Three antibacterial proteins were isolated from acid extracts of channel catfish (Ictalurus punctatus) skin by cation exchange chromatography and reverse-phase high-pressure liquid chromatography. The molecular masses of the proteins were 15.5, 15.5 and 30 kD as determined by SDS-polyacrylamide gel electrophoresis. Mass spectrometry, amino acid composition and amino acid sequence data suggest that the most abundant protein is closely related to histone H2B. The H2B-like protein was inhibitory to Aeromonas hydrophila and Saprolegnia spp., which are important bacterial and fungal pathogens of fish. These findings suggest that histones may be important defensive molecules in fish.

In the presence of Ag(I) ions, the C-T and m(5)iC (5-methylisocytosine)-T base pairs showed comparable stability to the C-Ag(I)-C base pair, and the m(5)iC-C base pair was highly stabilized by the synergetic effect of Ag(I) coordination and possible hydrogen bonding.

No abstract

Cholesterol has been claimed to be involved in the generation and/or accumulation of amyloid beta protein (Abeta). However, the underlying molecular mechanisms have not been fully elucidated yet. Here, we have investigated the effect of membrane cholesterol content on gamma-secretase activity using Chinese hamster ovary cells stably expressing beta-amyloid precursor protein (APP) and either wild-type or N141I mutant-type presenilin 2. Cholesterol was acutely depleted from the isolated membrane by methyl-beta-cyclodextrin, and Abeta production was assessed in a cell-free assay system. Reduced cholesterol did not significantly alter the amounts of Abeta produced by either total cell membranes or cholesterol-rich low-density membrane domains. Even its extremely low levels in the latter domains did not affect Abeta production. This indicates that the membrane cholesterol content does not directly modulate the activity of gamma-secretase. To ascertain that gamma-secretase resides in cholesterol-rich membrane domains, low-density membrane domains were further fractionated with BCtheta (biotinylated theta-toxin nicked with subtilisin Carlsberg protease), which has recently been shown to bind selectively to rafts of intact cells. The membrane domains purified with BCtheta did indeed produce Abeta. These observations indicate that the gamma-cleavage required for generating Abeta occurs in rafts, but its activity is virtually cholesterol-independent.

No abstract

Three new isomalabaricane triterpenes, 29-hydroxystelliferin D (2), 3-epi-29-hydroxystelliferin E (3), and 3-epi-29-hydroxystelliferin A (4), were isolated from the marine sponge Stelletta globostellata. Their structures, including absolute stereochemistry, were determined on the basis of spectral data and chemical methods. Rat fibroblasts treated with 0.2 microM of 2-4 exhibited unusual morphological characteristics, followed by death in 5 days.

The expression and drug efflux activity of the ATP binding cassette transporters Cdr1p and Pdh1p are thought to have contributed to the recent increase in the number of fungal infections caused by Candida glabrata. The function of these transporters and their pumping characteristics, however, remain ill defined. We have evaluated the function of Cdr1p and Pdh1p through their heterologous hyperexpression in a Saccharomyces cerevisiae strain deleted in seven major drug efflux transporters to minimize the background drug efflux activity. Although both Cdr1p-and Pdh1p-expressing strains CDR1-AD and PDH1-AD acquired multiple resistances to structurally unrelated compounds, CDR1-AD showed, in most cases, higher levels of resistance than PDH1-AD. CDR1-AD also showed greater rhodamine 6G efflux and resistance to pump inhibitors, although plasma membrane fractions had comparable NTPase activities. These results indicate that Cdr1p makes a larger contribution than Phd1p to the reduced susceptibility of C. glabrata to xenobiotics. Both pump proteins were phosphorylated in a glucosedependent manner. Whereas the phosphorylation of Cdr1p affected its NTPase activity, the protein kinase A-mediated phosphorylation of Pdh1p, which was necessary for drug efflux, did not. This suggests that phosphorylation of Pdh1p may be required for efficient coupling of NTPase activity with drug efflux.

Abstract.To reveal the molecular mechanism of selective follicular atresia in porcine ovaries, we investigated the changes in the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (DR4) proteins and TRAIL mRNA in granulosa cells during follicular atresia. Immunohistochemical, Western immunoblotting and reverse transcription-polymerase chain reaction analyses (RT-PCR) revealed that significant increases in TRAIL protein and mRNA levels but not DR4 protein were changed during atresia. The RT-PCR product was confirmed to be porcine TRAIL by the cDNA sequence determination. An in vitro apoptosis inducing assay using cultured granulosa cells prepared from healthy follicles showed that TRAIL could activate caspase-3 and induce apoptotic cell death in the cells. The present findings confirm that TRAIL induces apoptosis in granulosa cells during atresia in porcine ovaries. Key words: Apoptosis, Follicular atresia, Granulosa cell, Porcine ovary, Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (J. Reprod. Dev. 49: [313][314][315][316][317][318][319][320][321] 2003) n mammalian ovaries, more than 99% of follicles undergo a degenerative process known as "atresia", and only a few follicles ovulate during ovarian follicular development [1][2][3][4][5][6][7]. We have investigated the molecular mechanism of selective follicular atresia in porcine ovaries, and have reported that follicular selection dominantly depends on granulosa cell apoptosis [8][9][10]. Unfortunately, we have little knowledge of the molecular mechanisms that control apoptotic cell death in granulosa cells during follicle selection.To date, at least four cell death ligand-receptor systems, tumor necrosis factor-α (TNFα), Fasligand (also called APO-1/CD95-ligand), TNFrelated apoptosis-inducing ligand (TRAIL; also called APO-2 ligand) and APO-3 ligand, have been found [11]. TRAIL was initially identified as the homologic gene of TNFα. The biological roles of TRAIL are not yet fully understood. TRAIL has been reported to induce apoptosis in various tumor cells but not in normal cells [12][13][14]. However, a r ece nt s t ud y r ev ealed t hat T R A IL ind uc es apoptosis in normal hepatocytes of human, but not in those of rat, mouse or rhesus monkey, indicating that there are species-specific differences in the mode of action of TRAIL and the receptor systems [15]. Moreover, our preliminary experiments showed that TRAIL and its receptor proteins, death receptor