Amplification of epidermal growth factor receptor (EGFR) gene and oncogenes in human gastric carcinomas

DNAs from 37 human gastric carcinomas and seven lymph node metastases were analyzed for alterations of the epidermal growth factor receptor (EGFR) gene and oncogenes by the Southern blot hybridization method. The probes used were EGFR gene, c-Ha-ras, v-Ki-ras, N-ras, c-myc, v-myb, v-fos, c-erbB-2, v-erbA, v-abl and v-fes. Amplification of the EGFR gene was detected in only one poorly differentiated adenocarcinoma. Amplifications of c-myc gene and c-erbB-2 gene were each observed in two well differentiated adenocarcinomas. One of these tumors had coamplification of c-erbB-2 and c-erbA genes but there were no amplifications nor rearrangements of other oncogenes. The poorly differentiated adenocarcinom with amplified EGFR gene also showed enhanced expression of EGFR gene by Northern blot analysis and additionally had strong synchronous immunoreactivity for EGFR and EGF. Supported in part by Grants-in Aid for Cancer Research from the Ministry of Education, Science and Culture of Japan and for Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan     

Normothermic Cardiac Arrest and Cardiopulmonary Resuscitation: A Mouse Model of Ischemia-Reperfusion Injury

Acute Kidney Injury (AKI) is a common, highly lethal, complication of critical illness which has a high mortality^1-4 and which is most frequently caused by whole-body hypoperfusion.^5,6 Successful reproduction of whole-body hypoperfusion in rodent models has been fraught with difficulty.^7-9,9,10 Models which employ focal ischemia have repeatedly demonstrated results which do not translate to the clinical setting, and larger animal models which allow for whole body hypoperfusion lack access to the full toolset of genetic manipulation possible in the mouse.^11,12 However, in recent years a mouse model of cardiac arrest and cardiopulmonary resuscitation has emerged which can be adapted to model AKI.¹³ This model reliably reproduces physiologic, functional, anatomic, and histologic outcomes seen in clinical AKI, is rapidly repeatable, and offers all of the significant advantages of a murine surgical model, including access to genetic manipulative techniques, low cost relative to large animals, and ease of use. Our group has developed extensive experience with use of this model to assess a number of organ-specific outcomes in AKI.^14,15     

Amyloplast-Localized SUBSTANDARD STARCH GRAIN4 Protein Influences the Size of Starch Grains in Rice Endosperm

