Purification and characterization of an angiotensin I‐converting enzyme inhibitory peptide derived from porcine troponin C

To search for a novel angiotensin I‐converting enzyme (ACE) inhibitory peptide, porcine skeletal troponin was hydrolyzed with pepsin. This hydrolysate showed ACE inhibitory activity, and was applied to various kinds of chromatography to separate an active peptide. Analysis using a protein sequencer identified this peptide as RMLGQTPTK (9mer). This sequence was estimated to occur at the 44–52 position of troponin C, and its 50% inhibitory protein concentration (IC₅₀) was 34 µM. RMLGQTP (7mer), a partial peptide of 9mer, showed activity with an IC₅₀ of 503 µM. RP‐HPLC analysis of a reaction mixture of 9mer and ACE showed that 9mer was slowly hydrolyzed by ACE. On the other hand, 7mer was rapidly hydrolyzed by ACE. Activity of 9mer was reduced as its hydrolysis by ACE proceeded. To estimate the resistance of 9mer to digestive proteases after oral administration, it was reacted with pepsin, α‐chymotrypsin, or trypsin. In each of these reaction mixtures, a significant amount of 9mer remained as a substrate after digestion. Remaining ACE inhibitory activity was close to that of 9mer. These results suggest that 9mer might not be digested after oral administration, because of its relatively high resistance to digestive proteases. Therefore, 9mer might be expected to work well in vivo as an ACE inhibitor.     

A bovine myeloid antimicrobial peptide (BMAP‐28) kills methicillin‐resistant Staphylococcus aureus but promotes adherence of the bacteria

The cathelicidin family is one of the several families of antimicrobial peptides (AMPs). A bovine myeloid antimicrobial peptide (BMAP‐28) belongs to this family. Recently, the emergence of drug‐resistant bacteria such as methicillin‐resistant Staphylococcus aureus (MRSA) has become a big problem. AMPs are expected to be leading compounds of new antibiotics against drug‐resistant bacteria. In this study, we focused on the activity of BMAP‐28 against bacterial cell surfaces. First, we observed morphological change of MRSA caused by BMAP‐28 using a scanning probe microscope. We also studied activities of BMAP‐28 against adherence of S. aureus to fibronectin, collagen type I, collagen type IV. We confirmed whether BMAP‐28 can bind to lipoteichoic acid (LTA) of S. aureus. BMAP‐28 was indicated as damaging the cell surface of MRSA. In a particular range of concentrations, BMAP‐28 promoted adherence of S. aureus against fibronectin and collagens. It was revealed that BMAP‐28 and LTA of S. aureus bound with each other. Our study showed the potential of BMAP‐28 which can damage MRSA and interact with LTA of S. aureus but promote its adherence in some concentrations. This study provides new points of which to take notice when we use AMPs as medicines.     

Midgut juice of Plutellaxylostella highly resistant to Bacillusthuringiensis Cry1Ac contains a three times larger amount of glucosinolate sulfatase which binds to Cry1Ac compared to that of susceptible strain

Midgut juice of Plutellaxylostella strain PXR which is resistant to Cry1Ac was biochemically characterized relative to the susceptible PXS strain. The midgut juice of PXR (PXR-Juice) was shown to process Cry1Ac protoxin to 60 kDa active toxin with the same processing pattern as that of juice from PXS (PXS-Juice) in SDS–PAGE. PXS larvae which were given the Cry1Ac toxin pre-processed with PXR-Juice were killed with the same rate as that with Cry1Ac pre-activated by trypsin. PXR-Juice was found to contain three times larger amount of 66 kDa protein (P66) than PXS-Juice and the N-terminal amino acid sequence of P66 was matched to that of glucosinolate sulfatase in data base search. The protein band of P66 was coincided with the band of p-nitro phenyl sulfatase activity in zymogram. P66 purified to homogeneity in SDS-PAGE bound to Cry1Ac and soybean agglutinin, and K_D for Cry1Ac was estimated to be 718 nM with surface plasmon resonance analysis. Using purified sulfatase, K_m and V_max were estimated and involvement of the enzyme in the PXR resistance was discussed.     

Diversification of Multipurpose Plant, <Emphasis Type="Italic">Perilla Frutescens</Emphasis>

The traditional Asian crop, Perilla frutescens has multiple uses. There are specialized cultivars for seed oil and for medicinal use, as well as wild/weedy forms growing in various habitats. Based on selective characteristics of leaf odor, anthocyanin pigmentation, seed hardness and seed diameter, the diversity of this species was investigated to clarify the intraspecific differentiation. P. frutescens was divided into five groups by the combination of three qualitative characteristics: leaf odor, anthocyanin pigmentation and seed hardness. Most of the plants cultivated for oil belonged to one group, while medicinal plants belonged to three other groups. Wild/weedy forms were in the last group. The five groups could not be distinguished by seed diameter. Though the plants cultivated for oil tended to have larger seeds than the medicinal and wild/weedy plants, there was no boundary either between the two crops, or among various phenotypes of P. frutescens.     

