Origin of the plastid in the anomalously pigmented dinoflagellate Gymnodinium mikimotoi (Gymnodiniales, Dinophyta) as inferred from phylogenetic analysis based on the gene encoding the large subunit of form I-type RuBisCO

The dinoflagellate Gymnodinium mikimotoi Miyake et Kominami ex Oda possesses an anomalously pigmented plastid which contains 19′‐hexanoyloxyfucoxanthin, 19′‐butanoyloxyfucoxanthin and fucoxanthin instead of peridinin as the major carotenoids. Previously, we have shown that the plastid of G. mikimotoi belongs to the rhodoplast lineage as inferred from phylogenetic analyses based on the amino acid sequences deduced from psbA and psaA and the nucleotide sequence of the plastid small subunit ribosomal RNA. Furthermore, in the present study, we cloned and sequenced an additional representative plastid gene, rbcL, encoding the large subunit of ribulose 1–5 bisphosphate carboxylase/oxygenase (RuBisCO LSU) from G. mikimotoi. The amino acid sequence deduced from the rbcL gene of G. mikimotoi apparently revealed the conventional form I RuBisCO LSU, which is present in most photosynthetic organisms, and not the divergent form II existing in typically pigmented dinofl age Nates with plastids containing peridinin as the main carotenoid. This finding supports the hypothesis that the origins of the plastids in G. mikimotoi and peridinin‐type dinoflagellates are not related to each other. Molecular phylogenetic analysis based on the amino acid sequence deduced from the rbcL gene further showed that the plastid of G. mikimotoi belongs to the rhodoplast lineage. In particular, G. mikimotoi clustered with haptophytes in the phylogenetic tree. From this result, two hypotheses with respect to the origin of the plastid in G. mikimotoi can be proposed: G. mikimotoi may have engulfed a haptophyte‐like cell (tertiary symbiosis) or englulfed a rhodophyte‐like cell that was closely related to the origin of the plastid in the haptophyte (secondary symbiosis).     

Non-seasonal clade-specificity and subclade microvariation in symbiotic dinoflagellates (Symbiodinium spp.) in Zoanthus sansibaricus (Anthozoa: Hexacorallia) at Kagoshima Bay, Japan

While much work has investigated the genetic diversity of symbiotic dinoflagellate genus Symbiodinium Freudenthal in cnidarians, investigations into such diversity over temporal scales (seasonal and/or annual) remain scarce. Here, we have sequenced the internal transcribed spacer of ribosomal DNA (ITS‐rDNA) of Symbiodinium from samples of designated Zoanthus sansibaricus Carlgren (Anthozoa: Hexacorallia) colonies collected for 12 months (August 2004–July 2005) at a high latitude non‐reefal coral community at Sakurajima, Kagoshima Bay, Japan (31°35′N, 130°35′E). Our results show that despite large ocean temperature changes (15.0–29.0°C) throughout the one‐year experimental period, Z. sansibaricus colonies contained only clade C Symbiodinium from many different subclade C1/C3‐related novel types not previously reported. While no temporal changes in clade‐level associations were seen, there were consistent and extremely large amounts (145 unique sequences out of 153 total obtained sequences) of genotypic microvariation observed in our obtained sequences. Despite Z. sansibaricus acquiring Symbiodinium horizontally and the presence of various other Symbiodinium clades (A, G) and subclades (e.g. C15 and derived subclades) in the immediate environment, Z. sansibaricus at Sakurajima specifically associates with subclade C1/C3‐related Symbiodinium. While subclades C1/C3 have been found in a variety of different environments and are believed to be ancestral, ‘generalist’ types of Symbiodinium, C1/C3‐related clades such as seen here may be more adapted to specialized niches. We theorize that specific and year‐round association with many different types of subclade C1/C3‐related Symbiodinium helps Z. sansibaricus to survive in the fluctuating Sakurajima environment.     

Carbendazim-induced abnormal development of the acrosome during early phases of spermiogenesis in the rat testis

Effects of a single, high dose of orally administered carbendazim (100 mg/kg) on acrosome formation in the early phases of spermiogenesis were examined by electron microscopy and immunocytochemistry up to day 7.5 post-treatment. No obvious abnormality of acrosome development was noted in the Golgi phase spermatids on day 1.5 post-treatment. On day 3, step 1 spermatids were seen in stage III seminiferous tubules. In stage V tubules at this post-treatment interval, direct connections between the trans-side saccules of the Golgi stacks and the outer acrosomic membranes were observed in step 5 spermatids. Similar direct connections between these two organelles were also observed in the advanced round spermatids in later stages at days 4.5 and 7.5. On day 4.5, step 1 and 3 spermatids were seen in stage V tubules. On day 7.5, round spermatids with various abnormalities of acrosome development were observed in stage VII tubules, in addition to the discontinuous and granular acrosomes reported previously. These features were not observed in testes of control animals. In the immunocytochemical analysis using an antibody mMN7 that recognizes a protein delivered from the Golgi apparatus to the acrosome, spermatids exposed to carbendazim showed various abnormal immunostaining patterns in the acrosomes. On the other hand, strong immunoreactivity was observed in the Golgi saccules connecting to the acrosomes. These results suggest that in testis treated with carbendazim acrosome development is impaired during the early phases of spermiogenesis, and material supply from the Golgi apparatus to the acrosome is perturbed, which is a possible cause of the abnormal development. Received: 31 March 1998 / Accepted: 28 May 1998     

Cell Surface Galactosylation Is Essential for Nonsexual Flocculation in Schizosaccharomyces pombe

We have isolated fission yeast mutants that constitutively flocculate upon growth in liquid media. One of these mutants, the gsf1 mutant, was found to cause dominant, nonsexual, and calcium-dependent aggregation of cells into flocs. Its flocculation was inhibited by the addition of galactose but was not affected by the addition of mannose or glucose, unlike Saccharomyces cerevisiae FLO mutants. The gsf1 mutant coflocculated with Schizosaccharomyces pombe wild-type cells, while no coflocculation was found with galactose-deficient (gms1Δ) cells. Moreover, flocculation of the gsf1 mutant was also inhibited by addition of cell wall galactomannan from wild-type cells but not from gms1Δ cells. These results suggested that galactose residues in the cell wall glycoproteins may be receptors of gsf1-mediated flocculation, and therefore cell surface galactosylation is required for nonsexual flocculation in S. pombe.     

