High sensitivity of fibroblast cell lines derived from LEC rats to heat treatment

A fibroblast cell line derived from LEC rat was approximately twofold more sensitive to heat treatment at 45 degrees C than were that from WKAH rat in terms of heating time required to attain 50% loss of survival in a colony forming assay. The present study was carried out for understanding the mechanism underlying the higher sensitivity of LEC rat cells to heat treatment. Although apoptosis was not found in WKAH rat cells, the percentages of apoptotic cells in LEC rat cells significantly increased after heat treatment. LEC rat cells showed significantly lower sensitivity in induction of cell death and apoptosis to ceramide, a lipid signaling molecule that is associated with heat-induced apoptosis, than did WKAH rat cells. SP600125, an inhibitor of JNK suppressed the induction of cell death in both heated LEC and WKAH rat cells, but SB203580, an inhibitor of p38 mapk, did not. The relative surviving fractions of heated LEC and WKAH rat cells in the presence of both SB203580 and SP600125 were higher than those of cells in the presence of SP600125 alone. The amounts of hsp70 protein in WKAH rat cells increased from 4 to 12 hr after heat treatment, but did not in LEC rat cells. These results suggest that higher thermosensitivity in the fibroblast cell line from LEC rat is due to low inducibility of hsp70 protein after heat treatment.     

Inhibition of replication induces non-apoptotic cell death in fibroblast cell lines derived from LEC rats

Hydroxyurea (HU), an anticancer drug, inhibits ribonucleoside diphosphate reductase and reduces pool sizes of deoxyribonucleoside triphosphate (dNTP). The reduction of dNTP results in inhibition of DNA replication. The cytotoxic effect of HU was investigated using fibroblast cell lines from LEC rats. LEC rat cells showed significantly higher sensitivity to HU than did cell lines from control WKAH rats. No significant differences were observed between the percentages of apoptotic cells in either LEC or WKAH rat cells that had been treated with HU and those that had not been treated with HU. LEC rat cells also showed significantly higher sensitivity to aphidicolin, which blocks DNA synthesis by inhibiting DNA polymerase alpha, than did WKAH rat cells. In both LEC and WKAH rat cells, intensified bands of p53 protein were observed immediately after treatment with HU. Although the high level of p53 protein persisted in WKAH rat cells until 6 hr post-incubation time after treatment with HU, the level of p53 protein had decreased at 6 hr post-incubation time in LEC rat cells. When the cells were X-irradiated in the absence or presence of HU, the ratio of the surviving fraction without HU to that with HU only slightly increased after X-irradiation in WKAH rat cells. In contrast, the ratio in LEC rat cells significantly increased after X-irradiation in a dose-dependent manner.     

A monoclonal antibody‐based ELISA for the analysis of the insecticide flucythrinate in environmental and crop samples

A competitive enzyme‐linked immunosorbent assay (ELISA) has been developed for the detection of the insecticide flucythrinate in environmental and food samples. Two types of haptens, the acid moiety that is the hydrolyzed product of flucythrinate, and the carboxylated propyl derivative of the alcohol moiety, were used to prepare monoclonal antibodies (MAbs). Five MAbs, which raised against the former hapten, were reactive with flucythrinate. Among them, MAb F1A27‐4 showed the highest activity toward flucythrinate, and did not cross‐react with other pyrethroids such as cycloprothrin, fenvalerate, fluvalinate, etofenprox and silafluofen. The assay conditions of indirect competitive ELISA with MAb F1A27‐4 were studied to optimize the detection of flucythrinate in environmental and food samples. Incubation at 4 °C in the assay buffer, pH 8, with 300 mM sodium chloride improved the sensitivity. The addition of rabbit serum albumin or rabbit antiserum and the presence of 50 ml litre^?1 of methanol reduced matrix effects of the samples. Under optimized conditions, the ELISA detected flucythrinate spiked in water, soil, and extracts of apple and tea samples down to 10 mg litre^?1, 0.2 mg litre^?1, 0.3 mg litre^?1 and 0.3 mg litre^?1, respectively. The mean recovery and CV ranged from 91% to 120% and from 5% to 12%, respectively. The ELISA results in apple samples correlated well with those from LC–MS analysis (r² = 0.99, n = 12). © 2001 Society of Chemical Industry     

H2O2 production by copper amine oxidase, a component of the ecto-apyrase (ATPase)-containing protein complex(es) in the pea cell wall, is regulated by an elicitor and a suppressor from Mycosphaerella pinodes

