Ascidian sperm glycosylphosphatidylinositol-anchored CRISP-like protein as a binding partner for an allorecognizable sperm receptor on the vitelline coat

Although ascidians are hermaphroditic, many species including Halocynthia roretzi are self-sterile. We previously reported that a vitelline coat polymorphic protein HrVC70, consisting of 12 EGF (epidermal growth factor)-like repeats, is a candidate allorecognition protein in H. roretzi, because the isolated HrVC70 shows higher affinity to nonself-sperm than to self-sperm. Here, we show that a sperm 35-kDa glycosylphosphatidylinositol-anchored CRISP (cysteine-rich secretory protein)-like protein HrUrabin in a low density detergent-insoluble membrane fraction is a physiological binding partner for HrVC70. We found that HrVC70 specifically interacts with HrUrabin, which had been separated by SDS-PAGE and transferred onto a nitrocellulose membrane. HrUrabin has an N-linked sugar chain, essential for binding to HrVC70. HrUrabin mRNA is expressed in the testis but not in the ovary, and the protein appears to be localized on the surface of sperm head and tail. Anti-HrUrabin antibody, which neutralizes the interaction between HrUrabin and HrVC70, potently inhibited fertilization and allorecognizable sperm-binding to HrVC70-agarose. However, no significant difference in the binding ability of HrUrabin to HrVC70 was observed in autologous and allogeneic combinations by Far Western analyses. These results indicate that sperm-egg binding in H. roretzi is mediated by the molecular interaction between HrUrabin on the sperm surface and HrVC70 on the vitelline coat, but that HrUrabin per se is unlikely to be a direct allorecognition protein.     

Vertebrate genomes code excess proteins with charge periodicity of 28 residues

All amino acid sequences derived from 248 prokaryotic genomes, 10 invertebrate genomes (plants and fungi) and 10 vertebrate genomes were analysed by the autocorrelation function of charge sequences. The analysis of the total amino acid sequences derived from the 268 biological genomes showed that a significant periodicity of 28 residues is observable for the vertebrate genomes, but not for the other genomes. When proteins with a charge periodicity of 28 residues (PCP28) were selected from the total proteomes, we found that PCP28 in fact exists in all proteomes, but the number of PCP28 is much larger for the vertebrate proteomes than for the other proteomes. Although excess PCP28 in the vertebrate proteomes are only poorly characterized, a detailed inspection of the databases suggests that most excess PCP28 are nuclear proteins.     

Drought induction of Arabidopsis 9-cis-epoxycarotenoid dioxygenase occurs in vascular parenchyma cells

The regulation of abscisic acid (ABA) biosynthesis is essential for plant responses to drought stress. In this study, we examined the tissue-specific localization of ABA biosynthetic enzymes in turgid and dehydrated Arabidopsis (Arabidopsis thaliana) plants using specific antibodies against 9-cis-epoxycarotenoid dioxygenase 3 (AtNCED3), AtABA2, and Arabidopsis aldehyde oxidase 3 (AAO3). Immunohistochemical analysis revealed that in turgid plants, AtABA2 and AAO3 proteins were localized in vascular parenchyma cells most abundantly at the boundary between xylem and phloem bundles, but the AtNCED3 protein was undetectable in these tissues. In water-stressed plants, AtNCED3 was detected exclusively in the vascular parenchyma cells together with AtABA2 and AAO3. In situ hybridization using the antisense probe for AtNCED3 showed that the drought-induced expression of AtNCED3 was also restricted to the vascular tissues. Expression analysis of laser-microdissected cells revealed that, among nine drought-inducible genes examined, the early induction of most genes was spatially restricted to vascular cells at 1 h and then some spread to mesophyll cells at 3 h. The spatial constraint of AtNCED3 expression in vascular tissues provides a novel insight into plant systemic response to drought stresses.     

Serological survey of Toxoplasma gondii in wild waterfowl in Chukotka, Kamchatka, Russia and Hokkaido, Japan

