A Species-Specific Cluster of Defensin-Like Genes Encodes Diffusible Pollen Tube Attractants in Arabidopsis

Genes directly involved in male/female and host/parasite interactions are believed to be under positive selection. The flowering plant Arabidopsis thaliana has more than 300 defensin-like (DEFL) genes, which are likely to be involved in both natural immunity and cell-to-cell communication including pollen–pistil interactions. However, little is known of the relationship between the molecular evolution of DEFL genes and their functions. Here, we identified a recently evolved cluster of DEFL genes in A. thaliana and demonstrated that these DEFL (cysteine-rich peptide [CRP810_1]) peptides, named AtLURE1 peptides, are pollen tube attractants guiding pollen tubes to the ovular micropyle. The AtLURE1 genes formed the sole species-specific cluster among DEFL genes compared to its close relative, A. lyrata. No evidence for positive selection was detected in AtLURE1 genes and their orthologs, implying neutral evolution of AtLURE1 genes. AtLURE1 peptides were specifically expressed in egg-accompanying synergid cells and secreted toward the funicular surface through the micropyle. Genetic analyses showed that gametophytic mutants defective in micropylar guidance (myb98, magatama3, and central cell guidance) do not express AtLURE1 peptides. Downregulation of the expression of these peptides impaired precise pollen tube attraction to the micropylar opening of some populations of ovules. Recombinant AtLURE1 peptides attracted A. thaliana pollen tubes at a higher frequency compared to A. lyrata pollen tubes, suggesting that these peptides are species-preferential attractants in micropylar guidance. In support of this idea, the heterologous expression of a single AtLURE1 peptide in the synergid cell of Torenia fournieri was sufficient to guide A. thaliana pollen tubes to the T. fournieri embryo sac and to permit entry into it. Our results suggest the unique evolution of AtLURE1 genes, which are directly involved in male–female interaction among the DEFL multigene family, and furthermore suggest that these peptides are sufficient to overcome interspecific barriers in gametophytic attraction and penetration.     

Chromosome aberration frequencies and chromosome instability in mice after long-term exposure to low-dose-rate gamma-irradiation

Chronological changes of chromosome aberration rates related to accumulated doses in chronically exposed humans and animals at a low-dose-rate have not been well studied. C3H female specific pathogen-free mice (8 weeks of age) were chronically irradiated. Chromosome aberration rate in mouse splenocytes after long-term exposure to low-dose-rate (LDR) gamma-rays was serially determined by conventional Giemsa method. Incidence of dicentrics and centric rings increased almost linearly up to 8000 mGy following irradiation for about 400 days at a LDR of 20 mGy/day. Clear dose-rate effects were observed in the chromosome aberration frequencies between dose rates of 20 mGy/day and 200 Gy/day. Furthermore, the frequencies of complex aberrations increased as accumulated doses increased in LDR irradiation. This trend was also observed for the incidences of micronuclei and trisomies of chromosomes 5, 13 and 18 in splenocytes, detected by micronucleus assay and metaphase fluorescence in situ hybridization (FISH) method, respectively. Incidences of 2-4 micronuclei and trisomy increased in mouse splenocytes after irradiation of 8000 mGy at a LDR of 20 mGy/day. These complex chromosome aberrations and numerical chromosome aberrations seem to be induced indirectly after radiation exposure and thus the results indicate that continuous gamma-ray irradiation for 400 days at LDR of 20 mGy/day induced chromosomal instability in mice. These results are important to evaluate the biological effects of long-term exposure to LDR radiation in humans.     

The roles of amino acid residues at positions 216 and 219 in the structural stability and metabolic functions of rat cytochrome P450 2D1 and 2D2

