Pre‐dispersal seed predation of bayberry Myrica rubra by Thiotricha pancratiastis (Lepidoptera: Gelechiidae) on Yakushima Island,Japan

We investigated pre‐dispersal seed predation by insects in a bayberry Myrica rubra Sieb. et Zucc. (Myricaceae) on Yakushima Island, Japan. To clarify the patterns of seed fate and predation, all fruit that fell into seed traps were collected to allow any insect larvae within the fruit to emerge, and the fruit were finally dissected to determine whether or not they had been attacked by insect predators. Two lepidopteran species, Thiotricha pancratiastis (Meyrick) (Gelechiidae) and Neoblastobasis spiniharpella Kuznetzov & Sinev (Blastobasidae), emerged from the fruits. Thiotricha pancratiastis is the major seed predator of M. rubra, attacking the fruits intensively during the primary stage of fruit development. Thiotricha pancratiastis had been known as a foliage feeder (leaf miner) of M. rubra, but we revealed that the insect is also an important seed predator of the bayberry.     

Involvement of aberrant DNA methylation on reduced expression of lysophosphatidic acid receptor-1 gene in rat tumor cell lines

Lysophosphatidic acid (LPA) is a bioactive phospholipid that stimulates cell proliferation, migration, and protects cells from apoptosis. It interacts with specific G protein-coupled transmembrane receptors. Recently, it has been reported that alterations of LPA receptor expression might be important in the malignant transformation of tumor cells. Therefore, to assess an involvement of DNA methylation in reduced expression of the LPA receptor-1 (lpa1) gene, we investigated the expression of the lpa1 gene and its DNA methylation patterns in rat tumor cell lines. Both rat brain-derived neuroblastoma B103 and liver-derived hepatoma RH7777 cells used in this study indicated no expression of lpa1. For the analysis of methylation status, bisulfite sequencing was performed with B103 and RH7777 cells, comparing with other lpa1 expressed cells and normal tissues of brain and liver. The lpa1 expressed cells and tissues were all unmethylated in this region of lpa1. In contrast, both B103 and RH7777 cells were highly methylated, correlating with reduced expression of the lpa1. Treatment with 5-aza 2'-deoxycytidine induced expression of lpa1 gene in B103 and RH7777 cells after 24h. In RH7777 cells treated with 5-aza 2'-deoxycytidine, stress fiber formation was also observed in response to LPA in RH7777 cells, but not in untreated RH7777 cells. These results suggest that aberrant DNA methylation of the lpa1 gene may be involved in its reduced expression in rat tumor cells.     

Caenorhabditis elegans WASP-interacting protein homologue WIP-1 is involved in morphogenesis through maintenance of WSP-1 protein levels

Mammalian WASP and N-WASP are involved in reorganization of the actin cytoskeleton through activation of the Arp2/3 complex and in regulation of cell motility or cell shape changes. In the present study, we identified WASP-interacting protein homologue (WIP)-1 in Caenorhabditis elegans. WIP-1 contains the domains and sequences conserved among mammalian WIP family proteins. Yeast two-hybrid analysis detected a physical interaction between WIP-1 and WSP-1, the sole homologue of WASP/N-WASP in C. elegans. Western analysis of embryo lysates showed that RNA interference (RNAi) treatment for wip-1 decreased levels of WSP-1 protein, and wsp-1(RNAi) treatment decreased levels of WIP-1 protein. However, wsp-1 mRNA levels were not decreased in wip-1(RNAi)-treated embryos, and wip-1 mRNA levels were not decreased in wsp-1(RNAi)-treated embryos. Furthermore, disruption of WIP-1 by RNAi resulted in embryonic lethality with morphologic defects in hypodermal cell migration, a process known as ventral enclosure. This phenotype was similar to that observed in RNAi experiments for wsp-1. Immunostaining showed that WIP-1 was expressed by migrating hypodermal cells, as was WSP-1. This expression during ventral enclosure was reduced in wip-1(RNAi)-treated embryos and wsp-1(RNAi)-treated embryos. Our results suggest that C. elegans WIP-1 may function in hypodermal cell migration during ventral enclosure by maintaining levels of WSP-1.     

Ribosome Modulation Factor,an Important Protein for Cell Viability Encoded by the Polyamine Modulon

We searched for proteins whose synthesis is enhanced by polyamines at the stationary phase of cell growth using an Escherichia coli polyamine-requiring mutant in which cell viability is greatly decreased by polyamine deficiency. The synthesis of ribosome modulation factor (RMF) was strongly enhanced by polyamines at the level of translation at the stationary phase of cell growth. In rmf mRNA, a Shine-Dalgarno (SD) sequence is located 11 nucleotides upstream of the initiation codon AUG. When the SD sequence was moved to the more common position 8 nucleotides upstream of the initiation codon, the degree of polyamine stimulation was reduced, although the level of RMF synthesis was markedly increased. Polyamine stimulation of RMF synthesis was found to be caused by a selective structural change of the bulged-out region of the initiation site of rmf mRNA. The decrease in cell viability caused by polyamine deficiency was prevented by the addition of a modified rmf gene whose synthesis is not influenced by polyamines. The results indicate that polyamines enhance cell viability of E. coli at least in part by enhancing RMF synthesis.     