Blue native PAGE analysis for cell wall proteins from pea epicotyls demonstrated that cell wall-associated ecto-apyrase (ATPase) formed a large protein complex(es) ranging from 450 to 900?kDa; one of the components of the complex was copper amine oxidase (CuAO), which catalyzes the oxidation of amines with the subsequent generation of ammonia and hydrogen peroxide. CuAO activity was coordinately regulated in vitro with ATP-hydrolyzing activity by an elicitor and a suppressor from Mycosphaerella pinodes. Moreover, treatment of cell wall proteins with the suppressor caused the appearance of the apyrase monomer. On the basis of these results, M. pinodes may target the apyrase-containing protein complex(es) of the host to attenuate cell wall-based, extracellular defense(s) including the production of hydrogen peroxide.     

Black root rot of cucurbits caused by Phomopsis sclerotioides in Japan and phylogenetic grouping of the pathogen

Although the causal agent of black root rot of Cucurbitaceae in Japan has been proposed as Phomopsis sclerotioides, the species identification of the pathogen has remained inconclusive because of a lack of spore formation. We confirmed that a Japanese isolate of Phomopsis sp. obtained from a diseased pumpkin root produced pycnidia containing α spores in sterilized bean pods. In phylogenetic analyses of rDNA-ITS regions, nine Japanese Phomopsis sp. isolates from melon, watermelon grafted onto bottle gourd, and pumpkin diagnosed with black root rot, formed a single clade with P. sclerotioides standard isolates. We identified the causal agent of the black root rot of melon, pumpkin, bottle gourd, and watermelon in Japan as P. sclerotioides and propose the Japanese name “Phomopsis-negusare-byo” for the disease. Patterns of random amplified polymorphic DNA (RAPD) of these Japanese isolates were also similar to those of P. sclerotioides, thus supporting the species identification. However, mycelial incompatibilities were found for many combinations among these P. sclerotioides isolates, suggesting some genotypic variations of this fungus in Japan at a level that the RAPD analyses cannot discriminate. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AB201430 to AB201444     

The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins

The first chordates appear in the fossil record at the time of the Cambrian explosion, nearly 550 million years ago. The modern ascidian tadpole represents a plausible approximation to these ancestral chordates. To illuminate the origins of chordate and vertebrates, we generated a draft of the protein-coding portion of the genome of the most studied ascidian, Ciona intestinalis. The Ciona genome contains approximately 16,000 protein-coding genes, similar to the number in other invertebrates, but only half that found in vertebrates. Vertebrate gene families are typically found in simplified form in Ciona, suggesting that ascidians contain the basic ancestral complement of genes involved in cell signaling and development. The ascidian genome has also acquired a number of lineage-specific innovations, including a group of genes engaged in cellulose metabolism that are related to those in bacteria and fungi.     

High sensitivity of thymocytes of LEC strain rats to induction of apoptosis by X-irradiation

It is known that physical disruption of cell contacts induces apoptosis of thymocytes. When thymocytes from LEC and WKAH rats were incubated in vitro at 37 degrees C for 0-6 hr and then the proportion of apoptotic cells was determined using a flow cytometer, it was found that the percentages of apoptotic thymocytes from both LEC and WKAH rats increased with incubation time and that the proportion of apoptotic cells from LEC rats was significantly higher than that from WKAH rats at each incubation time. The fact that cycloheximide, an inhibitor of protein synthesis, did not show significant inhibitory effects on induction of apoptosis of thymocytes indicates that induction of apoptosis during in vitro cultivation did not require de novo protein synthesis. When thymocytes from LEC and WKAH rats were X-irradiated in vitro at 4 and 8 Gy, the percentages of radiation-induced apoptotic cells increased with post-incubation time after X-irradiation in both LEC and WKAH rat thymocytes and the proportions of apoptotic cells from LEC rats were significantly higher than those from WKAH rat cells at 2 and 4 hr post-incubation after X-irradiation. When thymocytes from LEC and WKAH rats were X-irradiated in the presence of cycloheximide, the induction of apoptosis was substantially inhibited, indicating that radiation-induced apoptosis of thymocytes from LEC and WKAH rats required de novo protein synthesis. The present results showed high sensitivities of thymocytes of LEC rats to induction of apoptosis during in vitro cultivation and by X-irradiation.     

