Importance of Escherichia coli infection in ascites in broiler chickens shown by experimental production

Common commercial strain male broilers aged 14 days were intratracheally inoculated with 0.2 ml of 1.2 x 10(6) colony-forming units of Escherichia coli in nutrient broth and kept in a cool environment during the experiment. Ascites was produced in five surviving and two dead birds out of 50 but not in 50 mock-infected control birds. Among the 40 survivors that were infected, the erythrocyte packed cell volume (PCV) of the 10 birds with pericarditis was the same as in 21 grossly normal birds, although that of the four birds with enlarged right ventricle (RV) was high. The pericarditis caused by E. coli septicemia was not the primary cause of ascites. However, the PCV was high in some of the survivors with an enlarged RV without pericarditis, indicating overload due to the lung lesion. These data suggested that some of the birds with an enlarged RV, caused by supplying blood that was insufficiently oxygenated for the body size, suffered from ascites.     

Decolorization of kraft pulp bleaching effluent by a newly isolated fungus, Geotrichum candidum Dec 1

Kraft pulp bleaching effluent supplemented with glucose was decolorized by a newly isolated fungusGeotrichum candidum Dec 1 (Dec 1) that showed a wide decolorizing spectrum to synthetic dyes. When the glucose concentration in the effluent was 30 g/l, the color removal and the reduction of absorbable organic halogens were 78% and 43% after 7 days culture, respectively. The average molecular weight of colored substances measured by gel filtration chromatography was lowered to less than 3000 from 5600 after 7 days culture. As the contribution of extracellular enzymes such as peroxidase (DyP), manganese peroxidase, and laccase to the decolorization of the kraft pulp bleaching effluent was small, Dec 1 appears to have a different mechanism of decolorizing kraft pulp bleaching effluent when compared with enzymes used to decolorize synthetic dyes.     

Na?ve-like conversion enhances the difference in innate in vitro differentiation capacity between rabbit ES cells and iPS cells

Quality evaluation of pluripotent stem cells using appropriate animal models needs to be improved for human regenerative medicine. Previously, we demonstrated that although the in vitro neural differentiating capacity of rabbit induced pluripotent stem cells (iPSCs) can be mitigated by improving their baseline level of pluripotency, i.e., by converting them into the so-called “naïve-like” state, the effect after such conversion of rabbit embryonic stem cells (ESCs) remains to be elucidated. Here we found that naïve-like conversion enhanced the differences in innate in vitro differentiation capacity between ESCs and iPSCs. Naïve-like rabbit ESCs exhibited several features indicating pluripotency, including the capacity for teratoma formation. They differentiated into mature oligodendrocytes much more effectively (3.3–7.2 times) than naïve-like iPSCs. This suggests an inherent variation in differentiation potential in vitro among PSC lines. When naïve-like ESCs were injected into preimplantation rabbit embryos, although they contributed efficiently to forming the inner cell mass of blastocysts, no chimeric pups were obtained. Thus, in vitro neural differentiation following naïve-like conversion is a promising option for determining the quality of PSCs without the need to demonstrate chimeric contribution. These results provide an opportunity to evaluate which pluripotent stem cells or treatments are best suited for therapeutic use.     

Specific detection of potentially allergenic kiwifruit in foods using polymerase chain reaction

Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods.     

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.     

The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants

We report the draft genome sequence of the model moss Physcomitrella patens and compare its features with those of flowering plants, from which it is separated by more than 400 million years, and unicellular aquatic algae. This comparison reveals genomic changes concomitant with the evolutionary movement to land, including a general increase in gene family complexity; loss of genes associated with aquatic environments (e.g., flagellar arms); acquisition of genes for tolerating terrestrial stresses (e.g., variation in temperature and water availability); and the development of the auxin and abscisic acid signaling pathways for coordinating multicellular growth and dehydration response. The Physcomitrella genome provides a resource for phylogenetic inferences about gene function and for experimental analysis of plant processes through this plant's unique facility for reverse genetics.     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

Bioengineering of Leuconostoc mesenteroides glucansucrases that gives selected bond formation for glucan synthesis and/or acceptor-product synthesis

The variations in glucosidic linkage specificity observed in products of different glucansucrases appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. Various amino acid mutations near active sites of DSRBCB4 dextransucrase from Leuconostoc mesenteroides B-1299CB4 were constructed. A triple amino acid mutation (S642N/E643N/V644S) immediately next to the catalytic D641 (putative transition state stabilizing residue) converted DSRBCB4 enzyme from the synthesis of mainly α-(1→6) dextran to the synthesis of α-(1→6) glucan containing branches of α-(1→3) and α-(1→4) glucosidic linkages. The subsequent introduction of mutation V532P/V535I, located next to the catalytic D530 (nucleophile), resulted in the synthesis of an α-glucan containing increased branched α-(1→4) glucosidic linkages (approximately 11%). The results indicate that mutagenesis can guide glucansucrase toward the synthesis of various oligosaccharides or novel polysaccharides with completely altered linkages without compromising high transglycosylation activity and efficiency.