Immunomodulation of monocyte-derived dendritic cells through ligation of tumor-produced mucins to Siglec-9

Dendritic cells (DCs) play an essential role in the induction and maintenance of an effective immune response and express multiple siglecs. In the present study, we investigated whether or not the ligation of tumor-produced mucins with Siglec-9 expressed on immature DCs is related to escape from immunosurveillance in the tumor-bearing state.Expression of Siglec-9 was up-regulated on the development of monocytes into immature DCs and was decreased in mature DCs. Binding of various mucins and artificial glycopolymers carrying poly (NeuAc α2,6 LacNAc) or poly (NeuAc α2,3 LacNAc) to Siglec-9 was demonstrated by means of a plate assay. These mucins also bound to the surface of immature DCs. When immature DCs were treated with LPS in the presence of these mucins or artificial glycopolymers, the production of IL-12 was significantly reduced, but that of IL-10 was not. Furthermore, IL-12 production was decreased to a similar level on treatment with anti-Siglec-9 mAb. Mucins prepared from serum of cancer patients actually could bind to Siglec-9. These results suggest that Siglec-9 expressed on DCs is involved in immunoregulation through ligation with mucins in an epithelial cancer patient.     

Overexpressed proteins may act as mops removing their ligands from the host cells: A case study of calf PNP

Calf purine nucleoside phosphorylase (PNP) was overexpressed in Escherichia coli. The basic kinetic parameters of recombinant PNP were found to be similar to the values published previously for non-recombinant PNP from calf spleen. However, upon titration of the recombinant enzyme with the tight-binding multisubstrate analogue inhibitor DFPP-DG, endothermic as well as exothermic signals were obtained. This was not the case for PNP isolated from calf spleen for which only the endothermic process was observed. Further calorimetric titrations of the recombinant and non-recombinant enzyme with its potent and moderate ligands, and studied involving partial inactivation of the enzyme, lead to the conclusion that a part of the recombinant enzyme forms a complex with its product, hypoxanthine, although hypoxanthine was not present at any purification stage except for its natural occurrence in E. coli cells. Binding of hypoxanthine is accompanied with a large negative change of the free enthalpy, and therefore the replacement of this compound by DFPP-DG yields positive heat signal. Our data obtained with calf PNP indicate that similar processes - moping of ligands from the host cells - may take place in the case of other proteins with high overexpression yield.     

Development of simple sequence repeat (SSR) markers and construction of an SSR-based linkage map in Italian ryegrass (Lolium multiflorum Lam.)

In order to develop simple sequence repeat (SSR) markers in Italian ryegrass, we constructed a genomic library enriched for (CA)n-containing SSR repeats. A total of 1,544 clones were sequenced, of which 1,044 (67.6%) contained SSR motifs, and 395 unique clones were chosen for primer design. Three hundred and fifty-seven of these clones amplified products of the expected size in both parents of a two-way pseudo-testcross F₁ mapping population, and 260 primer pairs detected genetic polymorphism in the F₁ population. Genetic loci detected by a total of 218 primer pairs were assigned to locations on seven linkage groups, representing the seven chromosomes of the haploid Italian ryegrass karyotype. The SSR markers covered 887.8 cM of the female map and 795.8 cM of the male map. The average distance between two flanking SSR markers was 3.2 cM. The SSR markers developed in this study will be useful in cultivar discrimination, linkage analysis, and marker-assisted selection of Italian ryegrass and closely related species.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Cysteamine or beta-mercaptoethanol added to a defined maturation medium improves blastocyst formation of porcine oocytes after intracytoplasmic sperm injection

The present study was carried out to investigate the effect of adding 100 microM cysteamine (Cys) or 100 microM beta-mercaptoethanol (beta-ME) to a defined maturation medium on in vitro maturation (IVM), and fertilization and developmental competence of in vitro matured porcine oocytes following intracytoplasmic sperm injection (ICSI). The two control media for IVM culture were modified TCM199 containing 10% (v/v) porcine follicular fluid (pFF) or 0.05% (w/v) polyvinyl alcohol (PVA), and Cys or beta-ME was supplemented to the PVA-control medium. There was no significant difference in the proportions of in vitro matured oocytes among the four treatment groups (94.5-98.4%). The percentages of pronuclear formation (51.0-64.2%) after ICSI were also not significantly different among the four groups. The cleavage rate (72.8%) in the oocytes treated with Cys showed no significant difference compared with those of the two control media containing pFF (72.2%) or PVA (61.5%), but was higher (P<0.05) than that in the oocytes treated with beta-ME (56.3%). However, the rates of blastocyst formation of Cys (36.7%), beta-ME (27.1%) and pFF (31.4%) were higher (P<0.05) than that using the control medium containing PVA (15.6%). The mean cell number of blastocysts ranged from 42 to 52 among the four groups, without significant differences. In conclusion, the addition of Cys or beta-ME to a defined maturation medium enhanced blastocyst formation after ICSI, to a level similar to that achieved by adding pFF.