Immunohistochemical detection of polyunsaturated fatty acid oxidation markers in acetaminophen-induced liver injury in rats

To clarfity whether polyunsaturated fatty acid (PUFA) oxidation is involved in the mechanism of acetaminophen (APAP)-induced apoptotic cell death, the production and localization of PUFA oxidation markers N(ε)-propanoyl-modified lysine, N(ε)-hexanoyl-modified lysine, 4-hydroxyhexenal-modified histidine and crotonaldehyde-modified lysine were evaluated in the development of APAP-induced liver injury. The immunoexpression of these markers in the liver was examined up to 24 hr post-APAP intraperitoneal injection in rats (1 g/kg body weight). The histopathological changes in the liver appeared 3 hr after APAP injection and became exacerbated with time. Proapoptotic protein Bax immunoreactivity was first detected in the degenerative hepatocytes 3 hr after the injection and areas positively immunostained for Bax reached a peak level at 6 hr, and then decreased at 12 and 24 hr. There was a significant increase in the TUNEL-positive rate at 12 and 24 hr. Immunohistological expression of all these oxidation markers was first detected in the degenerative hepatocytes 3 hr after the injection, and earlier than the occurrence of hepatocyte apoptosis. Immunohistochemical expression of these markers were observed in almost all degenerative hepatocytes 3-24 hr after APAP injection. Areas positively immunostained for these markers reached a peak level at 6 hr, and then decreased at 12 and 24 hr. The results thus suggest that the generation of PUFA oxidation markers may be the signature of early events preceding the induction of liver cell apoptosis and thus useful for early detection of oxidative stress-related liver cell injury.     

Evidence for estrogen receptor expression during medullary bone formation and resorption in estrogen-treated male Japanese quails (Coturnix coturnix japonica)

The temporal expression of estrogen receptor (ER)-α and ER-β mRNA was examined in male Japanese quails. Femurs of quails receiving 17β-estradiol underwent RTPCR and histochemical analysis 1 to 15 days after treatment. Untreated quails were used as controls (day 0). Between days 0 and 5, cells lining the bone endosteal surface differentiated into osteoblasts, which in turn formed medullary bone. Expression of ER-α was already observed on day 0 and increased slightly during bone formation whereas ER-β was hardly detected throughout this process. After osteoclasts appeared on the medullary bone surface, this type of bone disappeared from the bone marrow cavity (days 7~15). ER-α expression simultaneously decreased slightly and ER-β levels remained very low. These results suggest that estrogen activity mediated by ER-α not only affects medullary bone formation but also bone resorption.     

Semen Collection and Polymerase Chain Reaction‐Based Sex Determination of Black‐Headed and Straw‐Necked Ibis

This study aimed to develop a polymerase chain reaction (PCR)‐based sexing and effective semen collection methods for black‐headed and straw‐necked ibis species. However, most birds are not sexually dimorphic, that is, the sexes appear similar. Therefore, the gender should be determined before semen collection. DNA was extracted from the blood samples of 11 black‐headed and 4 straw‐necked ibis. The sex was determined after PCR amplification of the EE0.6 region of W‐chromosome. The PCR products were separated using gel electrophoresis. A single band indicated the presence of the EE0.6 region and that the individual was a female, while no band indicated that the individual was a male. Further, the single bands from seven specimens were amplified. Semen collection was performed by massage or a combination of massage with electro‐ejaculation and was attempted during all four seasons. The semen was successfully collected in March from male straw‐necked ibis using the massage method. Limited motility, viability and concentration of straw‐necked ibis sperm were observed. The sperm length was 180 μm and that of the nucleus was 30 μm with acrosome located at the tip of the nucleus. Thus, the PCR‐based sexing proved to be an accurate molecular sexing method for black‐headed and straw‐necked ibis. Furthermore, we successfully collected semen and observed the stained sperm nucleus and acrosome of the straw‐necked ibis sperm. We propose that the use of this PCR methodology can be applied as a routine method for sex determination and semen collection in ibis species for future ecological research. However, considering our limited success, further studies on semen collection method are required.     