Glutathion peroxidase and glucose-6-phosphate dehydrogenase activities in bovine blood and liver

A total of 46 cattle, including 25 as control, 16 with glycogen degeneration and 5 with severe fatty degeneration were studied. Whole blood and liver tissue specimens were used to measure glutathione peroxidase (GSH-Px) and Glucose-6-Phosphate Dehydrogenase (G6PD) activities. The present study determined the value of these parameters in diagnosing glycogen and fatty degeneration in cattle from the point of the status of antioxidation and lipid peroxidation. The results showed a significant decrease in hepatic GSH-Px activity and a significant increase in hepatic G6PD activity in cases of fatty degeneration. On the other hand, there were no significant changes in erythrocytic and hepatic GSH-Px and G6PD activities in cases of glycogen degeneration. The results indicated lipoperoxidation process in the liver tissues increased in cases of fatty degeneration. Therefore, supplying animals suffering from fatty liver with sufficient quantities of nutrient antioxidants may be valuable when treatment is considered.     

Systemic spindle-cell proliferative disease in broiler chickens

The major organs and tissues of 24 broiler chickens (70 or 71 days old) suspected of spindle-cell proliferative disease (SPD) because of showing the tumorous lesions distributed throughout the body at meat inspection were collected for histopathological and immunohistochemical examination. Macroscopically, liver, spleen and cecal tonsil showed severe enlargement and white nodules or plaques were observed in the liver, spleen, kidney, intestine and bone marrow of the femur. All chickens were diagnosed with SPD based on the histopathological examination. The lesions of SPD were observed in the liver, spleen, kidney, heart, lung, pancreas, proventriculus, gizzard, duodenum, jejunum, ileum, rectum, cecal tonsil, bursa of Fabricius, bone marrow of the femur and skin. Hemangioma was observed in the lung of 1 bird. Eight 1-day-old specific pathogen-free chicks were inoculated intraperitoneally with 0.25 ml of a 20% homogenate of the affected spleens of three naturally occurring cases. One inoculated bird, necropsied at 10 weeks of age, macroscopically had a white nodule in the kidney and histopathologically had spindle-cell proliferative lesions, a pattern similar to that seen in the naturally occurring cases, in the liver, spleen, kidney, heart, lung, pancreas, proventriculus, duodenum, cecal tonsil and bone marrow of the femur, and was diagnosed with SPD. Immunohistochemically, significant positive reactions with a rabbit antiserum against avian leukosis virus antigens were detected in all spindle cells in the proliferative lesions of all examined SPD cases and in tumor cells of the hemangioma of a field case.     

Relationship between fat accumulation in the liver and energy intake, milk fat yield and blood metabolites in dairy cows

In the present study, 42 multiparous Holstein cows were used to investigate the relationship between fat accumulation in the liver and dry matter intake, milk yield and blood metabolites. Based on the percentage of fat in the liver cell at 2 weeks post‐parturition, the cows were classified into three groups. These groups were: (i) less than 10% of fat (normal group, n = 29); (ii) 10–20% of fat (mild group, n = 6); and (iii) more than 20% of fat (moderate group, n = 7). The bodyweight of the moderate group was high (771 kg) before calving. The sufficiency rates of total digestible nutrients (TDN) were remarkably decreased (approximately 65%) in early lactation. The milk fat yield and milk fat composition of the moderate group were higher (P < 0.05) than the other groups at 1 and 2 weeks post‐parturition. It was suggested that non‐esterified fatty acids (NEFA) mobilized from adipose tissues was directly used by the mammary gland for synthesis of milk fat. The percentage of bromsulfalein (BSP) retention of the moderate group was high (21.1%) at 30 min, and it showed that the BSP clearance function was significantly decreased. The concentrations of NEFA, β‐hydroxybutyric acid and glucose were appropriate indicators of energy status; however, aspartate aminotransferase, γ‐glutamyl transpeptidase and total bilirubin were not sensitive indicators of a moderate fatty liver. Thus, high‐yielding cows that calve in an overweight condition are more likely to develop excessive fat accumulation in the liver because of great mobilization from adipose tissues post‐parturition. In cows with a moderately fatty liver, a decrease in TDN sufficiency rates, an increase of milk fat yield and a reduction of liver function were observed in early lactation. The increase of serum NEFA and milk fat composition resulting from mobilization of adipose tissues helped to diagnose moderate fatty liver.