24(S)-hydroxycholesterol induces neuronal cell death through necroptosis, a form of programmed necrosis

24(S)-Hydroxycholesterol (24S-OHC) produced by cholesterol 24-hydroxylase expressed mainly in neurons plays an important physiological role in the brain. Conversely, it has been reported that 24S-OHC possesses potent cytotoxicity. The molecular mechanisms of 24S-OHC-induced cell death have not yet been fully elucidated. In this study, using human neuroblastoma SH-SY5Y cells and primary cortical neuronal cells derived from rat embryo, we characterized the form of cell death induced by 24S-OHC. SH-SY5Y cells treated with 24S-OHC exhibited neither fragmentation of the nucleus nor caspase activation, which are the typical characteristics of apoptosis. 24S-OHC-treated cells showed necrosis-like morphological changes but did not induce ATP depletion, one of the features of necrosis. When cells were treated with necrostatin-1, an inhibitor of receptor-interacting serine/threonine kinase 1 (RIPK1) required for necroptosis, 24S-OHC-induced cell death was significantly suppressed. The knockdown of RIPK1 by transfection of small interfering RNA of RIPK1 effectively attenuated 24S-OHC-induced cell death. It was found that neither SH-SY5Y cells nor primary cortical neuronal cells expressed caspase-8, which was regulated for RIPK1-dependent apoptosis. Collectively, these results suggest that 24S-OHC induces neuronal cell death by necroptosis, a form of programmed necrosis.     

Disentangling the paradox of insect phenology: are temporal trends reflecting the response to warming?

The strength and direction of phenological responses to changes in climate have been shown to vary significantly both among species and among populations of a species, with the overall patterns not fully resolved. Here, we studied the temporal and spatial variability associated with the response of several insect species to recent global warming. We use hierarchical models within a model comparison framework to analyze phenological data gathered over 40 years by the Japan Meteorological Agency on the emergence dates of 14 insect species at sites across Japan. Contrary to what has been predicted with global warming, temporal trends of annual emergence showed a later emergence day for some species and sites over time, even though temperatures are warming. However, when emergence data were analyzed as a function of temperature and precipitation, the overall response pointed out an earlier emergence day with warmer conditions. The apparent contradiction between the response to temperature and trends over time indicates that other factors, such as declining populations, may be affecting the date phenological events are being recorded. Overall, the responses by insects were weaker than those found for plants in previous work over the same time period in these ecosystems, suggesting the potential for ecological mismatches with deleterious effects for both suites of species. And although temperature may be the major driver of species phenology, we should be cautious when analyzing phenological datasets as many other factors may also be contributing to the variability in phenology.     

Anchorage-independent growth of mouse male germline stem cells in vitro

Spermatogenesis originates from a small number of spermatogonial stem cells that reside on the basement membrane and undergo self-renewal division to support spermatogenesis throughout the life of adult animals. Although the recent development of a technique to culture spermatogonial stem cells allowed reproduction of self-renewal division in vitro, much remains unknown about how spermatogonial stem cells are regulated. In this study, we found that spermatogonial stem cells could be cultured in an anchorage-independent manner, which is characteristic of stem cells from other types of self-renewing tissues. Although the cultured cells grew slowly (doubling time, approximately 4.7 days), they expressed markers of spermatogonia, and grew exponentially for at least 5 months to achieve 1.5 x 10(10) -fold expansion. The cultured cells underwent spermatogenesis following transplantation into the seminiferous tubules of infertile animals and fertile offspring were obtained by microinsemination of germ cells that had developed within the testes of recipients of the cultured cells. These results indicate that spermatogonial stem cells can undergo anchorage-independent, self-renewal division, and suggest that stem cells have the common property to survive and proliferate in the absence of exogenous substrata.     

The pH jump study of enzyme proteins. I. Liquefying alpha-amylase from Bacillus subtilis.

Rapid conformational changes due to pH jump were studied kinetically at 25 degrees mainly by the stopped-flow method using liquefying alpha-amylase from Bacillus subtilis [EC 3.2.1-.1, liquefying]. First, the conformational change due to a pH jump produced by mixing with alkali was monitored as a function of time at 245 nm through the ionization of phenolic hydroxyl groups of tyrosine residues which were originally buried and finally become exposed due to the pH jump. Three distinct phases of conformational change were clearly recognized by this method by varying the final pH values. Each phase involved the exposure of an essentially definite number of tyrosine residues, whose rate constant was crucially dependent on pH. Second, these phases of conformational change were subjected to examination in terms of the optical rotation change at 411 nm and the reversibility upon reverse pH jump with respect to conformational reconstitution, as observed through the protonation ofphenolic hydroxyl groups of ionized tyrosine residues and the enzyme activity. The first phase, which occurs above pH 12.5, involves no change in the optical rotation and is reversible as observed by the above two monitoring methods. In contrast, the other two phases, which are observed above pH 12.7, are accompanied by an optical rotation change and no appreciable reversibility was detected by these methods.     

