Inhibition of lysophospholipase D activity by unsaturated lysophosphatidic acids or seed extracts containing 1-linoleoyl and 1-oleoyl lysophosphatidic acid

Lysophospholipase D (lysoPLD), generating lipid mediator lysophosphatidic acid (LPA) from lysophosphatidyclcholine (LPC), is known to be inhibited by lysophosphatidic acids. Meanwhile, some plant lipids are known to contain lysophospholipids as minor components. Therefore, it is interesting to test whether edible seed samples, rich in phospholipids, may contain lysophospholipids, which express a strong inhibition of lysoPLD activity. First, the structural importance of fatty acyl group in LPAs was examined by determining the inhibitory effect of various LPAs on bovine lysoPLD activity. The most potent in the inhibition of lysoPLD activity was linoleoyl-LPA ( K i, 0.21 microM), followed by arachidonoyl-LPA ( K i, 0.55 microM), oleoyl-LPA ( K i, 1.2 microM), and palmitoyl-LPA ( K i, 1.4 microM), based on the fluoresecent assay. The same order of inhibitory potency among LPA analogs with different acyl chains was also found in the spectrophotometric assay. Subsequently, the extracts of 12 edible seeds were screened for the inhibition of lysoPLD activity using both spectrophotometric and fluorescent assays. Among seed extracts tested, the extract from soybean seed, sesame seed, or sunflower seed (30 mg seed weight/mL) was found to exhibit a potent inhibition (>80%) of lysoPLD activity. In further study employing ESI-MS/MS analysis, major LPA components in seed extracts were identified to be 1-linoleoyl LPA, 1-oleoyl LPA, and 1-palmitoyl LPA with 1-linoleoyl LPA being more predominant. Thus, the potent inhibition of lysoPLD activity by seed extracts might be ascribed to the presence of LPA with linoleoyl group rather than other acyl chains.     

Development and characterization of monoclonal antibodies to chicken interleukin-15

The chicken IL-15 gene was recently cloned and shown to encode a polypeptide with T cell growth factor activity similar to IL-2. To further characterize the chemical and biological properties of chicken IL-15, we generated a panel of monoclonal antibodies against bacterially expressed protein and characterized their binding specificities. All antibodies were reactive by ELISA with recombinant IL-15, but not IL-2, and identified a 15kDa recombinant chicken IL-15 by Western blot analysis. Two antibodies inhibited IL-15-induced proliferation of splenic lymphoblast cells. These monoclonal antibodies will be useful for further structural and immunological studies of chicken IL-15.     

Comparison and analysis of main effects,epistatic effects,and QTL × environment interactions of QTLs for agronomic traits using DH and RILs populations in rice

Two genetic linkage maps based on doubled haploid (DH) and recombinant inbred lines (RILs) populations, derived from the same indica-japonica cross ‘Samgang × Nagdong’, were constructed to analyze the quantitative trait loci (QTLs) affecting agronomic traits in rice. The segregations of agronomic traits in RILs population showed larger variations than those in DH population. A total of 10 and 12 QTLs were identified on six chromosomes using DH population and seven chromosomes using RILs population, respectively. Three stable QTLs including pl9.1, ph1.1, and gwp11.1 were detected through different years. The percentages of phenotypic variation explained by individual QTLs ranged from 8 to 18% in the DH population and 9 to 33% in the RILs population. Twenty-three epistatic QTLs were identified in the DH population, while 21 epistatic QTLs were detected in the RILs population. Epistatic interactions played an important role in controlling the agronomic traits genetically. Four significant main-effect QTLs were involved in the digenic interactions. Significant interactions between QTLs and environments (QE) were identified in two populations. The QTLs affecting grain weight per panicle (GWP) were more sensitive to the environmental changes. The comparison and QTLs analysis between two populations across different years should help rice breeders to comprehend the genetic mechanisms of quantitative traits and improve breeding programs in marker-assisted selection (MAS).     

Isolation of a soil bacterium capable of biodegradation and detoxification of endosulfan and endosulfan sulfate

Endosulfan, an endocrine disrupting chemical, is a widely used cyclodiene organochlorine pesticide worldwide, and it blocks neuronal GABA(A)-gated chloride channels in mammals and aquatic organisms. Endosulfan and its metabolites, such as endosulfan sulfate, are persistent in environments and are considered as toxic chemicals. For bioremediation of endosulfan, in this study, an attempt was made to isolate an endosulfan and endosulfan sulfate degrading bacterium from endosulfan-polluted agricultural soil. Through repetitive enrichment and successive subculture using endosulfan or endosulfan sulfate as the sole carbon source, a bacterium KS-2P was isolated. The KS-2P was identified as Pseudomonas sp. on the basis of the results of a 16S rDNA sequencing analysis and MIDI test. The degradation ratios for endosulfan or endosulfan sulfate in minimal medium containing endosulfan (23.5 microg mL(-1)) or endosulfan sulfate (21 microg mL(-1)) were 52% and 71%, respectively. Our results suggest that Pseudomonas sp. KS-2P has potential as a biocatalyst for endosulfan bioremediation.     

