Ancient Mitochondrial DNA Reveals the Origin of Sus scrofa from Rebun Island, Japan

The Kabukai A site (5 to 8C A.D.) of the Okhotsk cultural area is on Rebun Island, a small island near the coast, north–northwest of Hokkaido, Japan. Specimens of Sus scrofa, called the Sakhalin pig, were discovered in five cultural layers at the Kabukai A site. Ancient DNA was extracted from the remains of 42 Sakhalin pig bones. Thirty-nine nucleotide sequences of the 574-bp mitochondrial DNA control region, estimated to have originated from at least 21 individuals, were amplified and analyzed phylogenetically. Nine distinct haplotypes (A1, A2, A3, B1, B2, C1, C2, D1, and D2) from this site were classified into four haplotype groups (A, B, C, and D) by parsimonious network analysis. Phylogenetic analysis of 9 ancient and 55 modern haplotypes indicated that the population of Sakhalin pigs at the Kabukai A site belonged to two distinct clusters; haplotype groups A and B formed a cluster comprised only of themselves, and haplotype groups C and D belonged to the cluster of one of the two genetic groups of Japanese wild boars uniquely distributed in the western part of Japan, including one northeast Mongolian wild boar. Analysis of the haplotype distribution among three archaeological sites and their historical transitions among the five layers reflecting the cultural periods at the Kabukai A site suggests that the Sakhalin pig populations were introduced from Sakhalin island and the Amur River basin in the northeastern Eurasian continent together with some cultural influences. Received: 18 April 2000 / Accepted: 24 November 2000     

Cell-penetrating peptide-conjugated lipid nanoparticles for siRNA delivery

Lipid nanoparticles (LNP) modified with cell-penetrating peptides (CPP) were prepared for the delivery of small interfering RNA (siRNA) into cells. Lipid derivatives of CPP derived from protamine were newly synthesized and used to prepare CPP-decorated LNP (CPP-LNP). Encapsulation of siRNA into CPP-LNP improved the stability of the siRNA in serum. Fluorescence-labeled siRNA formulated in CPP-LNP was efficiently internalized into B16F10 murine melanoma cells in a time-dependent manner, although that in LNP without CPP was hardly internalized into these cells. In cells transfected with siRNA in CPP-LNP, most of the siRNA was distributed in the cytoplasm of these cells and did not localize in the lysosomes. Analysis of the endocytotic pathway indicated that CPP-LNP were mainly internalized via macropinocytosis and heparan sulfate-mediated endocytosis. CPP-LNP encapsulating siRNA effectively induced RNA interference-mediated silencing of reporter genes in B16F10 cells expressing luciferase and in HT1080 human fibrosarcoma cells expressing enhanced green fluorescent protein. These data suggest that modification of LNP with the protamine-derived CPP was effective to facilitate internalization of siRNA in the cytoplasm and thereby to enhance gene silencing.     

Prerelease exposure to methyl eugenol increases the mating competitiveness of sterile males of the oriental fruit fly (Diptera: Tephritidae) in a Hawaiian orchard

Males of the oriental fruit fly, Bactrocera dorsalis (Hendel), are strongly attracted to methyl eugenol (ME), and recent work demonstrated that ingestion of this chemical enhances male mating success, apparently owing its role as a precursor in the synthesis of the male sex pheromone. The current study expanded upon earlier laboratory and field-cage experiments by assessing whether prerelease exposure to ME increased the mating competitiveness of mass-reared, sterile males in Hawaiian orchards. Releases of sterile males from a pupal color-based sexing strain were made weekly in two fruit orchards over 8 mo, with the sterile males at one site given ME for 24 h before release (treated) and the sterile males at the other site given no ME before release (control). Fruits were collected periodically during the study period, and eggs were dissected and incubated to score hatch rate. At both sites, releases of sterile males increased the proportion of unhatched eggs well above prerelease levels, but the incidence of egg sterility was consistently, and statistically, greater in the orchard receiving ME-exposed males. Computed over the entire release period, the average value of Fried's competitive index (that characterizes the mating success of sterile males relative to their wild counterparts) for ME-treated males was 3.5 times greater than that for control males, although this difference was not statistically significant. However, when computed over the period during which egg sterility values were elevated and stable, presumably when females inseminated before the releases were rare or absent, the competitive indices were significantly higher for ME-treated sterile males. The implications of these results for implementing the Sterile Insect Technique against this species are discussed.     

Phosphorylation of a Protein (pp56) is Related to the Regeneration of Rice Cultured Suspension Cells

Short-term cultured suspension cells of rice (Oryza sativa L.)are capable of regeneration, but not in long-term culture. Forclarification of the mechanism of regeneration, protein phosphorylationin short-term and long-term cultured suspension cells was comparedby two dimensional- polyacrylamide gel electrophoresis. A 56kDa protein having an isoelectric point of 4.5 was phosphorylatedin vitro in short-term cultured suspension cells, but was notphosphorylated after regeneration. This protein in longtermcultured suspension cells remained phosphorylated after transferto the regeneration medium. However, using an antibody raisedagainst this protein from short-term cultured suspension cells,it was always detected in long-term and short-term culturedsuspension cells after transfer to the regeneration medium.The partial amino acid sequence of the HPLC-purified proteinshowed homology to a calcium-binding protein from maize. Thephosphorylation of the 56 kDa protein (pp56) appears to be associatedwith the regeneration of cultured rice cells. (Received December 11, 1995; Accepted June 3, 1996)     

Cold stress changes the concanavalin A-positive glycosylation pattern of proteins expressed in the basal parts of rice leaf sheaths

Post-translational modifications such as glycosylation are important for changing the properties and functions of proteins. To analyze the importance of glycosylation during cold stress in rice, a proteomics approach was used. Proteins extracted from the basal part of rice leaf sheaths were separated by two-dimensional polyacrylamide gel electrophoresis, and subjected to lectin blot analysis using concanavalin A. From a total of 250 detected proteins, 22 reacted with the lectin, suggesting that they were N-glycosylated proteins. To determine how N-glycosylation of these proteins is affected by cold stress, rice seedlings were incubated at 5°C for 48 h, and proteins extracted from the basal parts of leaf sheaths were analyzed by the lectin blot assay. Cold stress changed the reactivity toward the lectin for 12 of the 22 glycoproteins. The identity of the 12 proteins was determined by protein sequencing and mass spectrometry with the majority of these glycoproteins being categorized as involved in energy production. Furthermore, calreticulin, one of the 12 glycoproteins, was also phosphorylated as a result of cold stress. These results indicate that cold stress of the basal parts of rice leaf sheaths changes the glycosylation and phosphorylation profiles of calreticulin, a key protein that regulates the quality control of other proteins.     

Synthesis and Bioactivity of Novel Acetylenic Compounds

The synthesis of novel acetylenic ketone compounds and anti-inflammatory and antimicrobial activities are herein described.