Analysis of the function of activin betaC subunit using recombinant protein

Activins, TGF-beta superfamily members, have multiple functions in a variety of cells and tissues. Additional activin beta subunit genes, betaC and betaE, have been identified in humans and rodents. To explore the role of activin betaC subunit, we generated recombinant human activin C using Chinese hamster ovary cells. Recombinant activin C from the conditioned medium was purified by consecutive hydrophobic, size-exclusion, and high performance liquid chromatography. SDS-PAGE and Western blot analysis of the purified protein revealed that activin C formed disulfide bridges. However, activin C had no effect on the proliferation of cultured liver cells. Furthermore, there were no significant differences in erythroid differentiation and follicle stimulating hormone secretion in vitro. It was also shown that immunoreactive bands indicated the hetrodimer of activin betaC, and inhibin alpha subunits were detected in the conditioned medium from the activin C-producing cells, which were stably transfected with inhibin alpha subunit cDNA. This suggests that activin betaC subunit may have been present and that it may exert its effect as inhibin C.     

Identification and characterization of foliar polyphenolic composition in sweetpotato (Ipomoea batatas L.) genotypes

Trials over two years were conducted using 1389 sweetpotato (Ipomoea batatas L.) genotypes collected from all over the world to characterize the polyphenolic composition in sweetpotato leaves. Wide variation was observed in relation to their total and individual leaf polyphenolic constituents. In all genotypes studied, the total polyphenol contents of sweetpotato leaf ranged from 1.42 to 17.1 g/100 g dry weight. The six different polyphenolic compounds were identified and quantified by NMR, FABMS, and RPHPLC analysis procedures. This is the first report of polyphenolic compositions in sweetpotato leaves. The relative levels of polyphenolic acids in sweetpotato leaves were as follows: 3,5-di-O-caffeoylquinic acid > 4,5-di-O-caffeoylquinic acid > chlorogenic acid (3-O-caffeoylquinic acid) > 3,4-di-O-caffeoylquinic acid > 3,4,5-tri-O-caffeoylquinic acid > caffeic acid. The highest 3,4,5-tri-O-caffeoylquinic acid and 4,5-di-O-caffeoylquinic acid occurred at 221 and 1183.30 mg/100 g dry weight, respectively.     

Normal calves produced after transfer of embryos cultured in a chemically defined medium supplemented with epidermal growth factor and insulin-like growth factor I following ovum pick up and in vitro fertilization in Japanese black cows

The objective of this study was to examine whether high concentrations of epidermal growth factor (EGF) and/or insulin-like growth factor I (IGF-I) would have a beneficial effect on bovine embryo development in vitro and to obtain normal calves by using an ovum pick up method and embryo culture in a chemically defined medium. When compared with controls, EGF (100 or 200 ng/ml) or IGF-I (50 or 100 ng/ml) significantly increased the rate of embryos that developed into blastocysts during an 8-day culture after the in vitro fertilization of oocytes obtained from ovaries from a slaughterhouse. IGF-I induced a dose-dependent increase in cell number in both the inner cell mass and the trophectoderm, whereas EGF stimulated proliferation only in the inner cell mass. A combination of EGF (100 ng/ml) and IGF-I (50 ng/ml) produced an additive effect, and embryos developed into blastocysts at a comparatively high rate (27.9%) compared with controls (12.0%). A similar rate of development was achieved using a combination of EGF and IGF-I in the culture of embryos following ovum pick up by ultrasound-guided transvaginal follicular aspiration and in vitro fertilization, and 5 blastocysts that developed after the culture were transferred into uteri; two embryos implanted, and normal calves were born. These results suggest that the combined use of EGF and IGF-I makes bovine embryo culture in a chemically defined medium a practical and useful procedure for producing blastocysts, and its application to embryo culture following ovum pick up and in vitro fertilization could be useful for producing normal calves.     

Carrier rate of the c.235G>C mutation in the bovine isoleucyl-tRNA synthetase (IARS) gene of Japanese Black cows at Kagoshima prefecture, Japan,and analysis of the metabolic profiling and reproductive performance of heterozygous cows

Bovine isoleucyl-tRNA synthetase (IARS) disorder, a major cause of weak calf syndrome, is caused by a homozygous missense (c.235G>C) mutation in the bovine IARS gene of Japanese Black (JB) cattle, which was identified in 2013. However, the extent to which the carrier rate has changed at Kagoshima prefecture, Japan, and whether the carrier status is associated with any clinical or reproductive problems, have yet to be ascertained. In this study, using a real-time polymerase chain reaction-based genotyping assay, we determined the carrier rate in a regional JB cow population at Kagoshima prefecture. Comparative analyses were performed on the metabolic profile test (MPT) results and reproductive performance data obtained for heterozygous carrier and homozygous wild-type cows. In 2009 and 2018, DNA samples were collected from 130 and 462 clinically healthy JB cows, respectively, in Kagoshima prefecture. MPT results and reproductive performance data were evaluated for 62 cows, comprising four heterozygous carriers and 58 wild-type cows. Genotyping revealed that the carrier rate was 6.9% in 2009 and 1.5% in 2018, the difference of which was statistically significant (P<0.005). There were no statistically significant differences between the carrier and wild-type cows with respect to either MPT results or reproductive performance, indicating that the carrier cows have necessary IARS activity to maintain minimal health and reproductive potential.     

