Antibodies against Toxoplasma gondii in the Pacific bottlenose dolphin (Tursiops aduncus) from the Solomon Islands

Serum samples from 58 Pacific bottlenose dolphins (Tursiops aduncus) from the Solomon Islands were tested for the IgG antibody to Toxoplasma gondii by the latex agglutination test (LAT), enzyme-linked immunosorbent assay (ELISA), and immunoblotting. The ELISA cut-off value was taken as OD > or = 0.276, and the final dilution ratio, recognized as positive, was represented by the end titer. In 25 of 58 samples, no antibody activity was detected by LAT and ELISA. In 8 of 58 samples, anti-T. gondii IgG antibodies were detected by both LAT and ELISA, with titers of greater than 1 : 64 and 1 : 160, respectively. By immunoblotting, the 8 serum samples producing higher titers showed specific antibody IgG binding to several antigens on the T. gondii lane, but not on the Neospora caninum lane. No specific bands were noted on the lanes for either parasite in the 25 serum samples for which no antibody activity was detected. The specific binding of IgG antibodies to T. gondii antigens observed for serum samples producing higher titers suggests that Pacific bottlenose dolphins from the Solomon islands are exposed to T. gondii.     

Two callose synthases,GSL1 and GSL5, play an essential and redundant role in plant and pollen development and in fertility

Callose, a β-1,3-glucan that is widespread in plants, is synthesized by callose synthase. Arabidopsis thaliana contains a family of 12 putative callose synthase genes (GSL1–12). The role of callose and of the individual genes in plant development is still largely uncertain. We have now used TILLING and T-DNA insertion mutants (gsl1-1, gsl5-2 and gsl5-3) to study the role of two closely related and linked genes, GSL1 and GSL5, in sporophytic development and in reproduction. Both genes are expressed in all parts of the plant. Sporophytic development was nearly normal in gsl1-1 homozygotes and only moderately defective in homozygotes for either of the two gsl5 alleles. On the other hand, plants that were gsl1-1/+ gsl5/gsl5 were severely defective, with smaller leaves, shorter roots and bolts and smaller flowers. Plants were fertile when the sporophytes had either two wild-type GSL1 alleles, or one GSL5 allele in a gsl1-1 background, but gsl1-1/+ gsl5/gsl5 plants produced an extremely reduced number of viable seeds. A chromosome with mutations in both GSL1 and GSL5 rendered pollen infertile, although such a chromosome could be transmitted via the egg. As a result, it was not possible to obtain plants that were homozygous for mutations in both the GSL genes. Pollen grain development was severely affected in double mutant plants. Many pollen grains were collapsed and inviable in the gsl1-1/gsl1-1 gsl5/+ and gsl1-1/+ gsl5/gsl5 plants. In addition, gsl1-1/+ gsl5/gsl5 plants produced abnormally large pollen with unusual pore structures, and had problems with tetrad dissociation. In this particular genotype, while the callose wall formed around the pollen mother cells, no callose wall separated the resulting tetrads. We conclude that GSL1 and GSL5 play important, but at least partially redundant roles in both sporophytic development and in the development of pollen. They are responsible for the formation of the callose wall that separates the microspores of the tetrad, and also play a gametophytic role later in pollen grain maturation. Other GSL genes may control callose formation at different steps during pollen development.     

Comprehensive approach to genes involved in cell wall modifications in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The plant cell wall is of supermolecular architecture, and is composed of various types of heterogeneous polymers. A few thousand enzymes and structural proteins are directly involved in the construction processes, and in the functional aspects of the dynamic architecture in Arabidopsis thaliana. Most of these proteins are encoded by multigene families, and most members within each family share significant similarities in structural features, but often exhibit differing expression profiles and physiological functions. Thus, for the molecular dissection of cell wall dynamics, it is necessary to distinguish individual members within a family of proteins. As a first step towards characterizing the processes involved in cell wall dynamics, we have manufactured a gene-specific 70-mer oligo microarray that consists of 765 genes classified into 30 putative families of proteins that are implicated in the cell wall dynamics of Arabidopsis. By using this array system, we identified several sets of genes that exhibit organ preferential expression profiles. We also identified gene sets that are expressed differentially at certain specific growth stages of the Arabidopsis inflorescence stem. Our results indicate that there is a division of roles among family members within each of the putative cell wall-related gene families.     

Characterization of ichthyocidal activity of Pfiesteria piscicida: dependence on the dinospore cell density

The ichthyocidal activity of Pfiesteria piscicida dinospores was examined in an aquarium bioassay format by exposing fish to either Pfiesteria-containing environmental sediments or clonal P. piscicida. The presence of Pfiesteria spp. and the complexity of the microbial assemblage in the bioassay were assessed by molecular approaches. Cell-free water from bioassays that yielded significant fish mortality failed to show ichthyocidal activity. Histopathological examination of moribund and dead fish failed to reveal the skin lesions reported elsewhere. Fish larvae within "cages" of variable mesh sizes were killed in those where the pore size exceeded that of Pfiesteria dinospores. In vitro exposure of fish larvae to clonal P. piscicida indicated that fish mortality was directly proportional to the dinospore cell density. Dinospores clustered around the mouth, eyes, and operculi, suggesting that fish health may be affected by their direct interaction with skin, gill epithelia, or mucous surfaces. Molecular fingerprinting revealed the presence of a very diverse microbial community of bacteria, protists, and fungi within bioassay aquaria containing environmental sediments. Some components of the microbial community were identified as potential fish pathogens, preventing the rigorous identification of Pfiesteria spp. as the only cause of fish death. In summary, our results strongly suggest (i) that this aquarium bioassay format, which has been extensively reported in the literature, is unsuitable to accurately assess the ichthyocidal activity of Pfiesteria spp. and (ii) that the ichthyocidal activity of Pfiesteria spp. is mostly due to direct interactions of the zoospores with fish skin and gill epithelia rather than to soluble factors.     

