Molecular discrimination and phylogeographic patterns of clones of the parthenogenetic gecko Lepidodactylus lugubris in the Japanese Archipelago

Vertebrates usually reproduce sexually in which males and females contribute their offspring genome and produce genetically diverse offspring. However, some of them are asexual without genetic contribution from males. The nocturnal gecko, Lepidodactylus lugubris, is all females and reproduces parthenogenetically. This gecko is known to consist of diploid and triploid clones in the tropical and subtropical regions, which can be identified by their dorsal marking patterns, ploidy, and protein polymorphism. This gecko is also distributed in the southern parts of Japan, and several clones have been reported. In this study, we investigated the origins and genetic diversity of Japanese L. lugubris by clonal discrimination using microsatellite and mitochondrial DNA analyses. A total of 748 individuals were collected from 21 islands of five island groups (Ogasawara, Okinawa, Miyako, Yaeyama and Daito Islands) and 17 clones were distinguished genetically. Mitochondrial cyt b sequences of these clones suggested that they were all closely related and differentiated recently. Clonal diversity was much higher (14 clones) in the Daito Islands than in the other island groups in which only one or two clones coexisted. Judging from the dorsal marking patterns and ploidy known so far, six clones were cosmopolitan and may be colonized from the outside of Japan. However, other 11 clones were endemic to the Daito Islands and explained by possible hybridization between the one female diploid clone and one male diploid clone because other 9 clones were triploid and all had the combinations of polymorphic microsatellite alleles of these female and male diploid clones. Although the males have never been recorded in the Daito Islands, males might appear in the past. These findings contribute to understanding of clonal diversity and dynamics of asexually reproducing animals. If diploid parthenogenetic geckos can produce triploid clones by mating with the diploid males, clonal diversity would increase rapidly in a small island, and such newly produced triploid clones would expand widely.     

Stable integration and conditional expression of electroporated transgenes in chicken embryos

The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.     

Gadd45a, the gene induced by the mood stabilizer valproic acid, regulates neurite outgrowth through JNK and the substrate paxillin in N1E-115 neuroblastoma cells

Valproic acid (VPA), a mood stabilizer and anticonvulsant, has a variety of neurotrophic functions; however, less is known about how VPA regulates neurite outgrowth. Here, using N1E-115 neuroblastoma cells as the model, we show that VPA upregulates Gadd45a to trigger activation of the downstream JNK cascade controlling neurite outgrowth. VPA induces the phosphorylation of c-Jun N-terminal kinase (JNK) and the substrate paxillin, while VPA induction of neurite outgrowth is inhibited by JNK inhibitors (SP600125 and the small JNK-binding peptide) or a paxillin construct harboring a Ser 178-to-Ala mutation at the JNK phosphorylation. Transfection of Gadd45a, acting through the effector MEKK4, leads to the phosphorylation of the JNK cascade. Conversely, knockdown of Gadd45a with siRNA reduces the effect of VPA. Taken together, these results suggest that upregulation of Gadd45a explains one of the mechanisms whereby VPA induces the neurotrophic effect, providing a new role of Gadd45a in neurite outgrowth.     

Sialic acid is an essential moiety of mucin as a hydroxyl radical scavenger

In this work, we examined the antioxidant role of mucin, a typical sialic acid containing high-molecular weight glycoprotein. The function of mucin as a hydroxyl radical (.OH) scavenger was characterized using bovine submaxillary gland mucin (BSM). Non-treated BSM effectively protected DNA from the attack of .OH; however, desialylated BSM lost this potential. Moreover, we estimated the scavenging effects of BSM against .OH generated by UV irradiation of hydrogen peroxide using ESR analysis. Our results indicate that BSM has .OH scavenging ability the and sialic acid in mucin is an essential moiety to scavenge .OH.     

Visualization of RecA filaments and DNA by fluorescence microscopy

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5' to 3' of ssDNA as dATP hydrolysis proceeded.     

The unique role of natural killer T cells in the response to microorganisms

Natural killer T (NKT) cells combine features of the innate and adaptive immune systems. Recently, it has become evident that these T cells have crucial roles in the response to infectious agents. The antigen receptor expressed by NKT cells directly recognizes unusual glycolipids that are part of the membrane of certain Gram-negative bacteria and spirochetes. Moreover, even in the absence of microbial glycolipid antigens, these T cells respond to innate cytokines produced by dendritic cells that have been activated by microbes. This indirect sensing of infection, by responding to cytokines from activated dendritic cells, allows NKT cells to react to a broad range of infectious agents.     

Identification and characterization of plasma kallikrein-kinin system inhibitors from salivary glands of the blood-sucking insect Triatoma infestans

Two plasma kallikrein-kinin system inhibitors in the salivary glands of the kissing bug Triatoma infestans, designated triafestin-1 and triafestin-2, have been identified and characterized. Reconstitution experiments showed that triafestin-1 and triafestin-2 inhibit the activation of the kallikrein-kinin system by inhibiting the reciprocal activation of factor XII and prekallikrein, and subsequent release of bradykinin. Binding analyses showed that triafestin-1 and triafestin-2 specifically interact with factor XII and high molecular weight kininogen in a Zn2+-dependent manner, suggesting that they specifically recognize Zn2+-induced conformational changes in factor XII and high molecular weight kininogen. Triafestin-1 and triafestin-2 also inhibit factor XII and high molecular weight kininogen binding to negatively charged surfaces. Furthermore, they interact with both the N-terminus of factor XII and domain D5 of high molecular weight kininogen, which are the binding domains for biological activating surfaces. These results suggest that triafestin-1 and triafestin-2 inhibit activation of the kallikrein-kinin system by interfering with the association of factor XII and high molecular weight kininogen with biological activating surfaces, resulting in the inhibition of bradykinin release in an animal host during insect blood-feeding.