4)/methanol (55 degrees C, 5 min). These results suggest that for the quantitative analysis of CLA in lipid samples by GC, proper methylation methods should be chosen on the basis of the chemical forms of CLA in samples.     

Cathepsin L: a predominant heat-activated proteinase in arrowtooth flounder muscle

Characterization of the autolytic profile of arrowtooth flounder (ATF) muscle indicated the involvement of heat-activated proteinases active at both acidic and alkaline pH values. Further assay of fish extract exhibited the maximum activity at 60 degrees C against casein used as a substrate at both pH 5.5 and 8.0. The maximum activity shifted to lower temperatures by the addition of urea with two distinctive patterns: activity reduction at pH 5.5 and activity enhancement at pH 8.0. The highest inhibition by E-64 indicated the proteinase belongs to the cysteine proteinase class. At pH 5.5, the proteinase hydrolyzed Z-Phe-Arg-NMec and all types of protein substrates tested at higher rate than that at pH 8.0. Activity bands, observed on the activity-stained substrate gels, indicated similar proteinases are responsible for the proteolytic activity observed at both pH values. When proteins of fish extract were separated by HPLC-SEC, only one proteolytic peak was observed at the retention time of 26 min with an estimated molecular weight of 39800 Da. The results implied cathepsin L is a predominant proteinase responsible for autolysis of ATF muscle at elevated temperatures.     

Structure elucidation and antifungal activity of an anthracycline antibiotic, daunomycin, isolated from Actinomadura roseola

The actinomycete strain Ao108 producing antifungal metabolites active against some plant pathogenic fungi was identified as Actinomadura roseola, based on the analyses of morphological and physiological characteristics. The antibiotic Da2B that showed a strong antifungal activity was isolated from the culture broth and mycelial mats of A. roseola strain Ao108 using various chromatographic procedures. On the basis of (1)H NMR, (13)C NMR, and 2-D NMR correlation data, the antibiotic Da2B was confirmed to have the structure of an anthracycline antibiotic, daunomycin. In vitro antimicrobial spectrum tests showed that the antibiotic Da2B had substantial inhibitory activity (10 microg mL(-)(1) of MICs) against mycelial growth of Phytophthora capsici and Rhizoctonia solani. The antibiotic also showed antiyeast activity against Saccharomyces cerevisiae, but the growth of Candida albicans was not affected. Antibacterial activity was found only against Gram-positive bacteria. In the further evaluation of in vivo efficacy, application of the antibiotic Da2B effectively inhibited the development of Phytophthora blight in pepper plants. However, the control efficacy of the antibiotic against Phytophthora infection was somewhat less than that of metalaxyl. The antibiotic Da2B did not show any phytotoxicity on pepper plants even at 500 microg mL(-)(1).     

Changes in soil properties and humic substances after long‐term amendments with manure and crop residues in dryland farming systems

After 16 years of periodical applications of either farmyard manure or crop wastes at two levels of mineral N fertilization to a Calcic Haploxeralf in the semiarid central Spain, we found significant changes in chemical fertility levels and in the concentration, chemical composition, and carbon mineralization rates of soil organic matter (SOM). The changes in SOM quality were related to significant improvements of soil physical properties, mainly aggregate stability and water retention. Such changes were related to the increased concentration of humic colloids in soil, the mineral N dose, and the type of organic matter applied. When compared with the control plots, the organic matter accumulated in the amended plots tended to be less transformed, and its total concentration and humification degree decreased with increasing external N‐inputs. Humic acids from the amended plots showed a more marked aliphatic character (mainly after N addition) than those from control plots. Farmyard manure led to a significant improvement of soil physical properties, but had a comparatively small effect in promoting biodegradation and humification of crop wastes. This could be due to the high biological stability of the manure used which, in semiarid Mediterranean fields, usually leads to an accumulation of little transformed SOM.     

Antioxidative, antimutagenic, and anticarcinogenic activities of rice bran extracts in chemical and cell assays

Ethanol-water (70:30 v/v) extracts from rice brans removed from seeds of two blackish-purple pigmented (Sanhaehyanghyulla and Suwon 415) and one nonpigmented (Chuchung) brown rice cultivars were evaluated for antioxidative, anti-tumor-promoting, and anticarcinogenic activities in chemical assays and in mammalian cells (human leukemia HL-60, marmoset B lymphoblastoid B95-8, and Chinese hamster V79 lung cells) by the following tests: inhibition of xanthine oxidase activity; chelation of ferrous ions; reduction of potassium ferricyanide; scavenging of superoxide anions, hydroxyl radicals, and intracellular peroxides; inhibition of 4-nitroquinoline N-oxide-induced mutagenesis; and inhibition of phorbol ester-induced tumor promotion. The extracts from the pigmented rice seeds had generally higher activities in all tests than did the extract from the nonpigmented variety. The results suggest that brans from pigmented rice varieties may provide a source of new natural antioxidants and anticarcinogens and that such rice cultivars with high antioxidative potential also provide a genetic resource for the development of new, improved rice cultivars that may make it possible to enhance both the nutritional and medical value of rice-based diets.