Deuterium-resistant algal cell line for D labeling of heterotrophs expresses enhanced level of Hsp60 in D2O medium

Fully deuterated components from autotrophic cell lysate are useful materials for labeling of heterotrophs with deuterium. To facilitate the faster production of deuterated algal lysate, we selected a mutant Chlorella strain that grows faster in heavy water than the wild type. The mutant DR-17 was found to have a higher level of Hsp60 and an elevated level of protein synthesis. We previously isolated a deuterium-resistant yeast cell line that was also found to express elevated level of Hsp70 (K. Unno, T. Kishido, M. Morioka, S. Okada, and N. Oku, Biol. Pharm. Bull. 26:799-802, 2003). This suggests that the overexpression of heat shock proteins is required to compensate for the deuterium isotope effect.     

Fanconi anemia protein FANCD2 promotes immunoglobulin gene conversion and DNA repair through a mechanism related to homologous recombination

Recent studies show overlap between Fanconi anemia (FA) proteins and those involved in DNA repair mediated by homologous recombination (HR). However, the mechanism by which FA proteins affect HR is unclear. FA proteins (FancA/C/E/F/G/L) form a multiprotein complex, which is responsible for DNA damage-induced FancD2 monoubiquitination, a key event for cellular resistance to DNA damage. Here, we show that FANCD2-disrupted DT40 chicken B-cell line is defective in HR-mediated DNA double-strand break (DSB) repair, as well as gene conversion at the immunoglobulin light-chain locus, an event also mediated by HR. Gene conversions occurring in mutant cells were associated with decreased nontemplated mutations. In contrast to these defects, we also found increased spontaneous sister chromatid exchange (SCE) and intact Rad51 foci formation after DNA damage. Thus, we propose that FancD2 promotes a subpathway of HR that normally mediates gene conversion by a mechanism that avoids crossing over and hence SCEs.     

Comparisons of food availability and group density of Japanese macaques in primary, naturally regenerated, and plantation forests

We compared food availability and group density of Japanese macaques in Yakushima, southern Japan, among primary forest and two habitats that had been disturbed by logging and had different regeneration histories. The study was conducted in an undisturbed national park, forest that was logged 7-18 years ago and later naturally regenerated, and forest that was logged 19-27 years ago and later planted with Japanese cedar (Cryptomeria japonica) trees. The plantation forest was primarily composed of large Cryptomeria japonica trees at low stand density, while the naturally regenerated forest was composed of many small trees. The total basal area and number of trees in the primary forest were comparable to those in the plantation forest. Annual fruit production was greatest in the naturally regenerated forest, intermediate in the primary forest, and negligible in the plantation forest. Herb availability was high in the naturally regenerated forest, but low in the primary and plantation forests. The group density of Japanese macaques was high in the naturally regenerated forest, intermediate in the primary forest, and low in the plantation forest. Since group size in the naturally regenerated forest was small, individual density was almost the same as in the primary forest. These results suggest that the effects of regeneration on macaques vary between the two habitats. The plantation forest consisted mostly of Cryptomeria japonica, which supplies only flowers as food in a limited season, and had a lower density of macaques. On the other hand, in the naturally regenerated forest, fruit production and herb availability were high (probably because of the enhanced light conditions after logging), and the density of macaques was as high as in the primary forest.     

Vitellogenin-immunohistochemistry in the liver and the testis of the medaka, Oryzias latipes, exposed to 17beta-estradiol and p-nonylphenol

Vitellogenin (VTG) produced in male fish has been used for a biomarker to study endocrine disrupters. However, the characteristics of VTG produced in male fish have not been studied well. In this study, we investigated the localization of VTG in the liver and the testis of male medaka (Oryzias latipes) treated with 17beta-estradiol (E2) and p-nonylphenol (NP). The male fish were exposed to 1 microg/L E2 and 500 microg/L NP for 1-12 days. Control groups were kept in water including only vehicle. The frozen sections of the liver and the testis were stained with immunohistochemical methods using an antiserum against medaka VTG as the first antibody. In the E2 and NP treated liver, the hepatocytes showed immunoreactivity. In particular, the cytoplasm close to the cell membrane surrounding the sinusoids was strongly immunopositive. In the testis of both treatments, the interstitial tissues and the cells (spermatocytes) in the seminiferous tubules were immunopositive. The concentration of VTG became gradually higher in both tissues with longer treatments. These results suggest that germ cells in the testis treated with E2 and NP are able to incorporate and accumulate VTG.     

Ubiquitylation of RAG-2 by Skp2-SCF links destruction of the V(D)J recombinase to the cell cycle

The periodic destruction of RAG-2 at the G1-to-S transition couples V(D)J recombination to the G0 and G1 cell cycle phases and coordinates RAG-mediated DNA cleavage with DNA repair by nonhomologous end joining. To define the mechanism by which this occurs, we reproduced cell cycle-dependent regulation of the V(D)J recombinase in a cell-free system. The ubiquitin-proteasomal pathway carries out destruction of RAG-2 in lysates of S phase cells and during S phase in vivo. Remarkably, the Skp2-SCF ubiquitin ligase, which plays a central role in cell cycle regulation through the destruction of p27, mediates ubiquitylation of RAG-2 in vitro and degradation of RAG-2 in vivo. The regulation of antigen receptor gene assembly by Skp2-SCF provides an unexpected and direct mechanistic link between DNA recombination and the cell cycle.     

Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.