Anucleated cryptophyte vestiges in the gonyaulacalean dinoflagellates Amylax buxus and Amylax triacantha (Dinophyceae)

Cryptophyte vestiges showing selective digestion of nuclei were found in the gonyaulacalean dinoflagellates Amylax buxus (Balech) Dodge and Amylax triacantha (Jörgensen) Sournia. They emitted bright yellow‐orange fluorescence (590‐nm emission) under epifluorescent microscopy and possessed U‐shaped plastids, suggesting the vestiges were active in photosynthesis. Under transmission electron microscopy, the plastid was characterized by a loose arrangement of two to three thylakoid stacks and included a stalked pyrenoid, as in the cryptophyte genus Teleaulax. Indeed, molecular data based on the plastid small‐subunit rRNA gene demonstrated that the vestiges in Amylax originated from Teleaulax amphioxeia. The stolen plastid (kleptoplastids) in Dinophysis is also derived from this cryptophyte species. However, in sharp contrast to Dinophysis, the plastid of the vestige in Amylax was surrounded by a double layer of plastid endoplasmic reticulum, and within the periplastidal area, a nucleomorph was retained. The vestiges also possessed mitochondria with characteristic plate‐like cristae, but lost the cell‐surface structure. The phagocytotic membrane of the dinoflagellates seemed to surround the cryptophytes right after the incorporation, but the membrane itself would probably be digested eventually. Remarkably, only one cryptophyte cell among 14 vestiges in a cell of A. buxus had a nucleus. This is the first recording of possible kleptoplastidy in gonyaulacalean dinoflagellates, and documents the strategy of a dinoflagellate involving the selective elimination of the cryptophyte nucleus.     

Motif-programmed artificial extracellular matrix

Motif-programming is a method for creating artificial proteins by combining functional peptide motifs in a combinatorial manner. This method is particularly well suited for developing liaison molecules that interface between cells and inorganic materials. Here we describe our creation of artificial proteins through the programming of two motifs, a natural cell attachment motif (RGD) and an artificial Ti-binding motif (minTBP-1). The created proteins were found to reversibly bind Ti and to bind MC3T3-E1 osteoblast-like cells. Moreover, although the interaction with Ti was not covalent, the proteins recapitulated several functions of fibronectin, and thus, could serve as an artificial ECM on Ti materials. Because this motif-programming system could be easily extended to create artificial proteins having other biological functions and material specificities, it should be highly useful for application to tissue engineering and regenerative medicine.     

GEF-H1 Mediated Control of NOD1 Dependent NF-κB Activation by Shigella Effectors

Shigella flexneri has evolved the ability to modify host cell function with intracellular active effectors to overcome the intestinal barrier. The detection of these microbial effectors and the initiation of innate immune responses are critical for rapid mucosal defense activation. The guanine nucleotide exchange factor H1 (GEF-H1) mediates RhoA activation required for cell invasion by the enteroinvasive pathogen Shigella flexneri. Surprisingly, GEF-H1 is requisite for NF-κB activation in response to Shigella infection. GEF-H1 interacts with NOD1 and is required for RIP2 dependent NF-κB activation by H-Ala-D-γGlu-DAP (γTriDAP). GEF-H1 is essential for NF-κB activation by the Shigella effectors IpgB2 and OspB, which were found to signal in a NOD1 and RhoA Kinase (ROCK) dependent manner. Our results demonstrate that GEF-H1 is a critical component of cellular defenses forming an intracellular sensing system with NOD1 for the detection of microbial effectors during cell invasion by pathogens.     

Cryptococcus neoformans inhibits nitric oxide synthesis caused by CpG-oligodeoxynucleotide-stimulated macrophages in a fashion independent of capsular polysaccharides

Cryptococcus neoformans is eradicated by macrophages via production of NO. Unmethylated CpG-ODN protect mice from infection with this fungal pathogen by inducing IFN-gamma. The present study was designed to elucidate the effect of C. neoformans on the synthesis of NO by alveolar macrophages. For this purpose, MH-S, an alveolar macrophage cell line, was stimulated with CpG-ODN in the presence of IFN-gamma. A highly virulent strain of C. neoformans with thick capsule suppressed the production of NO. Capsular polysaccharides were not essential for this suppression, because there was no difference between acapsular mutant (Cap67) and its parent strain. Physical or close interaction of Cap67 with MH-S was necessary, as shown by the loss of such effect when direct contact was interfered by nitrocellulose membrane. Similar effects were observed by disrupted as well as intact Cap67. Whereas the inhibitory effect of intact Cap67 was completely abrogated by heat treatment, disrupted Cap67 did not receive such influence. Finally, disrupted Cap67 did not show any inhibitory effect on the TLR9-mediated activation of NF-kappaB in a luciferase reporter assay with HEK293T cells, although the TLR4-mediated activation was suppressed. These results revealed that C. neoformans suppressed the synthesis of NO by CpG-ODN and IFN-gamma-stimulated macrophages in a fashion independent of capsular polysaccharides, although the precise mechanism remains to be elucidated.