Spindle formation and microtubule organization during first division in reconstructed rat embryos produced by somatic cell nuclear transfer

The present study was conducted to demonstrate the spindle formation and behavior of chromosomes and microtubules during first division in reconstructed rat embryos produced by somatic cell nuclear transfer (SCNT) with cumulus cell nuclei. To demonstrate the effect of oocyte aging after ovulation on the cleavage of SCNT embryos, micromanipulation was carried out 11, 15 and 18 h after injection of hCG. SCNT oocytes were activated by incubation in culture medium supplemented with 5 microM ionomycin for 5 min followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) in mR1ECM for 2-3 h. For immunocytochemical observation, the SCNT embryos were incubated with monoclonal anti-alpha-tubulin antibody and then fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG. Cleavage rates were significantly higher for oocytes collected after 15 and 18 h rather than for those collected 11 h after injection of hCG (56 and 53%, respectively vs. 28%; P<0.05). Premature chromosome condensation occurred before activation of the SCNT oocytes, but adequate spindle formation was only rarely observed. The distribution of microtubules in SCNT embryos after activation was different from those of fertilized and parthenogenic oocytes, i.e., a dense microtubule organization shaped like a ring was observed. Eighteen to 20 h post-activation, most SCNT embryos were in the 2-cell stage, but no nucleoli were clearly visible, which was quite different from the fertilized oocytes. In addition, first division with and without small cellular bodies containing DNA was observed in the rat SCNT embryos in some cases. The present study suggests that reorganization of transferred nuclei in rat SCNT embryos may be inadequate in terms of formation of the mitotic assembly and nucleolar reorganization.     

Host range and molecular analysis of Beet leaf yellowing virus,Beet western yellows virus-JP and Brassica yellows virus in Japan

Beet western yellows virus (BWYV; genus Polerovirus, family Luteoviridae) is one of the most important viruses causing yellowing disease of many field and vegetable crops. This study isolated different poleroviruses from sugar beet, spinach, radish and brassica in Japan, and identified them as BWYV-JP, Beet leaf yellowing virus (BLYV), Brassica yellows virus (BrYV) and BrYV-R (radish strain) based on host range and molecular analysis. Among over 100 plant species from 19 families inoculated with the vector Myzus persicae, about half of the species in 13 families were infected with some of these viruses. BLYV shared a similar host range to Beet mild yellowing virus (BMYV). These had a much more limited host range than BWYV-JP, which resembled BWYV-USA. The host range of BrYV was similar to that of Turnip yellows virus (TuYV). Phylogenetic analyses at the 5′ portion (replication-related gene) of the genome showed that BLYV, BMYV, BWYV (-JP and -USA) and Cucurbit aphid-borne yellows virus (CABYV) formed one large group, whereas BrYV and TuYV were grouped together. BLYV and BWYV were most closely related to each other, and were more closely related to CABYV than to BMYV. However, at the 3′ end (coat protein gene), BLYV and BWYV-JP formed a distinct group, separated from the BrYV group, which in turn was more closely related to BWYV-USA, BMYV, TuYV and Beet chlorosis virus, a group originating from outside Asia. Thus, this study presents host range differences and phylogeographical relationships of BWYV-like poleroviruses that are distributed worldwide.     

Recording cerebellar neuron activities in swimming goldfish

ABSTRACT:   Neuronal activities were investigated in the cerebellum of immobilized and swimming goldfish Carassius auratus . Extracellularly recorded neural activities of the cerebellum in immobilized goldfish were characterized and classified into five types. Based on the waveforms and recording depths, these five neural activity types were estimated to originate from three identified classes of cerebellar neurons: Purkinje cells, eurydendroid cells, and granule cells. Chronic recording of cerebellar neuron activities in unrestrained goldfish was performed for more than 100 h. During the chronic recordings, a submersible amplifier attached to the goldfish head, and a multielectrode array developed for the present study were used. Neuronal activities in the cerebellum of free-swimming fish could also be classified into five types as in the immobilized condition. Firing patterns of two neurons identified as Purkinje cells and eurydendroid cells were analyzed during turning movements of the goldfish. The firing patterns of these neurons changed in relation to turning movements. Although some improvements are required, the chronic recording method developed in the present study can be applied to further investigations concerning the direct relationship between brain neural activities and certain behavior.     

