Comparison between the gelsolin and adseverin domain structure.

Adseverin (74-kDa protein, scinderin) is a calcium- and phospholipid-modulated actin-binding protein that promotes actin polymerization, severs actin filaments, and caps the barbed end of the actin filament, with its NH2-terminal half retaining these properties (Sakurai, T., Kurokawa, H., and Nonomura, Y. (1991) J. Biol. Chem. 266, 4581-4585). Further proteolysis of this NH2-terminal half generated five fragments, and two of them (Mr 15,000 and 31,000) showed Ca(2+)-dependent binding to monomeric actin. The Mr 31,000 fragment especially caused actin filament fragmentation, although its severing activity was also inhibited by several acidic phospholipids as was found in adseverin and its NH2-terminal half. Amino acid sequencing demonstrated that the two fragments' NH2 terminus were blocked in the same manner as the NH2 terminus of adseverin, and thus these two fragments are possibly located at the NH2-terminal of the adseverin molecule. This would then indicate that NH2-terminal fragments had a Ca(2+)-sensitive actin-binding function that relates to actin severing. The other two fragments' NH2-terminal sequencing showed a similar homology to the amino acid sequences of gelsolin and villin. Based on these observations, we propose that adseverin has a functional domain structure similar to that of the gelsolin and villin core.     

Effects of gelsolin on human platelet cytosolic phosphoinositide-phospholipase C isozymes.

The effective resolution of human platelet cytosolic phosphoinositide-phospholipase C (PLC) revealed five distinct activity peaks by Q-Sepharose and heparin-Sepharose column chromatographies when assayed using phosphatidylinositol (PI) and phosphatidylinositol 4,5-bisphosphate (PIP2). The results of Western blotting analysis with various antibodies against PLC isozymes showed that peak-Ia (PLC-delta type), peak-Ib (PLC-gamma 1 type), and peak-IIc (PLC-beta type) and two unidentified activity peaks (PLC-IIa and PLC-IIb) were present in human platelet cytosol. A protein with guanosine 5'-3-O-(thio)triphosphate-binding activity was coeluted with the PLC-IIa and was purified to homogeneity. It exhibited 86- and 42-kDa polypeptide bands upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis which were identified as gelsolin and actin by immunostaining, respectively. Large amounts of gelsolin/actin (1:1) complex "gelsolin complex" were detected in the PLC-delta and PLC-gamma 1 fractions. The PLC-gamma 1 and the gelsolin complex were co-immunoprecipitated by the antibody raised against PLC-gamma 1. Furthermore, the partially purified bovine brain PLC-gamma 1 fraction also was found to be associated with the gelsolin complex and the association was released by the addition of 1% sodium cholate. This finding has prompted us to examine effects of the gelsolin complex and the free gelsolin on activities of the above PLC isoforms from platelet cytosol. The gelsolin complex did not affect the PIP2 hydrolyzing activities of all PLC isoforms. In contrast, the purified gelsolin inhibited distinctly PIP2 hydrolyses by PLC-Ia (delta), PLC-Ib (gamma 1), and PLC-IIa (unidentified), whereas the inhibitory effects for PLC-IIb (unidentified) and PLC-IIc (beta) were moderate. The inhibitory effect of gelsolin on PIP2-hydrolysis by PLC-gamma 1 was diminished by a large amount of PIP2 substrate. These results suggested that the inhibition of PLC by gelsolin is due to sequestration of substrate PIP2 by its competitive binding.     

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

l-2-Amino-4-methylpimelic acid: A new amino acid from Lactarius species

A PC survey revealed the occurrence of an unusual amino acid in fruit bodies of the mushroom Lactarius quietus Fr. and a related species. This was identified as l-2-amino-4-methylpimelic acid, which has not been previously reported as a naturally occurring compound.     

KIT (c-kit oncogene product) pathway is constitutively activated in human testicular germ cell tumors

We investigated the expression of KIT (product of c-kit oncogene), gain-of-function mutations, and activation of its downstream signal transduction in human testicular cancers. KIT was expressed in 88% (22/25) of seminomas and in 44.4% (4/9) of non-seminomas compared to adjacent normal testicular tissue. Nine of the KIT-expressing seminomas had mutations (40.9%; 9/22) in the c-kit gene; two cases in exon 11 and 7 cases in exon 17. Two of these mutations in exon 17 were novel, and the other seven mutations were identical to the already known gain-of-function mutations which cause activation of KIT without ligand stem cell factor. All of the mutant KIT and 53.8% (7/13) of wild-type KIT were phosphorylated (activated) and associated with phosphorylated phosphatidylinositol 3-kinase (PI3K). Akt was also phosphorylated in these seminomas, suggesting that the KIT-PI3K-Akt pathway is activated in seminoma. These findings suggest that the KIT-PI3K-Akt pathway is constitutively activated in testicular germ cell tumors, due to overexpression of KIT protein and/or gain-of-function mutations in the c-kit gene.     

