N-glycan structures of a recombinant mouse soluble Fcγ receptor II

N-glycans of a recombinant mouse soluble Fc receptor II (sFcRII) expressed in baby hamster kidney cells were released from glycopeptides by digestion with glycoamidase A (from sweet almond), and the reducing ends of the oligosaccharides were reductively aminated with 2-aminopyridine. The derivatized N-glycans were separated and structurally identified by a three-dimensional high-performance liquid chromatography (HPLC) mapping technique on three kinds of HPLC columns [Takahashi, et al. (1995) Anal. Biochem. 226: 139–46]. Eighteen different major N-glycan structures were identified, of which six were neutral (45%), five mono-sialyl (49%), one di-sialyl (4.6%), five tri-sialyl (1.1%), and one tetra-sialyl (0.3%). All N-glycan structures determined were complex type with fucosylation at the N-acetylglucosamine residue of the reducing end, and N-acetylneuraminic acid, when present, was -(2,3)-linked. The existence of a unique structure containing both N-acetylgalactosamine and -(2,3)-N-acetylneuraminic acid residues at the reducing ends, as below, was confirmed by MALDI-TOF mass spectrometry.     

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.     

A non-destructive screenable marker,OsFAST, for identifying transgenic rice seeds

The production of transgenic plants has contributed greatly to plant research. Previously, an improved method for screening transgenic Arabidopsis thaliana seeds using the FAST (Fluorescence-Accumulating-Seed Technology) method and FAST marker was reported. Arabidopsis seeds containing the FAST marker may be visually screened using a fluorescence stereomicroscope or blue LED handy-type instrument. Although the FAST method was originally designed for Arabidopsis screens, this study endeavors to adapt this method for the screening of other plants. Here, an optimized technology, designated the OsFAST method, is presented as a useful tool for screening transgenic rice seeds. The OsFAST method is based on the expression of the OsFAST-G marker under the control of a seed-embryo-specific promoter, similar to the Arabidopsis FAST-G marker. The OsFAST method provides a simple and non-destructive method for identifying transgenic rice seeds. It is proposed that the FAST method is adaptable to various plant species and will enable a deeper analysis of the floral-dip method.Key words: Oryza sativa, oleosin, seed, green fluorescent protein, transformation, screenable markerThe production of transgenic plants has significantly enhanced many areas of plant science research. Antibiotic/herbicide-resistance genes are traditionally used as screenable markers for the selection of transgenic plants. However, this approach does have disadvantages. First, antibiotics or herbicides occasionally inhibit the growth of transgenic plants, regardless of the incorporation of antibiotic- or herbicide-resistance genes¹ into the transgenic plants. Second, the identification of resistant transgenic plants requires that the seed population be sown onto plates containing antibiotics or herbicides. Third, the selection process is slow and labor intensive, often involving the screening of vast numbers of potentially transgenic seeds on selective plates.To overcome these disadvantages, an improved approach for selecting transgenic Arabidopsis thaliana, designated the FAST (Fluorescence-Accumulating-Seed Technology) method, was developed. This method employs the use of a fluorescent protein that is expressed in seeds and used as a visual screenable marker for the identification of transgenic seeds. The seed-specific protein oleosin, a family of oil-body-membrane proteins,² has an important role as a size regulator of oil bodies.³ AtOLE1, the most abundant oleosin, functions in the freezing tolerance of Arabidopsis seeds.⁴ A plasmid containing an AtOLE1-GFP fusion gene controlled by the AtOLE1 promoter was constructed and designated the FAST-G (Fluorescence-Accumulating-Seed Technology with OLE1-GFP) marker. Interestingly, Arabidopsis seeds containing the FAST-G marker emitted clear fluorescence under a fluorescence stereomicroscope or blue LED handy-type instrument. The transgenic seeds were visually identified by the seed fluorescence without the use of antibiotics or herbicides, thus indicating that the FAST method offers a nondestructive approach. The FAST marker permits the identification of homozygous seeds among the T2 population with a false discovery rate of less than 1% as a co-dominant screenable marker. In contrast to conventional methods using antibiotics or herbicides, the FAST method reduces the amount of time required to acquire homozygous transgenic plants from 7.5 months to 4 months. The fluorescence of the FAST-G marker was limited to a specific organ (i.e., in seeds) and a specific time (i.e., during dormancy), desirable characteristics of selectable and/or screenable markers. Furthermore, the FAST marker does not require sterile seeding and the handling of large numbers of plants.     

