Identification and characterization of a novel alternative splice variant of mouse GMx33alpha/GPP34

We have cloned and characterized a novel splice variant of mouse GMx33alpha/Golgi-associated protein of 34 kDa (GPP34), hereby designated GMx33alphaV/GPP34V. This splice variant skips the second and third exons, and the resulting frame shift generates a stop codon in the fourth exon. GMx33alphaV/GPP34V is comprised of 81 amino acid residues derived from the N-terminal end of the full length protein and corresponds to approximately one-third of the full length GMx33alpha/GPP34 sequence with a calculated molecular mass of 8900. In contrast to GMx33alpha/GPP34 mRNA which is expressed at similar levels in various tissues, GMx33alphaV/GPP34V mRNA was differentially expressed when examined by RT-PCR. Compared to other tissues, skeletal muscle showed relatively strong expression of GMx33alphaV/GPP34V mRNA. This splice variant cDNA was also detected in a human cell line.     

Inhibition of Rho-associated kinases disturbs the collective cell migration of stratified TE-10 cells

Background

The collective cell migration of stratified epithelial cells is considered to be an important phenomenon in wound healing, development, and cancer invasion; however, little is known about the mechanisms involved. Furthermore, whereas Rho family proteins, including RhoA, play important roles in cell migration, the exact role of Rho-associated coiled coil-containing protein kinases (ROCKs) in cell migration is controversial and might be cell-type dependent. Here, we report the development of a novel modified scratch assay that was used to observe the collective cell migration of stratified TE-10 cells derived from a human esophageal cancer specimen.

Results

Desmosomes were found between the TE-10 cells and microvilli of the surface of the cell sheet. The leading edge of cells in the cell sheet formed a simple layer and moved forward regularly; these rows were followed by the stratified epithelium. ROCK inhibitors and ROCK small interfering RNAs (siRNAs) disturbed not only the collective migration of the leading edge of this cell sheet, but also the stratified layer in the rear. In contrast, RhoA siRNA treatment resulted in more rapid migration of the leading rows and disturbed movement of the stratified portion.

Conclusions

The data presented in this study suggest that ROCKs play an important role in mediating the collective migration of TE-10 cell sheets. In addition, differences between the effects of siRNAs targeting either RhoA or ROCKs suggested that distinct mechanisms regulate the collective cell migration in the simple epithelium of the wound edge versus the stratified layer of the epithelium.

Electronic supplementary material

The online version of this article (doi:10.1186/s40659-015-0039-2) contains supplementary material, which is available to authorized users.     

Expression of engrailed-family genes in the jumping bristletail and discussion on the primitive pattern of insect segmentation

It has been shown that segmentation in the short-germ insects proceeds by a two-step mechanism. The anterior region is simultaneously segmented in a manner similar to that in Drosophila, which is apparently unique to insects, and the rest of the posterior region is segmented sequentially by a mechanism involving a segmentation clock, which is derived from the common ancestor of arthropods. In order to propose the evolutionary scenario of insect segmentation, we examined segmentation in the jumping bristletail, the basalmost extant insect. Using probes for engrailed-family genes for in situ hybridization, we found no sign of simultaneous segmentation in the anterior region of the jumping bristletail embryos. All segments except the anteriormost segment are formed sequentially. This condition shown in the jumping bristletail embryos may represent the primitive pattern of insect segmentation. The intercalating formation of the intercalary segment is assumed to be a synapomorphic trait shared among all insects after the branching of the jumping bristletail.     

The Extracellular A-loop of Dual Oxidases Affects the Specificity of Reactive Oxygen Species Release