Stable phylloplane colonization by entomopathogenic bacterium Pseudomonas fluorescens KPM-018P and biological control of Phytophagous ladybird beetles Epilachna vigintioctopunctata (Coleoptera: Coccinellidae)

An entomopathogenic bacterium was isolated from tomato leaves and used as a microbial agent to control larvae of phytophagous ladybird beetles Epilachna vigintioctopunctata. The isolate was identified as Pseudomonas fluorescens KPM-018P on the basis of its bacteriological characteristics. KPM-018P produced extracellular chitinase to form a transparent zone around their colonies by hydrolyzing chitin in a minimal medium. Pale-yellow colonies turned red after a change of incubation temperature. These characteristics were availed as markers for tracking KPM-018P. The bacteria produced biosurfactants that enabled the bacteria to stably colonize the hydrophobic leaf surface; they were recovered without any considerable decrease even after a suspension of KPM-018P was sprayed onto leaves. KPM-018P, transformed with the gfp gene and observed with fluorescence microscopy, stably dwelled in the junctions of epidermal cells of bacteria-sprayed leaves. Ingestion of KPM-018P-sprayed leaves by the larvae caused prompt death of these insects to eventually suppress their pupation. This method is thus effective for decreasing the population of larvae and adult insect pests in the subsequent generation. The study provides an experimental basis for the biocontrol of herbivorous insect pests using a leaf-inhabiting, entomopathogenic strain of P. fluorescens.     

Time lag and negative responses of forest greenness and tree growth to warming over circumboreal forests

The terrestrial forest ecosystems in the northern high latitude region have been experiencing significant warming rates over several decades. These forests are considered crucial to the climate system and global carbon cycle and are particularly vulnerable to climate change. To obtain an improved estimate of the response of vegetation activity, e.g., forest greenness and tree growth, to climate change, we investigated spatiotemporal variations in two independent data sets containing the dendroecological information for this region over the past 30 years. These indices are the normalized difference vegetation index (NDVI3g) and the tree‐ring width index (RWI), both of which showed significant spatial variability in past trends and responses to climate changes. These trends and responses to climate change differed significantly in the ecosystems of the circumarctic (latitude higher than 67°N) and the circumboreal forests (latitude higher and lower than 50°N and 67°N, respectively), but the way in which they differed was relatively similar in the NDVI3g and the RWI. In the circumarctic ecosystem, the climate variables of the current summer were the main climatic drivers for the positive response to the increase in temperatures showed by both the NDVI3g and the RWI indices. On the other hand, in the circumboreal forest ecosystem, the climate variables of the previous year (from summer to winter) were also important climatic drivers for both the NDVI3g and the RWI. Importantly, both indices showed that the temperatures in the previous year negatively affected the ecosystem. Although such negative responses to warming did not necessarily lead to a past negative linear trend in the NDVI3g and the RWI over the past 30 years, future climate warming could potentially cause severe reduction in forest greenness and tree growth in the circumboreal forest ecosystem.     

cis‐Regulatory analysis for later phase of anterior neuroectoderm‐specific foxQ2 expression in sea urchin embryos

The specification of anterior neuroectoderm is controlled by a highly conserved molecular mechanism in bilaterians. A forkhead family gene, foxQ2, is known to be one of the pivotal regulators, which is zygotically expressed in this region during embryogenesis of a broad range of bilaterians. However, what controls the expression of this essential factor has remained unclear to date. To reveal the regulatory mechanism of foxQ2, we performed cis‐regulatory analysis of two foxQ2 genes, foxQ2a and foxQ2b, in a sea urchin Hemicentrotus pulcherrimus. In sea urchin embryos, foxQ2 is initially expressed in the entire animal hemisphere and subsequently shows narrower expression restricted to the anterior pole region. In this study, as a first step to understand the foxQ2 regulation, we focused on the later restricted expression and analyzed the upstream cis‐regulatory sequences of foxQ2a and foxQ2b by using the constructs fused to short half‐life green fluorescent protein. Based on deletion and mutation analyses of both foxQ2, we identified each of the five regulatory sequences, which were 4–9 bp long. Neither of the regulatory sequences contains any motifs for ectopic activation or spatial repression, suggesting that later mRNA localization is regulated in situ. We also suggest that the three amino acid loop extension‐class homeobox gene Meis is involved in the maintenance of foxQ2b, the expression of which is dominant during embryogenesis.