Attachment structures in Rhizotrochus (Scleractinia): macro‐ to microscopic traits and their evolutionary significance

Marine sessile benthic organisms living on hard substrates have evolved a variety of attachment strategies. Rhizotrochus (Scleractinia, Flabellidae) is a representative azooxanthellate solitary scleractinian coral with a wide geographical distribution and unique attachment structures; it firmly attaches to hard substrates using numerous tube‐like rootlets, which are extended from a corallum wall, whereas most sessile corals are attached by stereome‐reinforced structures at their corallite bases. Detailed morphological and constructional traits of the rootlets themselves, along with their evolutionary significance, have not yet been fully resolved. Growth and developmental processes of spines in Truncatoflabellum and rootlets in Rhizotrochus suggest that these structures are homologous, as they both develop from the growth edges of walls and are formed by transformation of wall structures and their skeletal microstructures possess similar characteristics, such as patterns of rapid accretion and thickening deposits. Taking molecular phylogeny and fossil records of flabellids into consideration, Rhizotrochus evolved from a common free‐living ancestor and invaded hard‐substrate habitats by exploiting rootlets of spines origin, which were adaptive for soft‐substrate environments.     

Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector

We have developed a system for stable germline transformation in the silkworm Bombyx mori L. using piggyBac, a transposon discovered in the lepidopteran Trichoplusia ni. The transformation constructs consist of the piggyBac inverted terminal repeats flanking a fusion of the B. mori cytoplasmic actin gene BmA3 promoter and the green fluorescent protein (GFP). A nonautonomous helper plasmid encodes the piggyBac transposase. The reporter gene construct was coinjected into preblastoderm eggs of two strains of B. mori. Approximately 2% of the individuals in the G1 broods expressed GFP. DNA analyses of GFP-positive G1 silkworms revealed that multiple independent insertions occurred frequently. The transgene was stably transferred to the next generation through normal Mendelian inheritance. The presence of the inverted terminal repeats of piggyBac and the characteristic TTAA sequence at the borders of all the analyzed inserts confirmed that transformation resulted from precise transposition events. This efficient method of stable gene transfer in a lepidopteran insect opens the way for promising basic research and biotechnological applications.     

Dielectric relaxation of DNA in aqueous solutions.

Using a four-electrode cell and a new electronic system for direct detection of the frequency differences specturm of solution impedance, the complex dielectric constant of calf thymus DNA (M_r = 4 × 10⁶) in aqueous NaCl at 10°C is measured at frequencies ranging from 0.2 Hz to 30 kHz. The DNA concentrations are C_p = 0.01% and 0.05%, and the NaCl concentrations are varied from C_s = 10^?4 M to 10^?3 M. A single relaxation regions is found in this frequency range, the relaxation frequency being 10 Hz at C_p = 0.01% and C_s = 10^?3 M. At C_p = 0.05% it is evidenced that the DNA chains have appreciable intermolecular interactions. The dielectric relaxaton time τ_d at C_p = 0.01% agrees well with the rotational relaxation time estimated from the reduced visocisty on the assumption that the DNA is not representable as a rigid rod but a coiled chain. It is concluded that the dielectric relaxiatioinis ascribed to the rotation of the molecule. Observed values of dielectric increment and other experimental findings are reasonably explained by assuming that the dipole moment of DNA results from the slow counterion fluctuation which has a longer relaxation time than τ_d.     

Electrostatic engineering of the interface between heavy and light chains promotes antibody Fab fragment production

Monoclonal antibodies and antibody fragments are used for diverse diagnostic and therapeutic applications. We have investigated the secretory production of Fab fragments from insect cells cotransfected with plasmid vectors carrying heavy- and light-chain genes. In the present study, to promote the formation of the disulfide bond between the heavy and light chains, some positively charged amino acid residues were introduced near the cysteine residue for the disulfide bond at the C-terminus of C_L, while some negatively charged amino acid residues were added near the cysteine residue for the disulfide bond at the C-terminus of C_H1. This electrostatic steering led to an increase in Fab secretions from insect cells.     

Glycomics of a novel type-2 N-acetyllactosamine-specific lectin purified from the feather star, Oxycomanthus japonicus (Pelmatozoa: Crinoidea)

A lectin - designated OXYL for the purposes of this study that strongly recognizes complex-type oligosaccharides of serum glycoproteins - was purified from a crinoid, the feather star Oxycomanthus japonicus, the most basal group among extant echinoderms. OXYL was purified through a combination of anion-exchange and affinity chromatography using Q-sepharose and fetuin-sepharose gel, respectively. Lectin was determined to be a 14-kDa polypeptide by sodium dodecyl sulphate-polyacrylamide gel electrophoresis under reducing conditions. However, 14-kDa and 28-kDa bands appeared in the same proportion under non-reducing conditions. Gel permeation chromatography showed a 54-kDa peak, suggesting that lectin consists of four 14-kDa subunits. Divalent cations were not indicated, and stable haemagglutination activity was demonstrated at pH 4-12 and temperatures below 60°C. Surface plasmon resonance analysis of OXYL against fetuin showed k(ass) and k(diss) values of 1.4×10(-6)M(-1)s(-1) and 3.1×10(-3)s(-1), respectively, indicating that it has a strong binding affinity to the glycoprotein as lectin. Frontal affinity chromatography using 25 types of prydylamine-conjugated glycans indicated that OXYL specifically recognizes multi-antennary complex-type oligosaccharides containing type-2 N-acetyllactosamines (Galβ1-4GlcNAc) if α2-3-linked sialic acid is linked at the non-reducing terminal. However, type-1 N-acetyllactosamine (Galβ1-3GlcNAc) chains and α2-6-linked sialic acids were never recognized by OXYL. This profiling study showed that OXYL essentially recognizes β1-4-linkage at C-1 position and free OH group at C-6 position of Gal in addition to the conservation of N-acetyl groups at C-2 position and free OH groups at C-3 position of GlcNAc in N-acetyllactosamine. This is the first report on glycomics on a lectin purified from an echinoderm belonging to the subphylum Pelmatozoa.     

Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica

Acharan sulfate is a glycosaminoglycan (GAG), having the structure →4)-2-acetamido-2-deoxy-α- -glucopyranose(1→4)-2-sulfo-α- -idopyranosyluronic acid (1→, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3–5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.