Chemical and Thermal Properties of Freshwater Prawn (Macrobrachium rosenbergii) Meat

Chemical compositions and thermal properties of cultured freshwater prawn meat (FPM) were studied. FPM contained 83.2% protein (dry basis), 62.7% of which was myofibrillar protein. Pepsin-soluble collagen (PSC) and insoluble collagen (ISC) contents were 0.63 and 0.32%, respectively. Both collagens were similar to type V collagen from porcine placenta. Glutamic acid/glutamine, arginine, aspartic acid/?asparagine, and lysine were abundant amino acids in FPM. Glycine, proline, hydroxyproline, and aspartic acid/?asparagine were predominant in both collagens. FPM exhibited thermal transition temperatures (T_max) of 48.3 and 64.7°C, whereas T_max of PSC and ISC were 43.0 and 46.0°C, respectively. Textural changes in FPM during post-mortem storage on ice are plausibly dependent upon its compositional and thermal properties.     

Cloning and expression of a surface immunogenic protein in Streptococcus dysgalactiae isolated from fish and its application in enzyme‐linked immunosorbent assays to diagnose S. dysgalactiae infections in fish

Lancefield group C Streptococcus dysgalactiae (GCSD) causes severe necrotic lesions in the caudal peduncle in the genus Seriola farmed in Japan. To develop a sero‐diagnostic method for GCSD infection in farmed fish, we attempted to identify a surface immunogenic protein that induces an antibody after infection with GCSD by immunoblot analysis using sera collected from infected fish. A protein obtained from sodium dodecyl sulfate (SDS) extracts of GCSD was identified as S. dysgalactiae surface immunogenic protein (Sd‐Sip). Sd‐Sip exhibited more than 94% homology with a surface antigen or a hypothetical protein from S. dysgalactiae mammalian isolates at the nucleotide sequence level. Expression of the recombinant Sd‐Sip (rSd‐Sip) was confirmed by immunoblot analysis, that is, its reactivity to GCSD‐infected sera. Antibody detection ELISA using rSd‐Sip and their usefulness for diagnosis of GCSD infection were examined. GCSD‐infected sera collected from farmed amberjack, Seriola dumerili (Risso), showed strong reaction with immobilized rSd‐Sip. Meanwhile, sera immunized by other pathogenic bacteria of fish were showed ELISA values similar to those of non‐infected sera. These results of this study suggest that the antibody detection ELISA using rSd‐Sip is an effective diagnostic method for GCSD infection in fish.     

Studies on the green liver in cultured red sea bream fed low level and non-fish meal diets: Relationship between hepatic taurine and biliverdin levels

SUMMARY: The aim of this study was to investigate the cause of green liver in red sea bream fed substitute protein diets. Red sea bream Pagrus major was given either of the following diets for 28 weeks: (1) control diet (50% fish meal), (2) low level fish meal diet (15% fish meal), and (3) non-fish meal diet (0% fish meal). The green liver was observed in all groups tested, but the incidence was much higher in the experimental diet groups. The feeding of substitute protein diets reduced plasma triglyceride and cholesterol levels. However, there was no significant difference in plasma hepatic enzyme activities and plasma bile salts concentration among the treatments. Fish fed the substitute protein diets showed low hepatic taurine levels with an appearance of a biliverdin in the liver. Moreover, the proportion of ditaurobilirubin to total biliary bile pigments was significantly lower in fish fed the substitute protein diets. These data indicate that feeding of substitute protein diets did not induce any cholestatic hepatobiliary obstructions and that the low hepatic taurine level was one of the probable factors responsible for the occurrence of green liver in red sea bream fed substitute protein diets.     

Dual effects of reversed winter–spring temperatures on year‐to‐year variation in the recruitment of chub mackerel (Scomber japonicus)

To clarify the effects of temperature on the recruitment of chub mackerel (Scomber japonicus) in the North Pacific, we investigated the influence of winter surface temperature (WST) on spawners at the time of maturity around the spawning grounds and the influence of ambient spring temperature on larvae using estimated temperature (ET) obtained from particle tracking experiments. We found a significant positive correlation between ET approximately 10 days following hatching and the recruitment per spawning stock biomass (RPS) after 2000. The closer (more meandering) the Kuroshio Current (KC) axis was in relation to the spawning ground, the higher (lower) the spring surface temperature and the higher (lower) RPS was in the spawning ground. In contrast, WST inside KC near the maturity/spawning ground was significantly negatively correlated with RPS. A significant negative correlation between the temperatures in winter and spring was detected in the area after 2000, when the conditions of the Pacific decadal oscillation index and the stability of the Kuroshio Extension were synchronous, indicating that KC shifted northward during this time. The reversed temperature pattern was consistent with the winter–spring movement of KC axis in the offshore direction and was correlated with the winter–spring difference in the intensity of the Aleutian low. These results suggest that the annual variation in chub mackerel recruitment after 2000 was strongly affected by the combined effects of ambient temperature because of the reversal of conditions that occurred between winter and spring around the maturity/spawning ground, which was related to the KC path.     