Effects of reaggregated granulosa cells and oocytes derived from early antral follicles on the properties of oocytes grown in vitro

In this study, we examined the effects of reconstructed oocyte–granulosa cell complexes (OGCs) on the development of porcine oocytes derived from early antral follicles (EAFs; 0.5–0.7 mm in diameter). When denuded oocytes were cocultured with granulosa cells derived from other EAFs, the oocytes and granulosa cells aggregated to form OGCs after 2 days of culture. After 14 days of culture, we compared cell number, oocyte diameter, and oocyte chromatin configuration in unmanipulated (natural) OGCs, reconstructed OGCs, and OGCs collected from antral follicles (AFs, 3.0–6.0 mm in diameter). The diameters of oocytes from reconstructed OGCs grown in vitro were not different from those of oocytes from natural OGCs, although they were significantly smaller than those of oocytes from antral follicle (AF) OGCs. Oocyte chromatin configuration did not differ among the 3 OGC groups, but the oocyte nuclear maturation rate was lower in the reconstructed OGCs and higher in the AF OGCs. However, when the in vitro culture period for the reconstructed OGCs was extended by 2 days, the nuclear maturation rate of oocytes from reconstructed OGCs was similar to that of oocytes from natural OGCs. In addition, blastocysts were successfully obtained from oocytes from reconstructed OGCs. In conclusion, we established an innovative culture method that allows oocytes and granulosa cells from EAFs to reaggregate as reconstructed OGCs, which yield oocytes with the ability to develop to the blastocyst stage.     

Construction of a linkage map and identification of DNA markers linked to Fom-1, a gene conferring resistance to Fusarium oxysporum f.sp. melonis race 2 in melon

Resistance to Fusarium oxysporum f.sp. melonis race 2 is conferred by a single dominant gene, Fom-1 in melon. Here, we identified DNA markers tightly linked to Fom-1 that could be used for marker assisted selection in breeding programs. First, we developed 125 F₂ plants derived from the cross between melon lines P11 (fom-1fom-1) and MR-1 (Fom-1Fom-1). Using the F₂ population, we constructed a linkage map including 14 SSR markers which had not been mapped previously. Fom-1 was confirmed to be allocated to linkage group 7. Then, we identified four AFLP markers using bulked segregant analysis. The AFLP marker TAG/GCC-470 was completely linked to Fom-1 and other three markers were mapped near Fom-1. TAG/GCC-470 and TCG/GGT-400 were respectively converted to STS and CAPS markers. Usefulness of DNA markers was confirmed in the analysis with several melon cultivars and lines.     

The Chromosomal Control of Leaf Characteristics of Early-Stage Plants in Wheat （Triticum aestivum L.）

Rapid expansion of leaves of early-stage plants in wheat produced by chromosomal control of characteristics related to rapid expansion of the first six leaves of wheat were investigated using a set of single chromosome substitution lines under two different temperature regimes(TRs).Results from this study indicated that several chromosomes could be responsible for each of the four characteristics studied(including leaf width,leaf length,leaf elongation rate and leaf appearance index)and that different chromosomes may be responsible for the same characteristics at different TRs.Only a small number of substitutions that showed the most significant effects on leaf length were found among those that showed the most significant effects on leaf width,providing further evidence that these two characteristics are likely controlled by different sets of genes.Substitutions 4B,4D,and 5A are among those that were repeatedly detected to having significant effects on different leaf characteristics at the lower-TR and genes residing on these three chromosomes could be homologous.This possibility and its implication are discussed.     

Comparison of efficiency between differential evolution and evolution strategy: application of the LST model to the Be River catchment in Vietnam

Parameter calibration is an important step in the development of rainfall–runoff models. Recently, there has been a significant focus on automatic calibration. In this paper, two evolutionary optimization algorithms were applied to calibration of the long- and short-term runoff model (LST model) to simulate the daily rainfall–runoff process in the Be River catchment located in southern Vietnam. The differential evolution (DE) and evolution strategy (ES) algorithms were employed to optimize three objective functions: the Nash–Sutcliffe efficiency coefficient, root mean square error, and mean absolute error, which are indices for evaluating the simulation accuracy of the LST model. Hydrometeorological data for the periods 1985–1989 and 1990–1991 were used for calibration and validation, respectively. The LST model was calibrated for each objective function using five different parent and offspring population conditions. The results show that both the DE and ES algorithms are efficient methods for automatic calibration of the LST model. After 1000 generations, the best values of the fitness indices found by the DE technique were slightly better and more stable than those found by the ES technique in both calibration and validation. The average computation time for each generation using the DE algorithm was approximately two-thirds as long as that using the ES algorithm.