Starch is a biologically and commercially important polymer of glucose and is synthesized to form starch grains (SGs) inside amyloplasts. Cereal endosperm accumulates starch to levels that are more than 90% of the total weight, and most of the intracellular space is occupied by SGs. The size of SGs differs depending on the plant species and is one of the most important factors for industrial applications of starch. However, the molecular machinery that regulates the size of SGs is unknown. In this study, we report a novel rice (Oryza sativa) mutant called substandard starch grain4 (ssg4) that develops enlarged SGs in the endosperm. Enlargement of SGs in ssg4 was also observed in other starch-accumulating tissues such as pollen grains, root caps, and young pericarps. The SSG4 gene was identified by map-based cloning. SSG4 encodes a protein that contains 2,135 amino acid residues and an amino-terminal amyloplast-targeted sequence. SSG4 contains a domain of unknown function490 that is conserved from bacteria to higher plants. Domain of unknown function490-containing proteins with lengths greater than 2,000 amino acid residues are predominant in photosynthetic organisms such as cyanobacteria and higher plants but are minor in proteobacteria. The results of this study suggest that SSG4 is a novel protein that influences the size of SGs. SSG4 will be a useful molecular tool for future starch breeding and biotechnology.Plastids originated from the endosymbiosis of cyanobacteria and can differentiate into several forms depending on their intracellular functions during the plant life cycle (Sakamoto et al., 2008). The amyloplast is a terminally differentiated plastid responsible for starch synthesis and storage. Starch forms insoluble particles in amyloplasts, referred to as starch grains (SGs). SGs are easily visualized by staining with iodine solution, and they can be observed using a light microscope. SGs are observed in storage organs such as seed endosperm, potato (Solanum tuberosum) tubers, and pollen grains. Nonstorage tissues such as endodermis and root caps also contain SGs (Morita, 2010).Cereal endosperm accumulates high levels of starch in amyloplasts. The volume of SGs is approximately the same as the volume of amyloplasts that fill most of the intracellular space. SGs in rice (Oryza sativa) endosperm are normally 10 to 20 μm in diameter (Matsushima et al., 2010). One amyloplast contains a single SG that is assembled of several dozen smaller starch granules. Each starch granule is a sharp-edged polyhedron with a typical diameter of 3 to 8 μm. This type of SG is called a compound SG (Tateoka, 1962). For compound SGs, starch granules are assembled (but not fused) to form a single SG, which is easily separated by conventional purification procedures. By contrast, simple SGs contain a single starch granule. Simple SGs are produced in several important crops, such as maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and wheat (Triticum aestivum; Tateoka, 1962; Matsushima et al., 2010, 2013).The size of SGs in cereal endosperm is diverse. Maize and sorghum SGs have a uniform size distribution of approximately 10 μm in diameter (Jane et al., 1994; Matsushima et al., 2010; Ai et al., 2011). In barley and wheat, SGs of two discrete size classes (approximately 15−25 μm and less than 10 μm) coexist in the same cells (Evers, 1973; French, 1984; Jane et al., 1994; Matsushima et al., 2010). In Bromus species, intrageneric size variations of SGs are observed in which even phylogenetic neighbors develop distinctly sized SGs (Matsushima et al., 2013). The size of SGs can be controlled by manipulating the activity of starch synthetic enzymes using transgenic plants or genetic mutants (Gutiérrez et al., 2002; Bustos et al., 2004; Ji et al., 2004; Stahl et al., 2004; Matsushima et al., 2010). However, the molecular mechanism that controls the interspecific size variations of SGs has not been resolved.The SG occupies most of the amyloplast interior, because the SG is approximately the same size as the amyloplast. The size of amyloplasts may affect the size of SGs, or vice versa. Amyloplasts and chloroplasts both develop from proplastids. The size of chloroplasts is controlled by the chloroplast binary fission division machinery, especially by the ring structures that form at the division sites (Miyagishima, 2011). Proteins involved in the ring structures have been isolated, including Filamenting temperature-sensitive mutantZ (FtsZ), Minicell locusD (MinD), MinE, and ACCUMULATION AND REPLICATIONS OF CHLOROPLAST5 (ARC5). Arabidopsis (Arabidopsis thaliana) mutants that are defective in these proteins have defects in chloroplast division and contain enlarged and dumbbell-shaped chloroplasts. In contrast to the binary fission of chloroplasts, amyloplasts divide at multiple sites and generate a beads-on-a-string structure (Yun and Kawagoe, 2009). The inhibition of the chloroplast division machinery does not result in enlarged amyloplasts (Yun and Kawagoe, 2009).We recently developed a rapid method to prepare thin sections of endosperm (Matsushima et al., 2010). Using this method, SGs in mature endosperm can be easily and clearly observed. We performed genetic screening for rice mutants defective in SG morphology and size. One of the isolated mutants, substandard starch grain4 (ssg4), develops enlarged SGs in its endosperm. In this study, we characterized ssg4 phenotypes and identified the responsible gene. SSG4 encodes a protein containing 2,135 amino acid residues and an N-terminal plastid-targeted sequence. The domain of unknown function 490 (DUF490) was found at the C terminus of SSG4, where the ssg4 mutation was located. This suggests that SSG4 is a novel factor that influences the size of SGs and has potential as a molecular tool for starch breeding and biotechnology.     

The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids

The well-characterized secretory glycoprotein, rice (Oryza sativa) α-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal–dependent manner.     

