Molecular Phylogenetic Positions and Ultrastructure of Marine Gregarines (Apicomplexa) Cuspisella ishikariensis n. gen., n. sp. and Loxomorpha cf. harmothoe from Western Pacific scaleworms (Polynoidae)

Marine gregarines are unicellular parasites of invertebrates commonly found infecting the intestine and coelomic spaces of their hosts. Situated at the base of the apicomplexan tree, marine gregarines offer an opportunity to explore the earliest stages of apicomplexan evolution. Classification of marine gregarines is often based on the morphological traits of the conspicuous feeding stages (trophozoites) in combination with host affiliation and molecular phylogenetic data. Morphological characters of other life stages such as the spore are also used to inform taxonomy when such stages can be found. The reconstruction of gregarine evolutionary history is challenging, due to high levels of intraspecific variation of morphological characters combined with relatively few traits that are taxonomically unambiguous. The current study combined morphological data with a phylogenetic analysis of small subunit rDNA sequences to describe and establish a new genus and species (Cuspisella ishikariensis n. gen., n. sp.) of marine gregarine isolated from the intestine of a polynoid host (Lepidonotus helotypus) collected from Hokkaido, Japan. This new species possesses a set of unusual morphological traits including a spiked attachment apparatus and sits on a long branch on the molecular phylogeny. Furthermore, this study establishes a molecular phylogenetic position for Loxomorpha cf. harmothoe, a previously described marine gregarine, and reveals a new group of gregarines that infect polynoid hosts.     

Expression of a gene for plastid ω-3 fatty acid desaturase and changes in lipid and fatty acid compositions in light- and dark- grown wheat leaves

The leaves of monocotyledonous plants create a developmental sequence of cells and plastids from the base to the apical portion. We investigated fatty-acid and lipid compositions in successive leaf sections of light- and dark-grown wheat (Triticum aestivum L. cv. Chihoku) seedlings. The most notable change in the fatty acid composition was the increase of linolenic acid (18:3) with maturation of leaf cells, which occurred both in light- and dark-grown leaf tissues. In light-grown leaves, the increase of 18:3 with maturation was mainly attributed to the increase of monogalactosyldiacylglycerol (MGD) and also to the increase of the 18:3 level of MGD. In dark-grown leaves, the increase of 18:3 in the leaf apex was caused by the increase of the levels of MGD and digalactosyldiacylglycerol (DGD) and also by the increase of the 18:3 levels of within these two lipids. Since MGD and DGD are mainly found in plastid membranes, these findings indicate that both the synthesis of galactolipids and the formation of 18:3 these lipids take place during plastid development. The plastid ω-3 fatty acid desaturase is responsible for the formation of 18:3 in plastid membrane lipids. To investigate the regulation of desaturation, we isolated a gene for wheat plastid ω-3 fatty acid desaturase (TaFAD7). The mRNA level of TaFAD7 in light-grown leaves was much higher than that in dark-grown leaves. During the greening of etiolated leaves the level of TaFAD7 mRNA increased significantly, accompanied by an increase of the 18:3 level of total fatty acids. On the other hand, the levels of TaFAD7 mRNA were almost the same in all the leaf sections of both light- and dark-grown leaf tissues. These results suggest that the effect of the expression of the TaFAD7 gene on the increase of the 18:3 level is different between the leaf development under continuous light- or dark-conditions and the light-induced greening process of etiolated leaves. The increase of 18:3 content of MGD (or MGD and DGD) with maturation is apparently regulated not solely by the level of TaFAD7 mRNA.     

The characterization of the monoclonal antibody Th-10a, specific for a nuclear protein appearing in the S phase of the cell cycle in normal thymocytes and its unregulated expression in lymphoma cell lines

