Improved porcine model for Shiga toxin‐producing Escherichia coli infection by deprivation of colostrum feeding in newborn piglets

Porcine edema disease (ED) is a toxemia caused by enteric infection with Shiga toxin 2e (Stx2e)‐producing Escherichia coli (STEC). ED occurs most frequently during the weaning period and is manifested as emaciation associated with high mortality. In our experimental infection with a specific STEC strain, we failed to cause the suppression of weight gain in piglets, which is a typical symptom of ED, in two consecutive experiments. Therefore, we examined the effects of deprivation of colostrum on the sensitivity of newborn piglets to STEC infection. Neonatal pigs were categorized into two groups: one fed artificial milk instead of colostrum in the first 24 h after birth and then returned to the care of their mother, the other breastfed by a surrogate mother until weaning. The oral challenge with 10¹¹ colony‐forming units of virulent STEC strain on days 25, 26 and 27 caused suppression of weight gain and other ED symptoms in both groups, suggesting that colostrum deprivation from piglets was effective in enhancing susceptibility to STEC. Two successive STEC infection experiments using colostrum‐deprived piglets reproduced this result, leading us to conclude that this improved ED piglet model is more sensitive to STEC infection than the previously established models.     

Diversity and differentiation of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> in Indonesia

Analysis of the genetic structure of Indonesian Oryza sativa and O. rufipogon using neighbour-joining trees based on single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers revealed that O. sativa in Indonesia is separated from O. rufipogon. Accessions of O. sativa in this study were differentiated into two major groups, indica and tropical japonica, excluding some varieties. SSR and SNP markers revealed the high value of differentiation (F _ST) and genetic distance (D) between indica and tropical japonica and we discovered four loci by SNP markers and one locus by SSR markers that play a role in differentiation between indica and tropical japonica. Interestingly, genetic diversity (H) in O. rufipogon was lower than that in O. sativa, however H in O. rufipogon was the highest and H in tropical japonica was the lowest when O. sativa was divided into two groups. Inbreeding coefficient (Fst) showed evidences that gene flow (Nm) between species and within species might be one of the mechanisms related to the diversification and differentiation of Indonesian rice germplasm by asymmetric pattern between species and within O. sativa as revealed by SSR and SNP markers. In addition, we found evidences on stabilizing selection in Indonesian rice germplasm and they might be the reasons why Indonesian rice germplasm did not differentiate due to source location of landrace. However, we found a weak relation between SSR and SNP markers probably due to highly polymorphic in SSR and the different properties of both markers.     

Genetic organization of aromatic rice as revealed by RAPD markers: A case study in conserving crop genetic resources on farm

Summary On-farm conservation of landraces is one strategy to maintain the diversity of crop germplasm in local agro-ecosystems. The genetic structures of landraces are a key biological factor in on-farm conservation strategies. To accumulate a genetic understanding that will help establish a methodology for on-farm conservation, the genetic organization of landraces of aromatic rice in Namdinh province, Vietnam was analyzed using RAPD markers. Eighteen RAPD markers detected 38 genotypes among 320 aromatic rice samples growing at 23 sites of farmers' fields and in the experimental field that derived from 13 sites. Geographical variation was observed in the frequency of genotypes, whereas individual landraces could not be distinguished by RAPD markers. Genetic variation within a site was generally smaller than that among sites. The degree of genetic similarity of the plants in a site varied among sites, as did the number of genotypes. Changes in genetic structure over time were investigated using experimental populations each derived from approximately 30 plants from 13 farmers' fields. The differences detected by DNA markers between the genetic structural in the farmers' fields and those in experimental fields suggested that genetic drift is a major cause of these differences. The present study suggests that DNA markers are an essential means to monitor the genetic structures of heterogeneous landraces of rice, and are useful for selecting study sites for the on-farm conservation of genetic diversity as well as for successive monitoring.     

Improved malonaldehyde assay using headspace solid-phase microextraction and its application to the measurement of the antioxidant activity of phytochemicals

A modified malonaldehyde (MA) assay for antioxidant activity, which involves derivatization and headspace solid-phase microextraction (HS-SPME) was developed and validated. The recovery of MA as 1-methylpyrazole (product of MA and N-methylhydrazine) from a headspace of an aqueous solution containing MA, buffer, surfactant, and cod liver oil using HS-SPME with a PDMS/DVB fiber was 91.3 +/- 3.38%. MA was analyzed by a gas chromatograph with a nitrogen-phosphorus detector, and its detection limit was 0.0103 nmol/mL. The antioxidant activities of natural compounds were determined as the percentage inhibition of MA formed from cod liver oil oxidized by Fenton's reagents in the above aqueous solution. Sesamol inhibited MA formation most (86.1%), followed by eugenol (84.4%), capsaicin (80.7%), ethylvanillin (45.3%), and vanillin (31.6%) at a level of 50 microg/mL. This method did not require any organic solvents and is a simple, fast, and a highly sensitive method for MA determination.     

