Correlation between adherence of Erysipelothrix rhusiopathiae strains of serovar 1a to tissue culture cells originated from porcine kidney and their pathogenicity in mice and swine

Adherence of four virulent and four avirulent strains of Erysipelothrix rhusiopathiae, serovar 1a, to porcine kidney cell lines, PK-15 and ESK cells, was examined in an in vitro system. The virulent strains adhered well to the cells (range of means, 9.95 +/- 0.87-36.01 +/- 1.10 per cell). In contrast, the avirulent strains showed negligible adherence to the cells (range of means, 0.11 +/- 0.04-1.41 +/- 0.13 per cell). Pretreatment of bacteria with heat, trypsin, or antiserum resulted in a marked decrease in adherence. Scanning electron microscopic examination revealed that the bacteria attached directly to the microvilli of cells.     

Potassium behavior and clay mineral composition in the soil with low effectiveness of potassium application

ABSTRACT

Increasing exchangeable potassium (ExK) content in soil to an appropriate level is important to mitigate the transfer of radioactive cesium to crops. We focused on a buckwheat (Fagopyrum esculentum Moench) field with a low ExK content, despite the application of K, in Fukushima Prefecture, Japan (Field A), following the Tokyo Electric Power Company Fukushima Dai-ichi (No. 1) Nuclear Power Plant accident in March 2011. We examined the relationship between K concentration and clay mineral composition in the soil of Field A and compared the findings with another field in Fukushima Prefecture (Field B) to clarify whether K applied to the soil was leached or remaining fixed. Pot experiments showed that K concentration in water seepage from pots following irrigation was significantly lower in pots from Field A than in those from Field B. Soil ExK content after soybean cultivation was lower in soils of Field A than those of Field B. These results indicate that K applied to Field A was fixed in the soil. Analysis of clay mineral composition confirmed the distinctive vermiculitic nature of Field A soils. This clay mineralogy would be associated with the higher K fixation ability of Field A than Field B soils. This study demonstrated that K fixation in vermiculite was a factor preventing the increase in ExK content from K application to Field A.     

Embryo collection from pigs post‐pseudopregnancy induced by estradiol dipropionate

We aimed to define whether embryo collection carried out after pseudopregnancy was of similar outcome and quality as after artificial abortion. To induce pseudopregnancy, 30 gilts or sows were given 20 mg intramuscular estradiol dipropionate (EDP) 10–11 days after the onset of estrus. Ten additional pigs were inseminated artificially at natural estrus as a control group. Prostaglandin F_2α (PGF_2α) was administered twice with a 24 hr interval beginning 15, 20, or 25 days after EDP‐treatment (n = 10 per group) or between 23 and 39 days after artificial insemination in control pigs. Following this, all pigs were given 1,000 IU equine chorionic gonadotropin and 500 IU human chorionic gonadotropin (hCG) and then inseminated. Embryos were recovered 6 or 7 days after hCG treatment and outcome was recorded. There was no significant difference in the number of normal embryos collected from the pigs with PGF_2α initiated at different time points or from the control group. Embryonic developmental stages 7 days after hCG treatment also did not differ among groups. These results indicate that the use of EDP to induce pseudopregnancy, followed by PGF_2α administration to synchronize estrus for subsequent embryo harvest, is a suitable alternative to the artificial abortion method.     

Microdroplet In Vitro Fertilization Can Reduce the Number of Spermatozoa Necessary for Fertilizing Oocytes

Successful in vitro fertilization (IVF) in mice has been achieved using spermatozoa at concentrations specifically optimized for the experimental conditions, such as species and source of spermatozoa. Although IVF in mice is mostly performed using about 80–500 µl drops, it is expected that the number of spermatozoa used for insemination can be reduced by decreasing the size of the IVF drops. The present study was undertaken to examine the extent to which the number of spermatozoa used for IVF could be reduced by using small droplets (1 µl). We devised the experimental parameters using frozen–thawed spermatozoa from C57BL/6 mice in anticipation of broader applications to other mouse facilities. We found that as few as 5 spermatozoa per droplet could fertilize oocytes (1 or 3 oocytes per droplet), although the fertilization rates were low (13–15%). Practical fertilization rates (> 40%) could be achieved with frozen-thawed C57BL/6J spermatozoa, which are sensitive to cryopreservation, when 20 sperm per droplet were used to inseminate 3 oocytes. Even with spermatozoa from a very poor quality suspension (10% motility), about 25% of oocytes were fertilized. Our calculations indicate that the number of inseminated spermatozoa per oocyte can be reduced to 1/96–1/240 by this method. In two separate embryo transfer experiments, 60% and 47%, respectively, of embryos developed to term. Our microdroplet IVF method may be particularly advantageous when only a limited number of motile spermatozoa are available because of inadequate freezing-thawing or genetic reasons.     

A comparison of changes in stable isotope ratios in the epidermal mucus and muscle tissue of slow‐growing adult catfish

Although stable isotope analysis is a powerful tool for determining diet, migration patterns and the structure of food webs in aquatic systems, the slow response of isotopic ratios in the widely used muscle tissue often hampers this approach, particularly in slow‐growing or adult fishes. We conducted a diet‐switch experiment to compare the changes in the stable carbon isotope ratio (δ¹³C values) in the epidermal mucus and muscle tissue of five‐year‐old catfish (Silurus asotus). The isotope ratios in noninvasively sampled mucus changed more rapidly than those in the muscle tissue. As isotopic change in mucus was relatively rapid, this technique can be used over a finer timescale than traditional isotopic analyses using the muscle tissue. The isotopic change half‐life in our experimental condition was 200 days, which would not be short enough for some research purposes. Examining mucus along with the muscle would enable food habits of slow‐growing fishes to be determined over different timescales. However, the rate of isotopic change in the mucus was negatively affected by the size of fish and was slower than the previously reported rates in juvenile steelhead. These findings suggest that mucus turnover rates need to be determined prior to the field data interpretation.     