Nitrogen nutrition,leaf resistance,and leaf photosynthetic rate of the rice plant

The evidence that indicates a close association between nitrogen nutrition and the Photosynthetic rate of single rice leaves (Table 1) shows that the photosynthetic rate is related to the nitrogen content on a dry weight basis or on a leaf area basis, Photosynthesis of single leaves can be analyzed in terms of a series of diffusion resistances, i.e., stomatal, mesophyll, and carboxylation (1, 3, 6). Nitrogen nutrition may affect either one of these or more than two at the same time. Nitrogen nutrition affects mainly the mesophyll resistance, and the stomatal resistance to a lesser extent, in cotton, beans, and maize (10) while it affects both the stomatal and mesophyll resistance in sugar beet (8).     

Studies on the properties of organic matfer in buried humic horizon derived from volcanic ash

To investigate the relationship between the change of humus property and time factor, the humus composition in the buried humic horizons with the age from 28,000 years B.P. to the present was studied, according to the method presented by Kumada et al. (7).

The fH decreased with the elapse of time and in case of most of humus in the buried humic horizons with the age over 10,000 years, the fH became lower than 40 and were in the form of so-called ‘combined’ humus.

When 3,000-4,000 years have passed since the surface soil buried, the humic acid first moved . to ‘combined form.’ Second, after over 10,000 years, the fulvic acid moved to ‘combined form.’

The humification of ‘free’ humic acid proceeded with the elapse of time till around 5,000 years but of 'combined' humic acid till around 7,000 years. Both the humification-degree of ‘free’ humic acid after around 5,000 years and of ‘combined’ humic acid after around 7,000 years decreased. The humification-degree of ‘free’ and ‘combined’ humic acid from the samples with the age about 15,000 years B.P. to 28,000 years B.P. may be the same.     

Effect of nitrogen application during ripening period on photosynthesis and dry matter production and its impact on yield and yield components of semidwarf iniica rice varieties under water culture conditions

Abstract

A water culture pot experiment was conducted to analyze the effects of N application during the ripening period (RP) on photosynthesis, dry matter production, and its impact on grain ripening and yield in two semidwarf indica type varieties viz. Gui Zhao 2 (GZ) and BR3 (BR) compared with a japonica type variety Koganemasari (KO) under four N rates viz. 0 (N₀), 10 (N₁₀), 20 (N₂₀), and 40 (N₄₀) mg L^?1. Results showed that N application enabled to maintain a higher leaf area and delay leaf senescence in both indica and japonica type varieties but the decrease in the rate, of leaf area was higher in the former than in the latter and the rate was reduced with increasing N rates. Flag leaf photosynthesis and SPAD values of N treated plants were higher throughout the RP, showing the presence of a significant correlation either for each variety or all the varieties together. Higher photosynthetic rate was supported by higher leaf chlorophyll (SPAD value basis) content, stomatai conductance, and N concentration in leaf blades. Top dry matter content increased with increasing N rates mainly due to mean leaf area rather than NAR except for BR during RP but it was higher in KO than in GZ and BR. Reduction of shoot weight due to translocation of dry matter to panicles during RP was suppressed by N rates both in GZ and BR while shoot weight increased in the N-treated plants in KO. The dependency of KO on current photosynthates for panicle weight was found to be almost hundred percent while the contribution of stored carbohydrates in shoot before heading to panicle weight in GZ and BR was in the range of 4-27 and 33-54%, respectively and the rest was contributed by current photosynthates. The percentage increased with increasing N rates. Percentage of ripened grains (PRG) increased with increasing N rates in GZ and BR due to the increase in dry matter production and in the photosynthetic rates of apex leaves, despite the larger spikelet number and larger hull size. However, KO showed almost no variation although it had the highest PRG among the varieties. Brown rice yield followed the same pattern as that of PRG in GZ and BR and the highest yield was produced by BR followed by GZ and KO. These results suggest that N application during RP was more effective in increasing yield in the semidwarf indica type varieties than in the japonica type variety.