Estrone sulfate and progesterone profiles during late gestation in recipient cows transferred embryos produced by nuclear transfer and in vitro fertilization

The aim of present experiment was to evaluate the plasma concentrations of estrone sulfate (E(1)S) and progesterone (P(4)) during late gestation in recipient cows transferred embryos produced by nuclear transfer (NT) and in vitro fertilization (IVF). Blood samples were collected from recipients transferred embryos produced by NT (n=9) and IVF (n=13) at 160, 220, 240, 260 and 270 d of gestation and then at 5 d intervals until parturition. Plasma samples were analyzed for E(1)S and P(4) by ELISA. One NT and three IVF cows aborted between days 220 and 260 of gestation. Two NT and one IVF cow had prolonged gestation (over 290 d). One IVF cow had an overweight fetus (50 kg) after abortion (257 d). The patterns of changes in the concentrations of E(1)S during late gestation in the NT and IVF cows were almost identical. The NT and IVF cows that aborted had prolonged gestation and much higher E(1)S concentrations than the average. One NT cow aborted after 220 d of gestation and had a sudden high increase in its E(1)S concentration from 160 d to 220 d of gestation. The NT and IVF cows that had prolonged gestation also had significantly higher (P<0.05) P(4) concentrations than the average. These results raise the possibility that the E(1)S and P(4) profiles can be used to monitor some late gestational problems, such as higher birth weight, abortion and prolonged gestation.     

Development and evaluation of pyramiding lines carrying early or late heading QTLs in the indica rice cultivar ‘IR64’

The heading date is an important trait for determining regional and climatic adaptability in rice. To expand the adaptability of the indica rice cultivar ‘IR64’, we pyramided multiple early or late heading quantitative trait locus (QTLs) in the ‘IR64’ genetic background by crossing previously developed near-isogenic lines (NILs) with a single QTL for early or late heading. The effects of pyramiding QTLs were observed in three different climatic zones of the Philippines, Madagascar, and Japan. The early heading pyramiding lines (PYLs) headed 6.2 to 12.8 days earlier than ‘IR64’ while the late heading PYLs headed 18.8 to 27.1 days later than ‘IR64’. The PYLs tended to produce low grain yield compared to ‘IR64’. The low yield was not improved by combining SPIKE, which is a QTL that increases the number of spikelets per panicle. Conversely, ‘IR64-PYL(7+10)’ carrying Hd5 and Hd1 headed earlier, produced more tillers, and more panicles per m² than ‘IR64’, and mitigated the yield decrease in early heading. These results suggest that the effects of pyramided QTLs on heading date were consistent across various environments and PYLs could be used to enhance the adaptation of ‘IR64’ in other rice growing environments.     

Establishment of testicular and ovarian cell lines from Honmoroko (Gnathopogon caerulescens)

We succeeded to establish cell lines from endemic fish species Honmoroko Gnathopogon caerulescens, which inhabits Lake Biwa, the third oldest lake in the world. Two cell lines designated as RMT1 and RMO1 were established from testis and ovary of G. caerulescens, respectively. These cell lines were initially cultured in Leibovitz’s L-15 medium supplemented with fetal bovine serum (FBS), fish embryo extract, epidermal growth factor, and basic fibroblast growth factor. Further addition of forskolin and β-mercaptoethanol was required to establish and maintain these cell lines for more than 60 passages. RMT1 and RMO1 cells showed fibroblast- and epithelial-like morphology, respectively. From immunocytochemical staining and gene expression patterns, RMT1 cells showed a characteristic of testicular Sertoli cells and RMO1 cells did that of ovarian theca cells. Both RMT1 and RMO1 cells multiplied well in the medium supplemented with 10 % FBS at 28 °C and their minimum population doubling times were 24.4 and 28.8 h, respectively. At the 45th passage, most of the RMT1 and RMO1 cells had a hyperploid set of chromosomes (67.3 and 96.1 %, respectively). Cells with normal diploid chromosome set were not observed. RMT1 cells were transfected with an enhanced green fluorescent protein (EGFP) expression vector and human elongation factor 1 α promoter worked efficiently to express EGFP. In addition, EGFP-expressing cell lines were also established, suggesting that the cell lines could be utilized as an in vitro monitor system (biosensor) for the evaluation of endocrine disruptors which might affect gonadal function.     

Bovine herpesvirus-1 (BHV-1) recombinant expressing pseudorabies virus (PrV) glycoproteins B and C induces type 1 immune response in BALB/c mice

Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.     

Effect of regular walking exercise on glucose tolerance and insulin response to i.v. glucose infusion in growing beef steers

The purpose of the present paper was to investigate the effect of regular walking exercise on glucose tolerance and insulin response to i.v. glucose infusion in growing beef steers. Four crossbred beef steers walked on a treadmill during a 6 week exercise period (1.2 km/h, 1 h/day and 5 days/week). The changes in plasma glucose and insulin levels following glucose infusion were analyzed immediately prior to (bodyweight: 260.4 ± 24.2 kg) and after (295.7 ± 30.1 kg) the exercise period. The basal levels of plasma glucose (86.4 vs. 82.0 mg/dL, P = 0.040) and insulin (24.5 vs. 14.3 μU/mL, P = 0.016) were significantly lower after the exercise period. Further, the increase in the levels of plasma glucose (420.4 vs. 280.8 mg/dL, P < 0.001) and insulin (94.5 vs. 73.1 μU/mL, P = 0.028) following the glucose infusion decreased after the exercise period. The area under the curve of plasma glucose (108.8 vs. 62.9 mg/dL per min, P < 0.001) and insulin (53.6 vs. 29.7 μU/mL per min, P = 0.018) indicated more rapid clearance of exogenous glucose and less insulin secretion for glucose clearance after the exercise period. These results suggest that regular exercise improves glucose tolerance, with lower insulin response to glucose infusion in growing steers, as observed in rodents and humans.     

N-terminal domain including conserved flg22 is required for flagellin-induced hypersensitive cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Flagellin in Pseudomonas syringae is a potent elicitor of defense responses including hypersensitive cell death in dicot plants. The oligopeptides flg22 consisting of 22 conserved amino acids near the N-terminus of flagellins is reported to induce plant defense responses. Because glycosylation of the central domain of flagellin affects its elicitor activity, we investigated whether any peptide sequence in addition to flg22 is required for flagellin-induced hypersensitive reaction. A study of recombinant flagellin polypeptides indicated that the N-terminal domain including the conserved flg22 is required for flagellin-induced hypersensitive cell death in Arabidopsis thaliana.     

The genetic structure of populations of <Emphasis Type="Italic">Turnip mosaic virus</Emphasis> in Kyushu and central Honshu,Japan

The genetic structure of the populations of Turnip mosaic virus in Kyushu and central Honshu, Japan was assessed. The host specificity of isolates was determined, and their gene sequences compared utilizing a population genetic approach. Phylogenetic analysis of partial sequences revealed that 32 of 49 Honshu isolates (65%) collected during 1997–2001 belonged to the basal-BR group as did 23 of 64 isolates from Kyushu. All these basal-BR isolates infected both Brassica and Raphanus plants. However, analyses of the positions of recombination sites in five regions of the genome (one third of the full sequence) showed that at least four intra-lineage recombinants were present in these populations. These analyses showed that Kyushu and Honshu shared none of these subpopulations, and genetically distinct basal-BR populations were present in the two districts. We conclude that different basal-BR subpopulations had expanded into those districts. The nucleotide sequences are deposited in the DDBJ/EMBL/GenBank databases under accession numbers AB267281-AB267376.