Activation of sea urchin eggs by a channel-forming ionophore amphotericin B

Micromolar amounts of the channel-forming ionophore amphotericin B (AMB) can activate Hemicentrotus pulcherimus eggs. Except for protein synthesis, activation by AMB was fairly normal in artificial sea water. In the case of AMB activation, it was found that external Na⁺ is necessary to initiate the activation process. External Ca²⁺, however, is not necessarily required—other than for complete fertilization membrane formation. These results suggest that AMB may induce the release of Ca²⁺ required for activation from internal calcium stock, as has been reported previously by many authors, using A 23187.     

NLRP3 Mediates NF-κB Activation and Cytokine Induction in Microbially Induced and Sterile Inflammation

Nucleotide-binding domain and leucine-rich repeat-containing family, pyrin domain containing 3 (NLRP3) has recently emerged as a central regulator of innate immunity and inflammation in response to both sterile inflammatory and microbial invasion signals. Although its ability to drive proteolytic procaspase-1 processing has drawn more attention, NLPR3 can also activate NF-κB. To clarify the physiological relevance of this latter function, we examined the effect of NLRP3 on NF-κB activation and cytokine induction in RNA-interference-based NLRP3-knockdown cell lines generated from the human monocytic cell line THP-1. Knocking down NLRP3 reduced NF-κB activation and cytokine induction in the early stages of Staphylococcus aureus infection. Expression of cytokine genes induced by Staphylococcus aureus was not inhibited by a caspase-1 inhibitor, and did not occur through an autocrine mechanism in response to newly synthesized cytokines. We also demonstrated that NLRP3 could activate NF-κB and induce cytokines in response to sterile signals, monosodium urate crystals and aluminum adjuvant. Thus, NLRP3 mediates NF-κB activation in both sterile and microbially induced inflammation. Our findings show that not only does NLRP3 activate caspase-1 post-translationally, but it also induces multiple cytokine genes in the innate immune system.     

Warming off southwestern Japan linked to distributional shifts of subtidal canopy‐forming seaweeds

To assess distributional shifts of species in response to recent warming, historical distribution records are the most requisite information. The surface seawater temperature (SST) of Kochi Prefecture, southwestern Japan on the western North Pacific, has significantly risen, being warmed by the Kuroshio Current. Past distributional records of subtidal canopy‐forming seaweeds (Laminariales and Fucales) exist at about 10‐year intervals from the 1970s, along with detailed SST datasets at several sites along Kochi's >700 km coastline. In order to provide a clear picture of distributional shifts of coastal marine organisms in response to warming SST, we observed the present distribution of seaweeds and analyzed the SST datasets to estimate spatiotemporal SST trends in this coastal region. We present a large increase of 0.3°C/decade in the annual mean SST of this area over the past 40 years. Furthermore, a comparison of the previous and present distributions clearly showed the contraction of temperate species' distributional ranges and expansion of tropical species' distributional ranges in the seaweeds. Although the main temperate kelp Ecklonia (Laminariales) had expanded their distribution during periods of cooler SST, they subsequently declined as the SST warmed. Notably, the warmest SST of the 1997–98 El Niño Southern Oscillation event was the most likely cause of a widespread destruction of the kelp populations; no recovery was found even in the present survey at the formerly habitable sites where warm SSTs have been maintained. Temperate Sargassum spp. (Fucales) that dominated widely in the 1970s also declined in accordance with recent warming SSTs. In contrast, the tropical species, S. ilicifolium, has gradually expanded its distribution to become the most conspicuously dominant among the present observations. Thermal gradients, mainly driven by the warming Kuroshio Current, are presented as an explanation for the successive changes in both temperate and tropical species' distributions.     

Enzymatic Lysis of Living Candida Cells in the Presence of Chemical Agents and Antibiotics

The enzyme preparation from Streptomyces No. 8 displayed β-1-3-glucanase, chitinase and protease activities, and attacked heat-treated cells of Candida albicans. The preparation, however, attacked native cells of the same strain and other Candida strains when supplemented with appropriate concentration of sodium dodecyl sulfate or variotin. Neither of the agents attacked native cells when used alone. Usefulness of the combinational use of cell wall lytic enzymes and antibiotics for therapeutic or preventative purpose is discussed.     

Fatty acid 2-hydroxylase regulates cAMP-induced cell cycle exit in D6P2T Schwannoma cells

Sphingolipids are ubiquitous components of eukaryotic cells that regulate various cellular functions. In many cell types, a fraction of sphingolipids contain 2-hydroxy fatty acids, produced by fatty acid 2-hydroxylase (FA2H), as the N-acyl chain of ceramide [hydroxyl fatty acid (hFA)-sphingolipids]. FA2H is highly expressed in myelin-forming cells of the nervous system and in epidermal keratinocytes. While hFA-sphingolipids are thought to enhance the physical stability of specialized membranes produced by these cells, physiological significance of hFA-sphingolipids in many other cell types is unknown. In this study, we report novel roles for FA2H and hFA-sphingolipids in the regulation of the cell cycle. Treatment of D6P2T Schwannoma cells with dibutyryl-cAMP (db-cAMP) induced exit from the cell cycle with concomitant upregulation of FA2H. Partial silencing of FA2H in D6P2T cells resulted in 60–70% reduction of hFA-dihydroceramide and hFA-ceramide, with no effect on nonhydroxy dihydroceramide and ceramide. Under these conditions, db-cAMP no longer induced cell cycle exit, and cells continued to grow and divide. Immunoblot analyses revealed that FA2H silencing prevented db-cAMP-induced upregulation of cyclin-dependent kinase inhibitors p21 and p27. These results provide evidence that FA2H is a negative regulator of the cell cycle and facilitates db-cAMP-induced cell cycle exit in D6P2T cells.     

Effects of deafening on the temporal pattern of vocalizations in the budgerigarMelopsittacus undulatus

The physical characteristics of vocalization were examined in budgerigars (Melopsittacus undulatus) deafened at the age of about 28 days. Autocorrelation was used in the analysis, with 2 parameters: 1) inter-element intervals and 2) amplitudes of all sound elements of the vocalization. The deafened birds developed the temporal pattern specific to normal warble song in autocorrelograms. However, abnormalities were found in the frequency spectrum of elements. The temporal pattern of warble song might be an innate character, because auditory feedback is unnecessary for its development.     

Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase.