cFLIP regulates death receptor-mediated apoptosis in an ovarian granulosa cell line by inhibiting procaspase-8 cleavage

More than 99% of follicles in mammalian ovaries undergo atresia, but the mechanisms regulating the strict selection process are still unclear. Granulosa cell apoptosis is considered the trigger of follicular atresia, which occurs in advance of the death of an oocyte. Cellular FLICE-like inhibitory protein (cFLIP), a homologue of procaspase-8 (also called FLICE), is an intracellular anti-apoptotic protein. It is expressed in granulosa cells of porcine ovaries, where its levels decreases during follicular atresia. We hypothesized that cFLIP regulates granulosa cell apoptosis by acting as a pro-survival factor. In the present study, to further reveal the function of cFLIP in granulosa cells, we examined the anti-apoptotic mechanism of cFLIP using KGN, a human granulosa tumor cell line. Fas-mediated apoptosis was induced by co-treatment with anti-Fas antibody (CH-11), which acts as an agonist of Fas-ligand, and cycloheximide (CHX). When cFLIP was stably expressed in KGN cells following transfection of an expression vector, the Fas-mediated apoptosis was inhibited. Suppression of cFLIP by small interfering RNA (siRNA) spontaneously induced cell death. Silencing of cFLIP promoted cleavage of procaspase-8, and the cell death caused by cFLIP siRNA was completely blocked by a caspase-8 inhibitor (Z-IETD-FMK), indicating that cFLIP regulates apoptosis in KGN cells by inhibiting cleavage of procaspase-8. In conclusion, cFLIP is an essential pro-survival factor for granulosa cells, and it prevents granulosa cell apoptosis by inhibiting procaspase-8 activation.     

Application of supercooling to long-term storage of fish meat

Fish meat was brought to a supercooled state through slow cooling, and changes in the texture, histology, and protein composition of the meat were investigated. The groups whose storage temperature were lowered by 1.0°C per day (the 1.0°C group) and 0.5°C per day (the 0.5°C group) began to freeze in the vicinity of −3.5 and −5.0°C, respectively. The freezing point depended on the fish species; the lowest freezing point was −8.5°C, for the red sea bream in the 1.0°C group. The breaking strength tended to decrease more slowly in the 1.0°C group, but the collagen fibers collapsed more rapidly in the 1.0°C group. In SDS electrophoresis, a slight change in the banding patterns was observed, but the relationship between this observation and changes in histology and physical properties was unclear. This study shows that it is possible to produce a supercooled state in fish meat, and demonstrates that supercooling is a potential new storage method that lowers the temperature without generating ice crystals.     

Behavioural analysis of a nonionic detergent in the kraft pulp washing process using cryo-time-of-flight secondary ion mass spectrometry and cryo-scanning electron microscopy

In order to understand the kraft pulp decolouring mechanism on using a nonionic detergent, the pulp washing process and the resulting pulp handsheets were investigated by examining the brightness, kappa number, thioacidolysis product yield, and dewatering efficiency in the pressing sheet making process. The pulp decolouring could be attributed to a decrease in the lignin content and an improvement in the dewatering efficiency. Furthermore, the detergent distribution in the aqueous pulp suspension obtained during the pulp washing process was visualised using cryo-time-of-flight secondary ion mass spectrometry/scanning electron microscopy (cryo-TOF-SIMS/SEM). The detergent was clearly observed at the transverse surface of the pulp fibre cell wall and was also detected in the lumen of the fibres, suggesting the permeation of the detergent into the pulp fibre cell wall. Based on these results, the pulp decolouring mechanism can be proposed as follows: the detergent permeates into the pulp fibre cell wall and promotes the solution-exchange between the inside and the outside parts of the fibre cell wall, finally washing away the chromophoric substances such as lignin and its degradation products owing to the enhanced dewatering efficiency.     

Detection of Tobamoviruses from Soils by Non-precoated Indirect ELISA

A method for detecting tobamoviruses from field soils was developed using non-precoated indirect enzyme-linked immunosorbent assay (Id-ELISA). Absorbance values in Id-ELISA were relatively low after directly applying Pepper mild mottle virus (PMMoV)-infested soil extract. However, heat treating the soil extract before application greatly enhanced the absorbance values. The heat treatment was essential for the Id-ELISA detection of tobamoviruses from infested soil, although the efficiency of virus recovery varied depending on the properties of soil. The number of local lesions in the infectivity assay was consistent with the absorbance values in Id-ELISA. Moreover, the absorbance values in Id-ELISA were correlated with the incidence of soil transmission of PMMoV. Thus, Id-ELISA combined with heat treatment is a practical technique for the diagnosis of infestation with Tobamovirus in field soils, Gray Lowland soil and Sand-dune Regosol. Received 4 October 1999/ Accepted in revised form 9 December 1999     

Dysfunction of erythropoietin-producing interstitial cells in the kidneys of ICR-derived glomerulonephritis (ICGN) mice

Anemia is a major secondary symptom in chronic renal disorder (CRD), but the precise cause of insufficient production of erythropoietin (EPO) remains unclear owing to the controversial localization of EPO-producing cells in the kidneys. The ICR-derived glomerulonephritis (ICGN) mouse, a new hereditary nephrotic mouse, is an appropriate model of anemia associated with CRD. By using an amplified in situ hybridization technique, we detected and counted the renal EPO-producing cells under both normoxic and hypoxic conditions. The expression levels of renal EPO mRNA were quantified and oxygen gradients were also assessed immunohistochemically. Amplified in situ hybridization clarified that EPO-producing cells were peritubular interstitial cells in the middle region of renal cortex in both ICR and ICGN mice. Hypoxia (7% O2) induced low oxygen tension in proximal tubular epithelial cells of renal cortex, and increased the expression of EPO mRNA and the number of EPO-producing cells in both ICR and ICGN mice. However, hypoxia did not increase the serum EPO levels in ICGN mice. The ICGN mouse is a good model for anemia associated with CRD, and the suppression of EPO protein production in the renal EPO-producing cells is considered to be a potential cause of anemia associated with CRD.     

The storage characters related to indica/japonica diferentiation of rice

The decline of rice quality and germination ability in the process of storage was a big problem to food production and nanagement. Gas and temperature control was used to decrease storage losing, but selecting germplasm with good storage characters was rare. In practice, it was found that storage characters of indica were better than that of japonica.