A monoclonal antibody (Th-10a) specific for the nuclear protein appearing in the S phase of the cell cycle in normal mouse thymocytes was derived by immunizing Wistar rats with a murine thymic lymphoma (TIGN), and its isotype was rat IgG_2a and had κ light chain. Immunohistochemical staining of frozen sections of B10.Thy1.1 newborn thymus and embryonic intestine revealed that this monoclonal antibody reacted strongly with the nuclear proteins of subcortical thymocytes and the basal layer of the mucosa, where many cells were dividing, but not with that of the thymic medullary area. To evaluate the expression of the nuclear proteins during the cell cycle in detail, the results of an immunofluorescence analysis of the thymocytes from hydroxyurea-treated B10 mice using Th-10a monoclonal antibody were compared with those of DNA synthesis of these cells with the use of the FITC-conjugated anti-BrdUrd monoclonal antibody. The results indicated that the nuclear protein detected by Th-10a monoclonal antibody was highly expressed in the S phase of normal thymocytes, while the cells in G₁, G₂ and M phases exhibited a low level of the expression. Moreover, the variations in expression of the nuclear proteins in the thymocytes at different times after hydroxyurea treatment were observed to correspond with the frequency of DNA synthesizing cells. In contrast, the high level and unregulated expression of the nuclear protein detected by Th-10a monoclonal antibody was observed throughout the cell cycle of the mouse lymphoma cell lines examined. Since Th-10a monoclonal antibody does not react with the nuclear proteins derived from human, hamster or rat proliferating cells, this antibody may recognize a murine specific epitope of the nuclear protein. To further characteize the nuclear proteins, we extracted them from normal thymocytes or thymic lymphomas, and analysed them by immunoblotting or***     

Elevation of soluble interleukin-2 receptor levels in nasal allergy

To investigate soluble IL-2 receptor (sIL-2R) levels in nasal allergy, the sera and nasal secretions from patients with nasal allergy and from healthy subjects were subjected to a double-epitope enzyme-linked immunosorbent assay. Significant elevation of sIL-2R concentrations in the sera and nasal secretions was observed in the allergy patients (n = 26) compared with those of healthy subjects (n = 9). IL-2R-positive (CD25(+)) cells were observed in the crust formed in an allergic nasal mucosa. The concentration of sIL-2R in the sera correlated neither with the eosinophil count of the peripheral blood count nor with clinical severity. The concentration of sIL-2R in the nasal secretions was significantly higher compared with that in the sera from allergic patients (p < 0.01), whereas no significant difference was observed between sIL-2R levels in the sera and nasal sections from normal subjects. These findings indicate that sIL-2R plays an essential role in allergic processes by regulating IL-2R-positive cells recruited into the nasal mucosa.     

Heterocapsa circularisquama sp. nov. (Peridiniales, Dinophyceae): A new marine dinoflagellate causing mass mortality of bivalves in Japan

Heterocapsa circularisquama Horiguchi sp. nov. is described from Ago Bay, central Japan. The dinoflagellate produced large-scale red tides in the bays of central and western Japan and caused mass mortality of bivalves, notably the pearl oysters. The cell is small and is composed of a conical epitheca and a hemi-spheroidal hypothecs. The chloroplast is single and is connected to the single pyrenoid. The nucleus is elongated and is located in the left side of the cell. Thecal plate arrangement has been determined as: Po, cp, 5′, 3a, 7″, 6c, 5s, 5″′, 2″″. Heterocapsa circularisquama is morphologically very similar to Heterocapsa illdefina and it is almost impossible to distinguish these two species at light microscopical level. The characteristics which can be used to distinguish these two species are the morphology of body scales and the ultrastructure of the pyrenoid matrix. The body scales of H. circularisquama possess six radiating ridges on the circular basal plate; no such ridges can be observed on the roughly triangular basal plate of the scales of H. illdefina. Furthermore, the scales of the latter species possess substantially shorter spines compared to those of H. circularisquama. The pyrenoid matrix of H. circularisquama is hardly perforated by cytoplasmic tubules, while in H. tlldefina the pyrenoid matrix is always penetrated by many cytoplasmic tubules. Based on the arrangement of thecal plates, morphology of body scales, and ultra-structure of the pyrenoid, I am placing H. circularisquama sp nov. into the genus Heterocapsa.     

Roscoffia minor sp. nov. (Peridiniales, Dinophyceae): A new, sand-dwelling, armored dinoflagellate from Hokkaido, Japan

A new, sand-dwelling, armored dinoflagellate, Roscoffia minor sp. nov., is described from Ishikari beach, Hokkaido, Japan. The dinoflagellate has been collected from sand samples taken both near the water's edge and further upshore (25 m from the water's edge at a depth of 1 m), indicating that it is a true sand-dwelling species. Roscoffia minor is heterotrophic and lacks both a chloroplast and an eye-spot. The cell consists of a flattened cap-shaped epitheca and a large hemispheroidal hypotheca, and it is quite different from cells of the typical armored dinoflagellates. The thecal plate formula is: Po, 3′, la, 5″, 3c, 3s, 5″, 1″″. Its distinct cell shape and the thecal plate arrangement indicate affinity to the monotypic genus Roscoffia. Roscoffia minor is distinguished from Roscoffia capitata, the type species, by its smaller size and the possession of a finger-like apical projection. The thecal arrangement of the epitheca is similar to those of the members of the family Podolampaceae, while the hypothecal arrangement is the same as that of members of the subfamily Diplopsalioideae (family Congruentidiaceae). The organism seems to be positioned somewhere intermediate between these two families, but the family to which this dinoflagellate should be affiliated could not be determined.     