Seed abortion of ‘Tosa-Buntan’ pummelo pollinated with soft-X-irradiated pollens

Cross-pollination was performed with soft-X-irradiated hyuga-natsu pollens (1000 Gy) for ‘Tosa-Buntan’ pummelo (Citrus grandis (L.) Osbeck). This resulted in the transformation of large and complete seeds into small and empty ones (practically seedless). Although fruit set, fruit retention, total soluble solids content (TSS) and titratable acidity of the juice were not affected, decrement in the fruit size was observed. Two weeks after the pollination, endosperm cell division with free nuclei began in both the non-irradiated and irradiated pollen treatment conditions. Seven weeks after pollination, endosperm division with the cell wall occurred in the non-irradiated pollen treatment conditions; however, the endosperm development ceased in most ovules that underwent the irradiated pollen treatment, and the ovules remained in their free nuclear stage. The delayed degeneration of the ovules, following successful fertilization and commencement of endosperm cell division, allow these seedless fruits to be categorized as pseudo-parthenocarpic.     

Effect of high temperature stress on the reproductive growth of strawberry cvs. ‘Nyoho’ and ‘Toyonoka’

High temperatures are known to reduce fruit size and fruit weight in strawberry, but cultivar differences in the response to high temperature stress during the reproductive stage up to the second inflorescence have not been sufficiently reported. We examined the effect of two day/night temperature regimes on fruit set and fruit growth in two cultivars, ‘Nyoho’ and ‘Toyonoka’. A high day/night temperature of 30/25 °C reduced the number of inflorescences, flowers, and fruits in both cultivars compared with plants grown at 23/18 °C. The percentage of fruit set in ‘Nyoho’ was not significantly different between the two temperature treatments, while that in ‘Toyonoka’ was much lower at 30/25 °C than at 23/18 °C. Days to ripening was shorter at 30/25 °C than at 23/18 °C, and no cultivar differences were observed. Fresh weight of primary, secondary, and tertiary fruits was greater at 23/18 °C than at 30/25 °C in both cultivars, and no cultivar differences were observed, except in tertiary fruits. The diameter of fruits from all positions was also reduced at 30/25 °C in both cultivars. Relative growth rates of fruits showed two peaks in both cultivars and in both temperature treatments. Both peaks appeared earlier at 30/25 °C than at 23/18 °C. Percentage of fruit set at 30/25 °C in the second inflorescence was also significantly lower in ‘Toyonoka’ than in ‘Nyoho’. These results indicate that high temperature stress negatively affects the reproductive process in strawberry and that plant response to high temperature stress is cultivar-related in such responses.     

Differentiation in competitive ability and cold tolerance between diploid and tetraploid cultivars in Lolium perenne

In a diploid grass, perennial ryegrass (Lolium perenne L.), many autotetraploid cultivars have been released and widely used in many cool temperate countries. In this study, competitive ability and cold tolerance, which seem to be important factors in determining geographical distribution and persistency of this species, were compared between six diploid and five autotetraploid cultivars. The tetraploid cultivars had significantly higher competitive ability and lower cold tolerance than the diploid cultivars. Furthermore, there was no overlap in the two traits between them. These results suggest that chromosome doubling has a larger effect on competitive ability and cold tolerance than does the genetic difference between the populations within a single ploidy level. This revised version was published online in August 2006 with corrections to the Cover Date.     

Identification of coumarins from lemon fruit (Citrus limon) as inhibitors of in vitro tumor promotion and superoxide and nitric oxide generation.

Three coumarins were isolated as siginificant inhibitors of tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus (EBV) activation in Raji cells from the peel of lemon fruit. They were identified as 8-geranyloxypsolaren (LE-1), 5-geranyloxypsolaren (bergamottin, LE-2), and 5-geranyloxy-7-methoxycoumarin (LE-3), respectively, by spectroscopic analysis. Three isolates had no potential O(2)-scavenging and markedly suppressed TPA-induced superoxide (O(2)(-)) generation in differentiated human promyelocytic HL-60 cells. Furthermore, LE-1 and LE-3 reduced both lypopolysaccharide (LPS) and interferon-gamma (IFN-gamma)-induced nitric oxide (NO) generation in mouse macrophage RAW 264.7 cells. Similarly, they were found to be NO generation inhibitors rather than scavengers by measuring the extracellular L-citrulline levels. The occurrence of these coumarins in a lemon fruit was abundant in the flavedo of the peel based on quantitative analysis using high-performance liquid chromatography (HPLC). The present study suggests that the coumarins in lemon fruit are promising chemopreventive agents by inhibiting radical generation.