Expression of a functional single-chain antibody against GA24/19 in transgenic tobacco.

An anti-gibberellin A24/19 single-chain Fv gene was constructed from gamma and kappa genes cloned from a hybridoma cell line producing monoclonal antibody against gibberellin A24/19, biosynthetic precursors of gibberellin A4/1 which are biologically active per se. The single-chain Fv gene was introduced into tobacco plants after the binding activity of the single-chain Fv expressed in Escherichia coli was confirmed. When the single-chain Fv expression is targeted to endoplasmic reticulum, the plants could accumulate the single-chain Fv protein with the antigen binding activity up to 3.6% of the total soluble protein. On the other hand, when the expression is targeted to cytosol, accumulation of the single-chain Fv protein was not detected at all. The dwarf phenotype of the transgenic plants expressing the single-chain Fv protein, together with the preliminary analytical data indicating a decreased level of gibberellin A1 in the dwarf transgenics, suggested that the single-chain Fv decreased the concentration of bioactive gibberellins by trapping and inhibiting the metabolism of gibberellin A24 and/or A19 to gibberellin A4 and/or A1.     

Characterization of human genomic DNA sequences homologous to the interleukin 2 cDNA

Southern blot analysis of human placental DNA under low stringency hybridization conditions revealed several DNA fragments hybridizable to the human interleukin 2 (IL-2) cDNA. Four phage clones carrying these IL-2 cDNA-like sequences were isolated and their structures analyzed. A DNA fragment derived from one of the clones gave the strongest hybridization signal. Sequence analysis of this fragment revealed the presence of a cluster of three DNA segments, i.e. 20 base pairs (bp), 57 bp and 18 bp in length and having about 85%, 80% and 83% homology to three different parts of the coding region of human IL-2 cDNA, respectively.     

Effects of selenium status and supplementary seleno-chemical sources on mouse T-cell mitogenesis

Although selenium is thought to be essential for various immune responses, the excess supplementation may have an adverse effect on certain immunological functions. The present study was designed to determine the effective chemical forms of selenium and their optimal levels on T-cell mitogenesis with splenic cells from mice given a selenium-deficient diet for 8 weeks to avoid effects of cellular selenium sources. Although selenium in tissues, except for spleen and thymus, was almost depleted by feeding selenium-deficient diet, the lymphoid organs still contained low levels of selenium. Both activities of cellular glutathione peroxidase (cGPx) and thioredoxin reductase (TR) in liver and splenic cells showed a tendency to decrease by selenium deficiency. However, splenic cells were tolerant against decrease of the selenoenzyme activities, and TR was also more tolerant than cGPx. T-cell proliferation of the selenium-insufficient splenic cells induced by concanavalin A was increased by addition of Na₂SeO₃, Na₂SeO₄, Na₂Se, seleno-dl-cystine, seleno-l-methionine and selenocystamine. Their promoting action was observed at levels lower than 0.1 μmol/L and was completely suppressed at the highest concentration (1 μmol/L), except for selenocystamine. Na₂SeO₃ was one of the efficient selenocompounds for the mitogenesis, which was concomitant with the significant induction of cGPx and TR. However, recovery of cGPx activity in the selenium-insufficient cells by supplementary Na₂SeO₃ was only partial, while TR activity was readily recovered from selenium deficiency. These results therefore indicate that only low levels of selenium is essential for T-cell mitogenesis even in selenium-insufficient splenic cells, and TR, which is readily recovered by Na₂SeO₃, may be the critical enzyme.     

Persistent High Numbers of Clostridium perfringens in the Intestines of Japanese Aged Adults

TSN agar was applicable for enumeration of Clostridium perfringens in fecal samples of adults but not in those of infants. It was demonstrated using TSN agar that some healthy aged adults had persistently carried C. perfringens at levels ranging from 10⁷ to 10⁹, while some others ranged from 10³ to 10⁶ per ml volume of fecal sample although all of these adults had the same diets. In the test for agglutinability of isolates of C. perfringens collected from two elderly adults, a younger adult and a baby, it was demonstrated that most of the isolates obtained from an aged adult of high levels for 19 months belonged the same serotype, while rapid alteration of serotypes could be observed in three other persons with high or low levels. In spite of as many as 10⁹ C. perfringens per ml of feces, no trace of α-toxin could be detected in the fecal samples. In in vitro tests, fecal suspension suppressed the production of α-toxin although it allowed the organism to grow sufficiently.