Superoxide production at phagosomal cup/phagosome through beta I protein kinase C during Fc gamma R-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcgammaR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. betaI PKC, epsilonPKC, and diacylglycerol kinase (DGK) beta dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47(phox), an essential cytosolic component of NADPH oxidase and a substrate for betaI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O(2)(-)) production was profoundly inhibited by G?6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O(2)(-) production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of betaI PKC is involved in O(2)(-) production, and that O(2)(-) production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKbeta plays a prominent role in regulation of O(2)(-) production during FcgammaR-mediated phagocytosis.     

mTORC1 serves ER stress-triggered apoptosis via selective activation of the IRE1-JNK pathway

Mammalian target of rapamycin (mTOR) has a key role in the regulation of an array of cellular function. We found that rapamycin, an inhibitor of mTOR complex 1 (mTORC1), attenuated endoplasmic reticulum (ER) stress-induced apoptosis. Among three major branches of the unfolded protein response, rapamycin selectively suppressed the IRE1-JNK signaling without affecting PERK and ATF6 pathways. ER stress rapidly induced activation of mTORC1, which was responsible for induction of the IRE1-JNK pathway and apoptosis. Activation of mTORC1 reduced Akt phosphorylation, which was an event upstream of IRE-JNK signaling and consequent apoptosis. In vivo, administration with rapamycin significantly suppressed renal tubular injury and apoptosis in tunicamycin-treated mice. It was associated with enhanced phosphorylation of Akt and suppression of JNK activity in the kidney. These results disclosed that, under ER stress conditions, mTORC1 causes apoptosis through suppression of Akt and consequent induction of the IRE1-JNK pathway.     

Discovery of heteroaryl sulfonamides as new EP1 receptor selective antagonists

4-({2-[Isobutyl(phenylsulfonyl)amino]-5-(trifluoromethyl)phenoxy}methyl)benzoic acid (1) is a functional PGE2 antagonist selective for EP1 receptor subtype. Analogs of 1, in which the phenyl-sulfonyl moiety has been replaced with more hydrophilic heteroarylsulfonyl moieties, exhibited more optimized antagonist activity, while some of them showed in vivo antagonist activity. Structure-activity relationship (SAR) studies are also presented.     

Trade‐off relationship between productivity and thallus toughness in Laminariales (Phaeophyceae)

Trade‐off relationships are considered key to understanding the mechanisms supporting the coexistence of multiple species within kelp beds. Thus, information on trade‐offs is expected to contribute to conservation of kelp bed diversity. To test the existence of a trade‐off between productivity and thallus toughness, thallus traits and relationships between the traits were examined for seven species of Laminariales including 24 populations. For each population, photosynthetic capacity per unit biomass (as A _mass) and nitrogen (i.e., photosynthetic nitrogen‐use efficiency, PNUE), nitrogen content (as N _mass), thallus mass per unit thallus area (as TMA) and force required to penetrate the thallus (as F _p, a common index of leaf toughness in land plants by punch test) were determined. A _mass increased with increasing N _mass. Blades with high N _mass showed high A _mass. These blades may invest a large proportion of nitrogen to the photosynthetic parts, and consequently exhibit high metabolic rates. Moreover, blades with high N _mass tended to be associated with low TMA, and N _mass decreased with increasing TMA. A significant negative correlation was observed between TMA and A _mass because of the linkage of high A _mass with high N _mass and high N _mass associated with low TMA, while a significant positive correlation was observed between TMA and F _p. The two correlations indicate the existence of a trade‐off between productivity and thallus toughness in Laminariales. PNUE showed a significant negative correlation with TMA, which also showed a significant positive correlation with F _p as the index of thallus toughness, and therefore a trade‐off relationship between productivity and thallus toughness.     

Involvement of the Type IX Secretion System in Capnocytophaga ochracea Gliding Motility and Biofilm Formation

Capnocytophaga ochracea is a Gram-negative, rod-shaped bacterium that demonstrates gliding motility when cultured on solid agar surfaces. C. ochracea possesses the ability to form biofilms; however, factors involved in biofilm formation by this bacterium are unclear. A type IX secretion system (T9SS) in Flavobacterium johnsoniae was shown to be involved in the transport of proteins (e.g., several adhesins) to the cell surface. Genes orthologous to those encoding T9SS proteins in F. johnsoniae have been identified in the genome of C. ochracea; therefore, the T9SS may be involved in biofilm formation by C. ochracea. Here we constructed three ortholog-deficient C. ochracea mutants lacking sprB (which encodes a gliding motility adhesin) or gldK or sprT (which encode T9SS proteins in F. johnsoniae). Gliding motility was lost in each mutant, suggesting that, in C. ochracea, the proteins encoded by sprB, gldK, and sprT are necessary for gliding motility, and SprB is transported to the cell surface by the T9SS. For the ΔgldK, ΔsprT, and ΔsprB strains, the amounts of crystal violet-associated biofilm, relative to wild-type values, were 49%, 34%, and 65%, respectively, at 48 h. Confocal laser scanning and scanning electron microscopy revealed that the biofilms formed by wild-type C. ochracea were denser and bacterial cells were closer together than in those formed by the mutant strains. Together, these results indicate that proteins exported by the T9SS are key elements of the gliding motility and biofilm formation of C. ochracea.     

Biodegradation of bisphenol A by cultured cells of Caragana chamlagu

The biological degradation of 2,2-bis(4-hydroxyphenol)propane (1; bisphenol A, BPA), a representative endocrine disruptor, was studied with plant-cultured cells of Caragana chamlagu. An initial BPA concentration of 425 microM in an aqueous solution was degraded by C. chamlagu at 25 degrees C for 2 days in the dark, and two intermediates were then completely dissipated after 10 days.