Immunohistochemical detection of apolipoprotein B-100 and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs

Accumulation of lipids and hyalinosis in the splenic arteries of aged dogs are frequently detected by routine histopathologic examinations. The purpose of this study was to pinpoint the localization of canine apolipoprotein B-100 (CApoB-100) and immunoglobulins (IgA, IgM, IgG) in the splenic arteries of aging dogs (n = 80) through the use of immunohistochemical techniques. CApoB-100 deposits were found in the subendothelial space, extracellular matrix, and atheromatous lesions in the tunica media of the arteries in dogs > or = 6 years of age. Foamy cytoplasm of the infiltrated macrophages was also CApoB-100 immunopositive. In dogs > or = 10 years of age, almost all central arteries were CApoB-100 immunopositive. Hyaline deposits within the wall were characterized by immunopositivity against canine IgA, IgM, IgG, and albumin. Lipid accumulation in splenic arteries may be an age-related lesion and a precursor of the atheromatous plaques associated with splenic hemorrhage and infarcts later in life. In addition, deposition of immunoglobulins, probably mediated by immune complexes, may play an important role in the development of canine vascular diseases similar to human disease.     

DNA methylation status of bovine blastocyst embryos obtained from various procedures

DNA methylation is an important factor for the regulation of gene expression in early embryos. It is well known that the satellite I sequence is more heavily methylated in bovine somatic cell nuclear transfer (NT-SC) embryos than in embryos derived from in vitro fertilization (IVF). However, the methylation status of bovine embryos obtained by other procedures is not well known. To clarify DNA methylation levels of bovine embryos obtained from various procedures, we examined satellite I sequences in bovine blastocyst (BC) embryos derived from NT-SC, NT using embryonic blastomeres (NT-EM), in vivo (Vivo), IVF and parthenogenetic treatment (PA). Furthermore, in order to evaluate the efficacy of DNA demethylation by the NT procedure, we determined the DNA methylation levels in bovine embryos in which NT was recapitulated (Re-NT). Although the DNA methylation levels in the NT-SC embryos were higher than those in the other embryos, the NT-EM embryos exhibited lower DNA methylation levels. The satellite I sequence in the NT-SC embryos was more demethylated than that in the donor cells. Although the DNA methylation level in the individual NT-SC embryos showed variation, the full-term developmental efficacy of these embryos were not different. These findings suggest that the methylation level of the satellite I sequence at the BC stage is not related to the abnormalities of bovine embryos produced by NT-SC. There was no difference in methylation levels between Re-NT and NT-SC embryos. Our results indicated that the DNA methylation status differed among embryos produced by various methods and that at least some of the demethylation of the donor cell genome occurred in the recipient cytoplast after NT-SC, but the demethylation ability of the NT procedure was noted in the first NT but not in the second NT.     

Biosynthesis of a syringyl 8-O-4′ neolignan in Eucommia ulmoides: formation of syringylglycerol-8-O-4′-(sinapyl alcohol) ether from sinapyl alcohol

To investigate the biosynthesis and stereochemistry of syringylglycerol-8-O-4′-(sinapyl alcohol) ether (SGSE), a syringyl 8-O-4′ neolignan, feeding experiments and enzyme assays using Eucommia ulmoides were carried out. Diastereoselective formation of erythro-SGSE was found. When [8-¹⁴C]sinapyl alcohol was administered to excised shoots of E. ulmoides, ¹⁴C was incorporated into free SGSE and SGSE glucosides. In stems, incorporation into (+)-erythro-[¹⁴C]SGSE (0.037%) with 9.1% enantiomeric excess (% e.e.) was found; incorporation into the threo isomer was not detectable. Erythro-[¹⁴C]SGSE glucosides (0.047%) dominated over threo forms (0.007%) with 74.0% diastereomeric excess (% d.e.); both diastereomers were levorotatory with 32.0% e.e. and 18.3% e.e., respectively. In leaves, higher incorporation into (−)-erythro-[¹⁴C]SGSE (0.500%, 15.9% e.e.) than into the threo isomer (0.206%, 7.4% e.e.) was observed (41.6% d.e.). (−)-Erythro-[¹⁴C]SGSE glucosides (1.692%, 25.0% e.e.) were produced at higher rates than threo isomers (0.177%, 16.4% e.e.) with 81.0% d.e. In incubations of a mixture of [8-¹⁴C]sinapyl and [8-¹⁴C]coniferyl alcohols with an insoluble enzyme preparation from stems of E. ulmoides, erythro-SGSE was preferentially produced. The highest % d.e. (82.8) was observed at 60 min with the (+)-erythro isomer (21.4% e.e.) and the (−)-threo form (4.3% e.e.).Part of this report was presented at the 52nd Annual Meeting of the Japan Wood Research Society, Gifu, April 2002, and the 47th Lignin Symposium, Fukuoka, October 2002