Studies on the Freeze Denaturation of Squid Actomyosin

Denaturation of squid actomyosin during frozen storage was studied by measuring solubility, viscosity, and ATPase activity, and by ultracentrifugal analysis and SDS-electrophoresis. The additive effect of sodium glutamate, well-known for its cryoprotective effect on the freeze denaturation of carp actomyosin, was also checked. When solutions (0.6M KC1) or suspesions (0.05M KC1) of the isolated squid actomyosin were stored at －20℃, the solubility, reduced viscosity and ATPase activity decreased with the length of frozen storage. The ultracentrifugal patterns showed that aggregation proceeded during frozen storage. Evidently, sodium glutamate prevented the freeze denaturation of squid actomyosin, as in the case of carp actomyosin. When the mantle muscle of squid was freeze-stored at －20℃ and extracted with 0.6M KC1, the amount of soluble actomyosin extractable from the frozen meat and the ATPase activity of the actomyosin were decreased only slightly even after a long freezing period. This differed from the results with isolated actomyosin

Mitochondria-independent induction of Fas-mediated apoptosis by MSSP.

Fas-mediated apoptosis has been proposed to play an important role in homeostasis. Fas triggers apoptosis after stimulation by its ligand FasL or the Fas ligand agonist anti-Fas antibody through a mitochondria-dependent or -independent pathway, and MSSP has been identified as a transcription factor that regulates the c-myc gene and was later found to positively or negatively regulate a variety of genes, including alpha-smooth actin, MHC class I, MHC class 2 and the thyrotropin receptor. We further found that expression of the Fas gene was repressed, resulting in abrogation of the Fas-mediated induction of apoptosis both in Mssp-knockout mice and primary thymocytes. MSSP was then found to stimulate promoter activity of the Fas gene by binding to a specific region. In this study, to identify the MSSP-dependent Fas-induced apoptosis pathway, primary fibroblasts from MSSP (+/+) and MSSP (-/-) cells were treated with the combination of interleukin 1-beta and interferon-gamma and expression of the Fas gene was examined. The results showed that the Fas gene was expressed at the same levels in the two cell types. Furthermore, when these cells were treated with the anti-Fas antibody, it was found that cytochrome C was not released in the cytosol and that activations of caspase 8 and caspase 3 occurred in primary fibroblasts from MSSP (+/+) cells but not from MSSP (-/-) cells. These results indicate that Fas-mediated apoptosis induced by MSSP occurs independently of mitochondria

DJ-1, a Target Protein for an Endocrine Disrupter, Participates in the Fertilization in Mice

DJ-1 was first identified as an activated ras-dependent oncogene product and was later also found to be an infertility-related protein affected by sperm toxicants such as ornidazole (OR) and epichlorohydrin. These findings suggest that DJ-1 has functions in both somatic cells and sperm. In this study, to determine the relationship between DJ-1 and an endocrine disrupter and to determine the functions of DJ-1 in sperm, in vitro fertilization experiments were carried out using eggs and sperm extracted from mice that had or had not been treated with OR. We found that the amount of DJ-1 in sperm and the efficiency of fertilization decreased with the increasing dose of OR to which the mice were exposed. The addition of an anti-mouse DJ-1 serum to sperm solution before the in vitro fertilization reaction with eggs resulted in a decrease in the efficiency of fertilization to about one-third of that when pre-immune serum was added to sperm solution, indicating that DJ-1 participates in the fertilization

Candida albicans Promotes the Antimicrobial Tolerance of Escherichia coli in a Cross-Kingdom Dual-Species Biofilm

Cross-kingdom multi-species biofilms consisting of fungi and bacteria are often resistant to antimicrobial treatment, leading to persistent infections. We evaluated whether the presence of Candida albicans affects the antibacterial tolerance of Escherichia coli in dual-species biofilms and explored the underlying mechanism. We found that the survival of E. coli in the presence of antibacterial drugs was higher in dual-species biofilms compared to single-species biofilms. This tolerance-inducing effect was observed in E. coli biofilms that were treated with a C. albicans culture supernatant. To explore the antibacterial tolerance-inducing factor contained in the culture supernatant and identify the tolerance mechanism, a heated supernatant, a supernatant treated with lyticase, DNase, and proteinase K, or a supernatant added to a drug efflux pump inhibitor were used. However, the tolerance-inducing activity was not lost, indicating the existence of some other mechanisms. Ultrafiltration revealed that the material responsible for tolerance-inducing activity was <10 kDa in size. This factor has not yet been identified and needs further studies to understand the mechanisms of action of this small molecule precisely. Nevertheless, we provide experimental evidence that Candida culture supernatant induces E. coli antibacterial tolerance in biofilms. These findings will guide the development of new treatments for dual-species biofilm infections