CLAC: a novel Alzheimer amyloid plaque component derived from a transmembrane precursor, CLAC-P/collagen type XXV

We raised monoclonal antibodies against senile plaque (SP) amyloid and obtained a clone 9D2, which labeled amyloid fibrils in SPs and reacted with approximately 50/100 kDa polypeptides in Alzheimer's disease (AD) brains. We purified the 9D2 antigens and cloned a cDNA encoding its precursor, which was a novel type II transmembrane protein specifically expressed in neurons. This precursor harbored three collagen-like Gly-X-Y repeat motifs and was partially homologous to collagen type XIII. Thus, we named the 9D2 antigen as CLAC (collagen-like Alzheimer amyloid plaque component), and its precursor as CLAC-P/collagen type XXV. The extracellular domain of CLAC-P/collagen type XXV was secreted by furin convertase, and the N-terminus of CLAC deposited in AD brains was pyroglutamate modified. Both secreted and membrane-tethered forms of CLAC-P/collagen type XXV specifically bound to fibrillized Abeta, implicating these proteins in beta-amyloidogenesis and neuronal degeneration in AD.     

The expression of iron homeostasis-related genes during rice germination

To characterize Fe homeostasis during the early stages of seed germination, a microarray analysis was performed. mRNAs extracted from fully mature seeds or seeds harvested 1–3 days after sowing were hybridized to a rice microarray containing approximately 22,000 cDNA oligo probes. Many Fe deficiency-inducible genes were strongly expressed throughout early seed germination. These results suggest that the demand for Fe is extremely high during germination. Under Fe-deficient conditions, rice produces and secretes a metal-cation chelator called deoxymugineic acid (DMA) to acquire Fe from the soil. In addition, DMA and its intermediate nicotianamine (NA) are thought to be involved in long distance Fe transport in rice. Using promoter-β-glucuronidase (GUS) analysis, we investigated the expression patterns during seed germination of the Fe deficiency-inducible genes OsNAS1, OsNAS2, OsNAS3, OsNAAT1, and OsDMAS1, which encode enzymes that participate in the biosynthesis of DMA, and the transporter genes OsYSL2 and OsIRT1, which are involved in Fe transport. All of these genes were expressed in germinating seeds prior to protrusion of the radicle. These results suggest that DMA and NA are produced and involved in Fe transport during germination. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Necrosis-Induced Sterile Inflammation Mediated by Interleukin-1α in Retinal Pigment Epithelial Cells

Endogenous danger signals released from necrotic cells contribute to retinal inflammation. We have now investigated the effects of necrotic cell extracts prepared from ARPE-19 human retinal pigment epithelial cells (ANCE) on the release of proinflammatory cytokines and chemokines by healthy ARPE-19 cells. ANCE were prepared by subjection of ARPE-19 cells to freeze-thaw cycles. The release of various cytokines and chemokines from ARPE-19 cells was measured with a multiplex assay system or enzyme-linked immunosorbent assays. The expression of interleukin (IL)–1α and the phosphorylation and degradation of the endogenous nuclear factor–κB (NF-κB) inhibitor IκB-α were examined by immunoblot analysis. Among the various cytokines and chemokines examined, we found that ANCE markedly stimulated the release of the proinflammatory cytokine IL-6 and the chemokines IL-8 and monocyte chemoattractant protein (MCP)–1 by ARPE-19 cells. ANCE-induced IL-6, IL-8, and MCP-1 release was inhibited by IL-1 receptor antagonist and by an IKK2 inhibitor (a blocker of NF-κB signaling) in a concentration-dependent manner, but was not affected by a pan-caspase inhibitor (Z-VAD-FMK). Recombinant IL-1α also induced the secretion of IL-6, IL-8, and MCP-1 from ARPE-19 cells, and IL-1α was detected in ANCE. Furthermore, ANCE induced the phosphorylation and degradation of IκB-α in ARPE-19 cells. Our findings thus suggest that IL-1α is an important danger signal that is released from necrotic retinal pigment epithelial cells and triggers proinflammatory cytokine and chemokine secretion from intact cells in a manner dependent on NF-κB signaling. IL-1α is therefore a potential therapeutic target for amelioration of sterile inflammation in the retina.     

(-)-Epigallocatechin-3-gallate suppresses growth of AZ521 human gastric cancer cells by targeting the DEAD-box RNA helicase p68

(-)-Epigallocatechin-3-gallate (EGCG), the most abundant and biologically active polyphenol in green tea, induces apoptosis and suppresses proliferation of cancer cells by modulating multiple signal transduction pathways. However, the fundamental mechanisms responsible for these cancer-preventive effects have not been clearly elucidated. Recently, we found that EGCG can covalently bind to cysteine residues in proteins through autoxidation and subsequently modulate protein function. In this study, we demonstrate the direct binding of EGCG to cellular proteins in AZ521 human gastric cancer cells by redox-cycle staining. We comprehensively explored the binding targets of EGCG from EGCG-treated AZ521 cells by proteomics techniques combined with the boronate-affinity pull-down method. The DEAD-box RNA helicase p68, which is overexpressed in a variety of tumor cells and plays an important role in cancer development and progression, was identified as a novel EGCG-binding target. Exposure of AZ521 cells to EGCG lowered the p68 level dose dependently. The present findings show that EGCG inhibits AZ521 cell proliferation by preventing β-catenin oncogenic signaling through proteasomal degradation of p68 and provide a new perspective on the molecular mechanism of EGCG action.     