Antibodies to Toxoplasma gondii were assayed by ELISA in 22 experimentally inoculated domestic ducks. In addition, a serological assay was carried out at Obihiro, Hokkaido, Japan, in 2004 and 2005, on 221 wild ducks of 5 species: Anas platyrhynchus (n = 111); A. poecilorhyncha (n = 27); A. acuta (n = 58); A. penelope (n = 16); and A. crecca (n = 9). Assays were also conducted using sera from 197 wild geese of 2 species, i.e., Anser albifrons (n = 162) and Ans. fabalis (n = 35). Birds were collected between 2003 and 2005 from 3 different areas: Lake Miyajima-numa, Hokkaido, Japan, regions around Anadyr city of Chukotka autonomous okrug, and Lake Makobetukoe, Kamchatka oblast, Russia. The ELISA cutoff value (OD) was > or =0.395 based on results from uninfected ducks; the final dilution ratio recognized as positive was represented by the end titer. The end titer in the experimentally infected ducks ranged from 1:400 to 1:3,200. Antibodies to T. gondii were found in 49 of the 221 wild duck samples from Japan: A. platyrhynchus (22/74); A. poecilorhyncha (2/15); A. penelope (3/16); A. acuta (4/58); and A. crecca (0/9), all in 2004. In 2005, T. gondii was found in A. platyrhynchus (13/37); and A. poecilorhyncha (5/12). Thirty-two of 197 wild goose samples were seropositive, i.e., Ans. albifrons (7/51) in 2004 and (11/72) in 2005 in Miyajima-numa, Japan and 9/39 in Chukotka, Russia as well as in Ans. fabalis (5/35) in Kamchatka, for which the end titer ranged from 1:100 to 1:3,200. In immunoblotting, the A. platyrhynchus samples showed specific IgG antibody binding to several antigens in the T. gondii lane, i.e., at 30 and 43 kDa, but not in the Neospora caninum lane. No specific bands were noted in samples for which antibody activity was not detected. These results suggest that wild waterfowl inhabiting Hokkaido, Chukotka, and Kamchatka may be exposed to T. gondii.     

A taxonomic study on erysipelothrix by DNA-DNA hybridization experiments with numerous strains isolated from extensive origins

The purpose of this study was to clarify the taxonomic relationship between all the serovars and species of the genus Erysipelothrix by performing DNA-DNA hybridization experiments, the customary criterion for separation of bacterial genospecies. A total of 93 strains were isolated from a wide variety of sources, including pigs affected with acute or chronic erysipelas, other diseased animals, healthy animals, fish, retail meats, and environmental materials from throughout the world during the period 1958 to 1996. The present data on phenotypic characterization and DNA relatedness values demonstrate that 24 strains (96%) of E. tonsillarum are avirulent for swine, whereas 39 strains (66%) of genomic E. rhusiopathiae induced generalized or local urticarial lesion in swine after intradermal inoculation. This observation suggests that genomic E. tonsillarum has little etiological significance. Three minor groups contained several strains which exhibited minimal association with each type strain of E. rhusiopathiae and E. tonsillarum. In conclusion, it was confirmed that members of the E. rhusiopathiae and E. tonsillarum groups resemble each other in regard to many phenotypic characteristics, but differ in their ability to produce acid from saccharose and in their pathogenicity for swine. The genus Erysipelothrix certainly contains two main species: E. rhusiopathiae and E. tonsillarum.     

Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme.

An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate. The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK). The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103810 Da). The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid. It had a molecular mass of 105 kDa and a pI value of 5.0. The maximum activity was observed at pH 8 and 55 degrees C in Tris-HCl buffer. The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum. The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx. 30% identity for both. High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes.     

Quantitative comparison of anti-fading mounting media for confocal laser scanning microscopy.

Fading is one of the major obstacles to reliable observation in fluorescence microscopy. Using a confocal laser scanning microscope (CLSM) coupled to a computer, we quantitatively measured fading of fluorescence to formulate an equation, evaluated the anti-fading ability of several anti-fading media, and restored the faded images to the original level according to this equation. NIH 3T3 cells were stained with fluorescein isothiocyanate (FITC)-phalloidin, mounted with several commercial and homemade anti-fade media, and observed with CLSM under repeated illumination. With any mounting medium, attenuation of fluorescence intensity at a certain pixel occurred stepwise and the decrease was proportional to the intensity of the previous scan. From these results, we formulated an equation that has three coefficients: anti-fading factor (A), indicating the ability to retard fading; fluorescent intensity at the first scan (EM(1)); and background fluorescence (B). The fluorescent intensity at a certain point following nth scan is given as EM(n) = EM(1) * A ((n-1)). This equation was available for restoring faded images to their original states, even after the image had faded to only 60% of its original intensity.     

Colon cancer cell adhesion to endothelial E-selectin inhibits detachment of endothelial cells through activation of beta(1)-integrin

Previous studies have implicated a role for E-selectin in carcinoma cell adhesion to vascular endothelium. We examined the role of colon cancer cell adhesion to vascular endothelium via E-selectin using adenoviral vector-mediated transfection in human umbilical vein endothelial cells (HUVECs). We found that the amount of HUVEC detachment from the gelatin matrix 24 h after LS-180 cell adhesion was inhibited only when the HUVECs were transduced with wild-type E-selectin, but not with a cytoplasmic domain truncated mutant E-selectin or the control Lac-Z vector. We also found that the adhesion of LS-180 cells to wild-type E-selectin transduced HUVEC-induced activation of beta(1)-integrin receptors without affecting MMP activity. These results indicate that colon cancer cell adhesion via E-selectin inhibits HUVEC detachment from the monolayer, at least in part by modulating beta(1)-integrin activity in HUVECs. In addition, they indicate the importance of the cytoplasmic domain of E-selectin with this phenomenon.