Escape from floral herbivory by early flowering in Arabidopsis halleri subsp. gemmifera

Natural selection on flowering phenology has been studied primarily in terms of plant–pollinator interactions and effects of abiotic conditions. Little is known, however, about geographic variation in other biotic factors such as herbivores and its consequence for differential selection on flowering phenology among populations. Here, we examine selection by floral herbivores on the flowering phenology of Arabidopsis halleri subsp. gemmifera using two adjacent populations with contrasting herbivory regimes. Intensive floral herbivory by the leaf beetle Phaedon brassicae occurs in one population, while the beetle is absent in another population. We tested the hypothesis that the two populations experience differential selection on flowering time that is attributable to the presence or absence of floral herbivory. A two-year field study showed that early flowering was favoured in the population under intensive floral herbivory, whereas selection for early flowering was not found in one year in the population where floral herbivory was absent. Selection for early flowering disappeared when the abundance of floral herbivores was artificially decreased in a field experiment. Thus, the heterogeneous distribution of P. brassicae was a major agent for differential selection on flowering time. However, flowering time did not differ between the two populations when plants were grown in the laboratory. The lack of genetic differentiation in flowering time may be explained by ongoing gene flow or recent invasion of P. brassicae. This study illustrates that the role of floral herbivory in shaping geographic variation in selection on flowering phenology may be more important than previously thought.     

Regulation of neuronal morphology by Toca-1, an F-BAR/EFC protein that induces plasma membrane invagination

Actin reorganization is important for regulation of neuronal morphology. Neural Wiskott-Aldrich syndrome protein (N-WASP) is an important regulator of actin polymerization and also known to be strongly expressed in brain. Recently, Toca-1 (transducer of Cdc42-dependent actin assembly) has been shown to be required for Cdc42 to activate N-WASP from biochemical experiments. Toca-1 has three functional domains: an F-BAR/EFC domain at the N terminus, an HR1 at the center, and an SH3 domain at the C terminus. The F-BAR/EFC domain induces tubular invagination of plasma membrane, while Toca-1 binds both N-WASP and Cdc42 through the SH3 domain and the HR1, respectively. However, the physiological role of Toca-1 is completely unknown. Here we have investigated the neural function of Toca-1. Toca-1 is strongly expressed in neurons including hippocampal neurons in developing brain at early times. Knockdown of Toca-1 in PC12 cells significantly enhances neurite elongation. Consistently, overexpression of Toca-1 suppresses neurite elongation through the F-BAR/EFC domain with a membrane invaginating property, suggesting an implication of membrane trafficking in the neural function of Toca-1. In addition, knockdown of N-WASP, to our surprise, also enhances neurite elongation in PC12 cells, which is in clear contrast to the previous report that dominant negative mutants of N-WASP suppress neurite extension in PC12 cells. On the other hand, knockdown of Toca-1 in cultured rat hippocampal neurons enhances axon branching a little but not axon elongation, while knockdown of N-WASP enhances both axon elongation and branching. These results suggest that a vesicle trafficking regulator Toca-1 regulates different aspects of neuronal morphology from N-WASP.     

A genome sequence-based approach to taxonomy of the genus Nocardia

The genus Nocardia includes both pathogens and producers of useful secondary metabolites. Although 16S rRNA analysis is required to accurately discriminate among phylogenetic relationships of the Nocardia species, most branches of 16S rRNA-based phylogenetic trees are not reliable. In this study, we performed in silico analyses of the genome sequences of Nocardia species in order to understand their diversity and classification for their identification and applications. Draft genome sequences of 26 Nocardia strains were determined. Phylogenetic trees were prepared on the basis of multilocus sequence analysis of the concatenated sequences of 12 genes (atpD-dnaJ-groL1-groL2-gyrB-recA-rpoA-secA-secY-sodA-trpB-ychF) and a bidirectional best hit. To elucidate the evolutionary relationships of these genes, the genome-to-genome distance was investigated on the basis of the average nucleotide identity, DNA maximal unique matches index, and genome-to-genome distance calculator. The topologies of all phylogenetic trees were found to be essentially similar to each other. Furthermore, whole genome-derived and multiple gene-derived relationships were found to be suitable for extensive intra-genus assessment of the genus Nocardia.