Effect of CD4+CD25+ T cell-depletion on acute lethal infection of mice with Trypanosoma congolense

Despite the immense socio-economic repercussions of African trypanosomosis (AT), there is currently no effective control measure against the disease. Characterization of mechanisms governing resistance and/or susceptibility to AT could suggest interventions that might lead to more effective disease control. The present study was designed in an attempt to address the possible role of CD4+CD25+ T cells during an acute lethal infection of mice with Trypanosoma congolense, the causative agent of AT in domestic animals, through selective depletion using anti-CD25 monoclonal antibody. Accordingly, CD4+CD25+ T-cell-depletion resulted in a significant reduction or delay in parasitemia, pathology, and mortality, as compared to controls. The apparent resistance in CD4+CD25+-T-cell-depleted mice correlated with a profound suppression of Th2 cytokines in vitro and in vivo, culminating in a net Th1 cytokine environment. Cumulatively, these findings suggest that CD4+CD25+ T-cell- depletion improves the trypanotolerance of highly susceptible BALB/c mice acutely infected with the lethal T. congolense.     

‘Niigata S3’ is a new strawberry cultivar suitable for forcing culture under low temperature and insolation conditions

‘Niigata S3’ is a new strawberry (Fragaria × ananassa Duch.) cultivar that is early flowering and possesses high soluble solid content and good coloration. It was selected from a cross between Kei812 (seed parent) and ‘Asuka-Ruby’ (pollen parent). The first harvest date of ‘Niigata S3’ was December 27, 34 days earlier than ‘Echigohime’ and 9 days earlier than ‘Asuka-Ruby’ (means of 2007 and 2008). The marketable yield of ‘Niigata S3’ was 85% of ‘Echigohime’, 107% of ‘Asuka-Ruby’, while the early yield was 145% of ‘Echigohime’, 85% of ‘Asuka-Ruby’ (based on 2007 and 2008 means). The shape of the fruit is long conical, and its skin color medium-red. The fruit skin hardness of ‘Niigata S3’ was 31.5 g/mm², which was harder than ‘Echigohime’, and its average soluble solid content was 11.4%, which was higher than the values for ‘Echigohime’ and ‘Asuka-Ruby’ (2008). Furthermore, ‘Niigata S3’ did not bear apical overripe fruit. This new cultivar is adaptable to the climatic conditions of Niigata, as well as other regions that experience low winter temperatures and insolation.     

Laboratory diagnosis of canine GM2-gangliosidosis using blood and cerebrospinal fluid.

In the present study, laboratory techniques were used to diagnose canine GM2-gangliosidosis using blood and cerebrospinal fluid (CSF) that can be collected noninvasively from living individuals. Lysosomal acid beta-hexosaminidase (Hex) was measured spectrofluorometrically using 4-methylumbelliferyl N-acetyl-beta-D-glucosaminide and 4-methylumbelliferyl 7-(6-sulfo-2-acetamido-2-deoxy-beta-D-glucopyranoside) as substrates. Main isoenzymes A and B of Hex in leukocytes were also analyzed using cellulose acetate membrane electrophoresis. GM2-ganglioside in CSF was detected and determined quantitatively by using thin-layer chromatography/enzyme-immunostaining method with anti-GM2-ganglioside antibody. In normal dogs, Hex activities could be determined in leukocytes, serum, and CSF and the total activities were markedly reduced in all the enzyme sources in a dog with Sandhoff disease. Electrophoresis of a leukocyte lysate from a normal dog showed that the Hex A and Hex B were not separated distinctively with formation of a broad band, whereas there were no bands in electrophoresis of a lysate from a dog with Sandhoff disease, showing a deficiency in the total enzyme activity. GM2-ganglioside could be detected and determined quantitatively in as little as 100 microl of canine CSE GM2-ganglioside in CSF in a dog with Sandhoff disease increased to 46 times the normal level. In conclusion, the methods in the present study are useful for diagnosis of canine GM2-gangliosidosis. These techniques enable definitive and early diagnosis of canine GM2-gangliosidosis even if tissues and organs cannot be obtained.     

Cell wall degradation of tropical and temperate forage grasses measured by nylon bag and in vitro digestion techniques

Four tropical and four temperate grasses were subjected to nylon bag fiber degradation study with fistulated Holstein cattle as well as in vitro digestion with broad spectrum cellulase, and the degradability of cell wall carbohydrate and lignin for the respective forage grasses were determined. The degradability of neutral detergent fiber and acid detergent fiber for the same plant materials appeared to be similar, but that of acid detergent lignin was constantly lower. As for the plant materials used in the present study, Italian ryegrass, orchardgrass and guineagrass showed high degradability of fibers in nylon bag incubation, while setaria, cogongrass and wheat showed smaller degradability. In both nylon bag incubation and in vitro digestion experiments, the forage samples showing relatively lower degradability exhibited notably lower disappearance of xylose than that of glucose or arabinose. It was also observed that dry matter degradability correlated negatively with the initial content of esterified and/or etherified p‐coumaric acid. These results were assumed to be caused by the difference in resistance towards rumen microbial degradation due to the concentration of lignin‐carbohydrate complex.