Quantitative investigation of Enteromyxum leei (Myxozoa: Myxosporea) infection and relative condition factor in cultured olive flounder Paralichthys olivaceus (Temminck and Schlegel)

Enteromyxum leei has been reported to cause emaciation disease in various fish species. To determine the effect of parasite intensity on cultured olive flounder Paralichthys olivaceus, we investigated the relationship between the relative condition factor (rCF = CF/standard CF × 100) and parasite load with quantitative polymerase chain reaction (qPCR) and the challenge test. A total of 57 cultured olive flounders were obtained from 11 fish farms and divided into five groups based on their rCF. We investigated the parasite intensity in the posterior intestine of the fish. The parasite load was closely matched to severe loss of body weight. In addition, olive flounders were inoculated either orally or anally with intestinal scrapings of infected fish or phosphate‐buffered saline. The fish were reared at natural water temperature and transferred to different tanks, and the water temperature was adjusted to 20°C after 6 weeks of inoculation. When the water temperature was increased to 20°C, the rCF decreased in the experimentally infected group. The results demonstrated that qPCR can be utilized to determine the relative abundance of E. leei in olive flounders and water temperature is an important factor to track the progress of the emaciation disease.     

The production and characterization of anti-bovine CD14 monoclonal antibodies

To characterize further the chemical and biological properties of bovine soluble (bos) CD14, a panel of ten murine monoclonal antibodies (mAb) reactive with recombinant (r) bosCD14 were produced. A sandwich ELISA, using murine mAb and rabbit polyclonal antibodies reactive with rbosCD14 was developed. All the mAb were reactive by ELISA with baculovirus-derived rbosCD14 and they recognized rbosCD14 (40 kDa) by western blot analysis. The mAb also identified by western blot sCD14 (53 and 58 kDa) in milk and blood and sCD14 (47 kDa) in a lysate of macrophages obtained from involuted bovine mammary gland secretions. Analysis by ELISA of whey samples after intramammary injection of lipopolysaccharide (LPS) (10 micro g) revealed increased sCD14 levels between 8 to 48 h after injection. Flow cytometric analysis showed that the mAb bound to macrophages isolated from involuted mammary gland secretions and mouse macrophages but not to swine or horse monocytes. Addition of anti-rbosCD14 mAb to monocytes stimulated with LPS reduced in vitro production of TNF-alpha. The anti-rbosCD14 antibodies generated in this study will be useful in studying CD14, an accessory molecule that contributes to host innate recognition of bacterial cell wall components in mammary secretions produced during mastitis.     

Ectopic expression of serotonin N-hydroxycinnamoyltransferase and differential production of phenylpropanoid amides in transgenic tomato tissues

Tomato contains high levels of amines such as serotonin and tyramine and is a suitable host to enhance phenylpropanoid amides (PAs), an important class of nutraceuticals with strong antioxidant activity and chemotherapeutic effects, by ectopic expression of the corresponding gene, serotonin N-hydroxycinnamoyltransferase (SHT). To assess whether ectopic overexpression of SHT cDNA under the control of the CaMV 35S promoter would enhance levels of PAs, we generated transgenic tomato plants and analyzed the levels of PAs. Transgenic tomato plants exhibited increased synthesis of PAs such as feruloylserotonin (FS), 4-coumaroylserotonin (CS), feruloyltyramine (FT), 4-coumaroyltyramine (CT), and feruloyloctopamine (FO) in 1-month-old leaves compared to the wild type. The increase and relative levels of PAs were even more apparent in 3-month-old leaves of transgenic tomato. When tomato leaves were challenged by wounding, levels of PAs in the best transgenic line increased by 3- and 10-fold for CS + FS and CT + FT, respectively. In contrast to leaves, tomato fruit only showed enhanced synthesis of CT + FT, whereas CS + FS levels were not enhanced. Regarding amine content, the levels of tyramine were much higher than those of serotonin in tomato leaves and fruits. The high levels of tyramine may contribute to the preferential production of CT + FT rather than CS + FS, although SHT enzyme shows the highest substrate affinity toward serotonin rather than tyramine.     

A Comparative Study of Fourier Transform Raman and NIR Spectroscopic Methods for Assessment of Protein and Apparent Amylose in Rice

Fourier‐transform Raman (FT‐Raman) spectroscopy and near‐infrared (NIR) reflectance spectroscopy were used to compare calibration models for determining rice cooking quality parameters such as apparent amylose and protein. Samples from two seasons were used in each calibration set. The laboratory values ranged from 4.89 to 12.48% for protein and from 0.2 to 25.7% for amylose. The data for both FT‐Raman and NIR were preprocessed with orthogonal signal correction (OSC) for standardization. For both spectroscopic methods, five models were optimized by partial least squares regression (PLSR) and by Martens' uncertainty regression (MUR), including no processing, smoothing, normalization, first derivative (D1), and second derivative (D2). Based solely on standard error of cross‐validation (SECV), the FT‐Raman method was superior to the NIR method for protein. For amylose, the FT‐Raman and NIR methods resulted in similar calibration statistics with a high precision, with the FT‐Raman requiring fewer factors. The best FT‐Raman models were generated from OSC preprocessing with MUR for protein (SECV 0.15%, five factors) and from OSC without MUR for amylose (SECV 0.70%, seven factors). The best NIR models were obtained with D2 transform of OSC spectra for protein (SECV 0.22%, four factors) and with OSC spectra for amylose (SECV 0.57%, 11 factors).