NADPH oxidase (Nox) family proteins produce superoxide (O₂^⨪) directly by transferring an electron to molecular oxygen. Dual oxidases (Duoxes) also produce an O₂^⨪ intermediate, although the final species secreted by mature Duoxes is H₂O₂, suggesting that intramolecular O₂^⨪ dismutation or other mechanisms contribute to H₂O₂ release. We explored the structural determinants affecting reactive oxygen species formation by Duox enzymes. Duox2 showed O₂^⨪ leakage when mismatched with Duox activator 1 (DuoxA1). Duox2 released O₂^⨪ even in correctly matched combinations, including Duox2 + DuoxA2 and Duox2 + N-terminally tagged DuoxA2 regardless of the type or number of tags. Conversely, Duox1 did not release O₂^⨪ in any combination. Chimeric Duox2 possessing the A-loop of Duox1 showed no O₂^⨪ leakage; chimeric Duox1 possessing the A-loop of Duox2 released O₂^⨪. Moreover, Duox2 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA2 showed enhanced O₂^⨪ release, and Duox1 proteins possessing the A-loops of Nox1 or Nox5 co-expressed with DuoxA1 acquired O₂^⨪ leakage. Although we identified Duox1 A-loop residues (His¹⁰⁷¹, His¹⁰⁷², and Gly¹⁰⁷⁴) important for reducing O₂^⨪ release, mutations of these residues to those of Duox2 failed to convert Duox1 to an O₂^⨪-releasing enzyme. Using immunoprecipitation and endoglycosidase H sensitivity assays, we found that the A-loop of Duoxes binds to DuoxA N termini, creating more stable, mature Duox-DuoxA complexes. In conclusion, the A-loops of both Duoxes support H₂O₂ production through interaction with corresponding activators, but complex formation between the Duox1 A-loop and DuoxA1 results in tighter control of H₂O₂ release by the enzyme complex.     

Growth inhibition and chromosomal instability of cultured marsupial (opossum) cells after treatment with DNA polymerase α inhibitor

The DNA replication mechanism has been well established for eutherian mammals (placental mammals such as humans, mice, and cattle), but not, to date, for metatherian mammals (marsupials such as kangaroos, koalas, and opossums). In this study, we found that dehydroaltenusin, a selective inhibitor of mammalian (eutherian) DNA polymerase α, clearly suppressed the growth of metatherian (opossum and rat kangaroo) cultured cells. In cultured opossum (OK) cells, dehydroaltenusin also suppressed the progression of DNA replication. These results suggest that dehydroaltenusin inhibits metatherian as well as eutherian DNA replication. Dehydroaltenusin treatment of OK cells engendered fluctuations in the numbers of chromosomes in the OK cells as well as inhibition of cell growth and DNA replication. This suggests that partial inhibition of DNA replication by dehydroaltenusin causes chromosomal instability in cultured cells.     

High molecular weight lectin isolated from the mucus of the giant African snail Achatina fulica

To understand better the host defense mechanisms of mollusks against pathogens, we examined the anti-microbial activity of mucus from the giant African snail Achatina fulica. Hemagglutination activity of the mucus secreted by the integument of snails inoculated with Escherichia coli was observed to increase and to cause hemagglutination of rabbit red blood cells. Purification of the snail mucus lectin by sequential column chromatography revealed that the relative molecular mass of the lectin was 350 kDa. The hemagglutination activity of the lectin was Ca(2+)-dependent and was inhibited by galactose. Growth arrest tests showed that the lectin did not inhibit bacterial growth, but did induce agglutination of gram-positive and gram-negative bacteria. Tissue distribution analyses using a polyclonal antibody revealed that the lectin was expressed in the tissues of the mantle collar. The lectin isolated from the mucus of the snail appeared to contribute to its innate immunity.     

Polarization-based brightness discrimination in the foraging butterfly, Papilio xuthus

The human eye is insensitive to the angular direction of the light e-vector, but several animal species have the ability to discriminate differently polarized lights. How the polarization is detected is often unclear, however. Egg-laying Papilio butterflies have been shown to see false colours when presented with differently polarized lights. Here we asked whether this also holds in foraging butterflies. After training individuals to feed on nectar in front of an unpolarized spectral light, we carried out three dual-choice tests, where the discrimination of (i) the spectral content, (ii) the light intensity, and (iii) the e-vector orientation were investigated. In the first test, the butterflies selected the trained spectrum irrespective of its intensity, and in the second test they chose the light with the higher intensity. The result of the e-vector discrimination test was very similar to that of the second test, suggesting that foraging butterflies discriminate differently polarized lights as differing in brightness rather than as differing in colour. Papilio butterflies are clearly able to use at least two modes of polarization vision depending on the behavioural context.