Novel prediction method of beer foam stability using protein Z, barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and yeast thioredoxin

Foam stability is an important quality trait of beer. Our previous results of two-dimensional gel electrophoresis (2DE) analyses of beer proteins implied a relationship between barley dimeric alpha-amylase inhibitor-1 (BDAI-1) and beer foam stability as judged by the NIBEM-T analyzer. To develop a novel prediction method of beer foam stability under different conditions of barley cultivar and malt modification, multiple linear regression analysis was applied. The spot intensities of major beer proteins on 2DE gel were quantified and used as explanatory variables. The foam stabilities of 25 beer samples each brewed from malt with different malt modification in one of the three cultivars (cultivars A, B, and C) were explained by the spot intensities of BDAI-1 at the 5% significance level ( r = 0.421). Furthermore, two other major protein spots (b0 and b5) were observed on the 2DE gels of Japanese commercial beer samples with different foam stability. Then, multiple regression for foam stability was calculated using these three spot intensities as explanatory variables. As a result, 72.1% of the beer foam stability in 25 beer samples was explained by a novel multiple regression equation calculated using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. To verify the validity of the multiple regression equation and the explanatory variables, the beer foam stability in practical beer samples was analyzed. As a result, 81.5% of the beer foam stability in 10 Japanese commercial beer samples was also explained by using spot b0 and BDAI-1 as positive explanatory variables and spot b5 as a negative variable. Mass spectrometry analyses followed by database searches revealed that protein spots b0 and b5 were identified as protein Z originated from barley and thioredoxin originated from yeast, respectively. These results confirm that BDAI-1 and protein Z are foam-positive factors and identify yeast thioredoxin as a possible novel foam-negative factor.     

The influence of barley malt protein modification on beer foam stability and their relationship to the barley dimeric alpha-amylase inhibitor-I (BDAI-I) as a possible foam-promoting protein

The foam stability of beer is one of the important key factors in evaluating the quality of beer. The purpose of this study was to investigate the relationship between the level of malt modification (degradation of protein, starch, and so on) and the beer foam stability. This was achieved by examining foam-promoting proteins using two-dimensional gel electrophoresis (2DE). We found that the foam stability of beer samples brewed from the barley malts of cultivars B and C decreased as the level of malt modification increased; however, the foam stability of cultivar A did not change. To identify the property providing the increased foam stability of cultivar A, we analyzed beer proteins using 2DE. We analyzed three fractions that could contain beer foam-promoting proteins, namely, beer whole proteins, salt-precipitated proteins, and the proteins concentrated from beer foam. As a result, we found that in cultivar A, some protein spots did not change in any of these three protein fractions even when the level of malt modification increased, although the corresponding protein spots in cultivars B and C decreased. We analyzed these protein spots by peptide mass finger printing using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. As a result, all of these spots were identified as barley dimeric alpha-amylase inhibitor-I (BDAI-I). These results suggest that BDAI-I is an important contributor to beer foam stability.     

Spontaneous well-differentiated pancreatic islet cell carcinoma with vascular invasion in a male F344 rat

This case report describes a case of spontaneous pancreatic islet cell carcinoma with vascular invasion in a 110-week-old male F344 rat. Histologically, a pancreatic nodule consisting of tumor cells and many blood-rich vessels, and covered with a fibrous capsule showed local invasion in the capsule and adjacent acinar tissues, encircling a large duct-like structure (DS). The tumor was composed of well-differentiated tumor cells resembling normal pancreatic islet cells, which had small round nuclei and eosinophilic cytoplasm. Mitotic figures were rare. Immunohistochemistry revealed that the tumor cells were positive for insulin. Although endothelial cells were not detected, the DS wall showed cells positive for α-smooth muscle actin and elastic fibers, suggesting that the DS is the pancreatic artery. This is a rare case of islet cell carcinoma consisting of well-differentiated tumor cells with invasion of the pancreatic artery in a rat.     

Highly metastatic ovarian yolk sac carcinoma in a rat

We investigated a highly metastatic ovarian yolk sac carcinoma in a 52-week-old female Crl:CD(SD) rat. Macroscopically, the present case had severe ascites, bilateral ovarian masses and numerous nodules in the abdominal and thoracic cavities. Histopathologically, these masses and nodules were generally composed of two types of cells mimicking a parietal and visceral yolk sac. The parietal cells were round to polygonal, contained eosinophilic droplets and were arranged in nests and cords in the eosinophilic matrix. Both the intracytoplasmic droplets and the matrix were stained positively with PAS. The visceral cells were cylindriform, and proliferated in papillary and tubular patterns and occasionally formed Shiller-Duval body-like structures. In the dissemination sites, the neoplastic cells proliferated on the surface of the various tissues and often infiltrated into deeper parts of the tissues. Immunohistochemically, both neoplastic cells were positive for α-fetoprotein and keratin, and the eosinophilic matrix was positive for laminin. Ultrastructurally, the parietal cells had dilated rough endoplasmic reticulums, which were filled with electron-lucent laminated structures. The visceral cells had poorly to moderately developed intracytoplasmic organelles and were interconnected with desmosomes. Taken together, the present tumor was diagnosed as yolk sac carcinoma arising from the ovary and was characterized by not only high metastasis but also invasive infiltration with biphasic proliferation of the parietal and visceral cells.