On the constancy of the evolutionary rate of cistrons

Summary The variations of evolutionary rates in hemoglobins and cytochrome c among various lines of vertebrates are analysed by estimating the variance. The observed variances appear to be larger than expected purely by chance.If the amino acid substitutions in evolution are the result of random fixation of selectively neutral or nearly neutral mutations, the evolutionary rate of cistrons can be represented by the integral of the product of mutation rate and fixation probability in terms of selective values around the neutral point. This integral is called the effective neutral mutation rate.The influence of effective population number and generation time on the effective neutral mutation rate is discussed. It is concluded that the uniformity of the rate of amino acid substitutions over diverse lines is compatible with random fixation of neutral or very slightly deleterious mutations which have some chance of being selected against during the course of substitution. On the other hand, definitely advantageous mutations will introduce significant variation in the substitution rate among lines. Approximately 10% of the amino acid substitutions of average cistrons might be adaptive and create slight but significant variations in evolutionary rate among vertebrate lines, although the uniformity of evolutionary rate is still valid as a first approximation.Contribution No. 813 from the National Institute of Genetics, Mishima, Shizuokaken 411 Japan. Aided in part by a grant-in-aid from the Ministry of Education, Japan.     

Analysis of outer membrane vesicle protein involved in biofilm formation of Helicobacter pylori

Helicobacter pylori is one of the most common causes of bacterial infection in humans. Infection with H. pylori is closely associated with gastritis and peptic ulcers and is a risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori forms biofilms on glass surfaces at the air–liquid interface in in-vitro batch cultures. We previously reported that strain TK1402 showed a strong biofilm-forming ability in vitro. We also suggested the outer membrane vesicles (OMV) produced by strain TK1402 might be related to its biofilm forming ability. In the present study, we analyzed the protein profile of the OMV produced by strain TK1402 and found a unique 22-kDa protein in TK1402 OMV cultured for 2–3 days. In addition, this protein could not be detected in the OMVs produced by other H. pylori strains. These results suggest that the 22-kDa protein is involved in effective biofilm formation by strain TK1402.     

Analysis of gamete membrane dynamics during double fertilization of Arabidopsis

Angiosperms have a unique sexual reproduction system called “double fertilization.” One sperm cell fertilizes the egg and another sperm cell fertilizes the central cell. To date, plant gamete membrane dynamics during fertilization has been poorly understood. To analyze this unrevealed gamete subcellular behavior, live cell imaging analyses of Arabidopsis double fertilization were performed. We produced female gamete membrane marker lines in which fluorescent proteins conjugated with PIP2a finely visualized egg cell and central cell surfaces. Using those lines together with a sperm cell membrane marker line expressing GCS1-GFP, the double fertilization process was observed. As a result, after gamete fusion, putative sperm plasma membrane GFP signals were occasionally detected on the egg cell surface adjacent to the central cell. In addition, time-lapse imaging revealed that GCS1-GFP signals entered both the egg cell and the central cell in parallel with the sperm cell movement toward the female gametes during double fertilization. These findings suggested that the gamete fusion process based on membrane dynamics was composed of (1) plasma membrane fusion on male and female gamete surfaces, (2) entry of sperm internal membrane components into the female gametes, and (3) plasmogamy.     

Larval dispersal dampens population fluctuation and shapes the interspecific spatial distribution patterns of rocky intertidal gastropods

Many marine benthic invertebrates pass through a planktonic larval stage whereas others spend their entire lifetimes in benthic habitats. Recent studies indicate that non‐planktonic species show relatively greater fine‐scale patchiness than do planktonic species, but the underlying mechanisms remain unknown. One hypothesis for such a difference is that larval dispersal enhances the connectivity of populations and buffers population fluctuations and reduces local extinction risk, consequently increasing patch occupancy rate and decreasing spatial patchiness. If this mechanism does indeed play a significant role, then the distribution of non‐planktonic species should be more aggregated – both temporally and spatially – than the distribution of species with a planktonic larval stage. To test this prediction, we compared 1) both the spatial and the temporal abundance–occupancy relationships and 2) both the spatial and the temporal mean–variance relationships of population size across species of rocky intertidal gastropods with differing dispersive traits from the Pacific coast of Japan. We found that, compared to planktonic species, non‐planktonic species exhibited 1) a smaller occupancy rate for any given level of mean population size and 2) greater variations in population size, both spatially and temporally. This suggests that the macroecological patterns observed in this study (i.e. the abundance–occupancy relationships and mean–variance relationships of population size across species) were shaped by the effect of larval dispersal dampening population fluctuation, which works over both space and time. While it has been widely assumed that larval dispersal enhances population fluctuations, larval dispersal may in fact enhance the connectively of populations and buffer population fluctuations and reduce local extinction risks.     

Embryonic Hematopoietic Progenitor Cells Reside in Muscle before Bone Marrow Hematopoiesis

In mice, hematopoietic cells home to bone marrow from fetal liver prenatally. To elucidate mechanisms underlying homing, we performed immunohistochemistry with the hematopoietic cell marker c-Kit, and observed c-Kit(+) cells localized inside muscle surrounding bone after 14.5 days post coitum. Flow cytometric analysis showed that CD45(+) c-Kit(+) hematopoietic cells were more abundant in muscle than in bone marrow between 14.5 and 17.5 days post coitum, peaking at 16.5 days post coitum. CD45(+) c-Kit(+) cells in muscle at 16.5 days post coitum exhibited higher expression of Gata2, among several hematopoietic genes, than did fetal liver or bone marrow cells. Colony formation assays revealed that muscle hematopoietic cells possess hematopoietic progenitor activity. Furthermore, exo utero transplantation revealed that fetal liver hematopoietic progenitor cells home to muscle and then to BM. Our findings demonstrate that hematopoietic progenitor cell homing occurs earlier than previously reported and that hematopoietic progenitor cells reside in muscle tissue before bone marrow hematopoiesis occurs during mouse embryogenesis.