HALOSTYLODINIUM ARENARIUM, GEN ET SP. NOV. (DINOPHYCEAE), A COCCOID SAND-DWELLING DINOFLAGELLATE FROM SUBTROPICAL JAPAN

A new genus and species of marine coccoid dinoflagellate from subtropical Japan, Halostylodinium arenarium Horiguchi et Yoshizawa-Ebata, gen. et sp. nov., is described. The dominant stage of the dinoflagellate is a nonmotile ovoidal to spheroidal cell with a distinct stalk. The stalk consists of an upper thick tubule, a lower thin tubule, and a discoidal holdfast. The dinoflagellate possesses a yellowish-brown chloroplast with multiple lobes radiating from a central pyrenoid. It reproduces by the formation of two motile cells, which swim for a short period and then transform directly into the stalked nonmotile cell. The stalk is produced during transformation from the apical stalk complex present in the apex of the motile cell. The apical stalk complex consists of a double-folded apical pore plate and doughnut-shaped holdfast-building material. The ultrastructure of the apical stalk complex is compared with those of Bysmatrum arenicola and Stylodinium littorale. Halostylodinium arenarium possesses delicate thecal plates, and the thecal plate formula is Po, 5', 2a, 7", 7c, 6s, 5"', 1p, 2"". A phylogenetic study based on the 18S ribosomal RNA gene did not show any clear affinities between this organism and any species included in the analysis.     

The bvg‐repressed gene brtA,encoding biofilm‐associated surface adhesin,is expressed during host infection by Bordetella bronchiseptica

Bordetella species display phase modulation between Bvg⁺ and Bvg^? phases. Because expression of known virulence factors is up‐regulated in the Bvg⁺ phase, bacteria in this phase are considered competent for infection. However, the Bvg^? phase is of negligible importance for infection. No studies have shown that bacterial factors specific to the Bvg^? phase (bvg‐repressed factors) are expressed in the course of Bordetella infection. In the present study, the gene brtA (B ordetella RT X‐family A dhesin), which is a typical bvg‐repressed gene but is expressed in B. bronchiseptica infecting hosts, was characterized. BrtA is composed of repeated pairs of the VCBS unit and dystroglycan‐type cadherin‐like unit, the von Willebrand Factor A domain, RTX motif and type I secretion target signal. It is herein demonstrated that BrtA is secreted by the type I secretion system and is essential for Ca²⁺‐dependent bacteria‐to‐substrate adherence, followed by biofilm formation. Although the contribution of BrtA to bacterial colonization of the rat trachea currently remains unclear, this is the first study to present concrete evidence for the expression of a bvg‐repressed gene during infection, which may provide a novel aspect for analyses of Bordetella pathogenesis.

     

Hemagglutinating activity of trypsinized Clostridium perfringens enterotoxin

Abstract The hemagglutinating activity of Clostridium perfringens enterotoxin (CPE) was studied after trypsin treatment. Untreated CPE did not show any hemagglutinating activity to human type A, B, and O, sheep, chicken, horse, guinea-pig, or rabbit erythrocytes. Trypsinized CPE resulted in a more than 100-fold increase in hemagglutinating activity with rabbit erythrocytes only. Other erythrocytes and trypsinized rabbit erythrocytes were not agglutinated at all. The hemagglutinating activity of CPE was also found on treatment with a lysine-specific proteinase. On the other hand, trypsinized CPE did not significantly increase the cytotoxic and enterotoxic activities. The binding reaction between trypsinized and rabbit erythrocytes was not inhibited by any mono-, di-, or polysaccharides, glycoproteins or ganglioside mixtures. These results suggest that the hydrolysis of bonds involving lysine residues is mainly required for hemagglutinating activity, and that the receptor for trypsinized CPE on rabbit erythrocytes is probably the protein moiety.