We examined the effects of the mutual substitution of amino acid residues at positions 216 and 219 between rat CYP2D1 and CYP2D2 on their microsomal contents and enzymatic functions using a yeast cell expression system and 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT) as a substrate. CYP2D1 has amino acid residues, leucine and valine, at positions of 216 and 219, respectively, whereas CYP2D2 has phenylalanine and aspartic acid at the same positions. In reduced carbon monoxide-difference spectroscopic analysis, the substitution of Asp-219 of CYP2D2 by valine markedly increased a peak at 450 nm and concomitantly decreased a peak at 420 nm, while the replacement of Phe-216 of CYP2D2 with leucine gave no observable change. The double substitution of Phe-216 and Asp-219 by leucine and valine, respectively, yielded a typical CYP spectrum. The substitution of Val-219 of CYP2D1 by aspartic acid decreased the CYP content to one-half, whereas the replacement of Leu-216 with phenylalanine did not have any effect. The double substitution of Leu-216 and Val-219 of CYP2D1 by phenylalanine and aspartic acid, respectively, diminished the CYP content by 90%. CYP2D1 catalyzed both 5-MeO-DIPT N-deisopropylation and O-demethylation at relatively low levels, while CYP2D2 catalyzed 5-MeO-DIPT O-demethylation efficiently. The substitution of the amino acid at position 216 substantially increased 5-MeO-DIPT oxidation activities of the two CYP2D enzymes. The replacement of the amino acid at position 219 increased the 5-MeO-DIPT O- and N-dealkylation activities of CYP2D1, whereas it decreased the 5-MeO-DIPT O-demethylation activity of CYP2D2. These results indicate that amino acid residues at positions 216 and 219 have important roles in the enzymatic functions of rat CYP2D1 and CYP2D2.     

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.     

Gametophytic pollen tube guidance

The concept of a pollen tube attractant was proposed in the late nineteenth century when pollen tubes were found to grow toward excised pistil tissues on medium. Since then, for about 140 years, plant biologists have tried to identify the pollen tube attractants. However, no molecule has been convincingly demonstrated to be the true attractant that actually controls the navigation of pollen tubes in the pistil. The past decade has seen substantial progress in this field in terms of our understanding of the various mechanisms of pollen tube guidance. It was suggested that diffusible pollen tube attractants might provide localized signals that affect the directional growth of the pollen tube, especially in the last phase of guidance by the target female gametophyte. Here, we review the mechanisms of pollen tube guidance, with special focus on the gametophytic guidance and the attractant. The necessary and appropriate conditions required by the true attractant will be discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.     

Population dynamics of Propsilocerus akamusi and Chironomus plumosus (Diptera: Chironomidae) in Lake Suwa in relation to changes in the lake's environment

Hydrobiologia - Lake Suwa is a shallow eutrophic lake in central Japan. We have investigated the long-term population dynamics of chironomids in this lake. The objective of this study was...     

Production of arachidonic acid byMortierella fungi

The growing interest in the application of arachidonic acid (ARA) in various fields of health and dietary requirements has elicited much attention on the industrial production of ARA-containing oil by the cultivation ofMortierella fungi. For the industrial production of ARA, various studies, such as isolation of a high-potential strain and optimization of culture conditions, have been conducted. Studies including the investigation of morphology are important because ARA is accumulated in the mycelia, and thus cultivation with high biomass concentration is essential for obtaining a high ARA yield. Combining the results derived from various studies, a high ARA yield was attained in an industrial fermentor. These ARA production techniques are applicable to the production of other polyunsaturated fatty acids (PUFAs), and will contribute to the improvement of fermentation technology especially in the field of fungal cultivation.     

Emission Mössbauer studies of some organocobalamins

Subtle changes in Mössbauer parameters are observed while going from methyl- to ethyl- to adenosylcobalamin, and also when the ‘base’ is detached from the cobalt. The observation of these changes demonstrates that the Co-C bond, among others, remains intact after the Auger event, accompanying the electron-capture decay of the cobalt-57.The differences between ethylcobalamin and the other two organocobalamins in the magnitude of the quadrupole splittings have been interpreted on the basis of the σ-donating tendency of the organic moiety and the CoC bond length. The latter is presumably determined by the steric hindrance offered to the group in approaching the cobalt atom.The ethyl- and adenosylcobalamins in their ‘base-off’ form exhibit a larger quadrupole splitting than the corresponding ‘base-on’ form. In the ‘base-off’ form, the cobalt atom is perhaps raised above the mean plane of the four equatorial nitrogen atoms of the corrin ring, which may result in the diminution of the delocalization of the 3d_π electron density. The higher population of d_π orbitals and the enhanced metallic character of the d_z2, resulting from shrink-age of the CoC bond length, enhances the magnitude of the quadrupole splitting.