Chromatographic separation of asparagine-linked oligosaccharides labeled with an ultravioletabsorbing compound,p-aminobenzoic acid ethyl ester

We have expanded on the suitability ofp-aminobenzoic acid ethyl ester as an ultraviolet-absorbing reagent [Wanget al., (1984) Anal Biochem 141:366–81] for the analysis of asparagine-linked oligosaccharides derived from glycoproteins. The oligosaccharides released from glycoproteins by hydrazinolysis/N-reacetylation were derivatized withp-aminobenzoic acid ethyl ester and the derivatives were purified and separated into neutral and acidic oligosaccharides on a PRE-SEP C₁₈ cartridge. The acidic oligosaccharides could be further separated into a few species by high-voltage paper electrophoresis. p-Aminobenzoic acid ethyl ester derivatives of neutral oligosaccharides were analyzed by gel permeation chromatography on Bio-Gel P-4 and HPLC on a silica-based amide column. The elution profile and the proportion of the oligosaccharides were in agreement with literature values. The overall yield of oligosaccharides from glycoproteins was approximately 70%. Fifty pmol of oligosaccharide were detectable on Bio-Gel P-4 and 4–5 pmol on HPLC.Abbreviations HPLC high performance liquid chromatography - NABEE p-aminobenzoic acid ethyl ester - FAB-MS fast-atom bombardment mass spectrometry - (GlcNAc)₂, (GlcNAc)₃, (GlcNAc)₄, (GlcNAc)₅ and (GlcNAc)₆ chito-oligosaccharides containing 2,3,4,5 and 6 residues ofN-acetylglucosamine     

Translation products of mRNA from infective larvae of Trichinella spiralis include an antigenic polypeptide

Total RNA was extracted from packed infective larvae of Trichinella spiralis by centrifugation through a 5.7 M caesium chloride cushion. Polyadenylated messenger RNA was separated from total RNA in an oligothymidylic acid-cellulose gel column. The in vitro translation of the mRNA, isolated from infective larvae of T. spiralis, was carried out using the rabbit reticulocyte cell-free translation system. Incorporation of 35S-methionine into the trichloroacetic acid precipitates in the lysate containing mRNA was 5 times greater than that in control. The translation products were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Many polypeptides with molecular weights of less than 100,000 were synthesized in the lysate. A T. spiralis positive mouse serum was mixed with translation products to form antigen-antibody complexes, which were then absorbed by Staphylococcus aureus Cowan 1 strain and analysed by autoradiography of SDS-PAGE. An antigenic polypeptide with a molecular weight of 48,000 was demonstrated to react specifically with IgG antibody in T. spiralis positive mouse serum. T. spiralis larvae were cultured in methionine-free medium containing 35S-methionine, and antigenic polypeptides in somatic extracts and ES products were compared with those in translation products by autoradiography of SDS-PAGE. Several polypeptides in ES products and somatic extracts reacted specifically with IgG antibodies in positive serum. Especially the polypeptide with a molecular weight of 48,000 in ES products strongly reacted with IgG antibody in positive serum.     

Restriction fragment length polymorphisms of Anisakinae larvae

The analysis of restriction fragment length polymorphisms (RFLPs) was applied to distinguish several kinds of Anisakinae larvae, Anisakis larvae (type I) collected from two different paratenic hosts, Anisakis larvae (type II) and Contracaecum larvae. The patterns of the two different paratenic host-derived DNA of Anisakis larva (I) were exactly the same in hybridized fragments generated by six endonucleases. The quite different patterns in RFLPs of genomic DNA were observed among the Anisakis larva (I), Anisakis larva (II) and Contracaecum larvae. The results suggest that the RFLPs analysis may be useful for distinguishing Anisakinae larvae and clarifying the relationships between Anisakis larvae and their adult worms.     

Toxicity of Microcystis species isolated from natural blooms and purification of the toxin.

Microcystis strains (2 toxic and 18 nontoxic to mice) were isolated from toxic waterblooms that had been collected from Lake Kasumigaura, Ibaraki Prefecture, Japan, in August 1985. Thirteen of the strains (2 toxic and 11 nontoxic) were Microcystis aeruginosa, 2 (nontoxic) were Microcystis wesenbergii, and the other 5 were difficult to identify. Six (1 toxic and 4 nontoxic M. aeruginosa and 1 M. wesenbergii) of these 20 strains were established as axenic cultures. A toxic and axenic strain of M. aeruginosa, K-139, was used to study the relationship between growth conditions and toxicity. Cells in early-to-mid-log phase showed the highest toxicity (50% lethal dose, 7.5 mg of cells per kg of mouse), and maximum toxicity was not affected by growth temperatures between 22 and 30 degrees C. Purification and characterization of the toxins from K-139 cells were also conducted, and at least two toxins were detected. One of the toxins (molecular mass, 980 daltons) has not been reported previously. The main target of the toxin in mice was the liver. Marked congestion and necrosis in the parenchymal cells around the central veins of the liver were observed microscopically in specimens that had been prepared from the mice with acute toxicity after injection with the toxin.     

Biological activities of chemically synthesized partial structure analogues of lipid A

Analogues of the nonreducing sugar part of lipid A were chemically synthesized and tested for biological activities such as Limulus amebocyte lysate gelation, interferon- and tumor necrosis factor-induction, lethal toxicity in galactosamine-sensitized mice, and pyrogenicity. A 4-O-monophosphorylglucosamine derivative possessing 2-N-3-tetradecanoyl-oxytetradecanoyl and 3-O-tetradecanoyl groups (GLA-27) exhibited all activities tested except for pyrogenicity. Alteration of the acyl substituents or dephosphorylation as well as acylation or phosphorylation of the 6-OH caused most activities of GLA-27 to diminish or disappear altogether. On the other hand, the biological activities expressed by GLA-27 were not significantly affected even when the glucosamine backbone was changed to 1-deoxy type, epimer type at C-3 (allose form), or 3-amino type. These results indicate that the acyl substituents and the phosphorylation positions rather than the backbone structures in these partial structure analogues of lipid A affect the expression of biological activities of endotoxin. The results also clearly indicate that some biological activities of endotoxin can be expressed separately from pyrogenicity.     

Structural requirements of lipid A responsible for the functions: a study with chemically synthesized lipid A and its analogues

To confirm the revised lipid A structure of Escherichia coli and to establish the structure responsible for its functions, biological activities of the synthetic compounds based on the presented structure of E. coli lipid A were investigated. Compound 506, 2-deoxy-6-O-(2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-3-O [(R)3-tetradecanoyloxytetradecanoyl]-beta-D-glucopyranosyl]-3-O-[(R) -3-hydroxytetradecanoyl]-2-[(R)-3-hydroxytetradecanoylamino]-alpha -D-glucopyranose 1,4'-bis(phosphate), exhibited activities identical to those of natural E. coli lipid A in eliciting Shwartzman reaction and tests on lethality, pyrogenicity, interferon- and tumor necrosis factor-inducing activities as well as in B-cell activating activity and Limulus amebocyte lysate gelating activity. With the exception of the Shwartzman reaction the monophosphorylated synthetic compounds at either the 1 or 4' position showed slightly lower activities than the compound with the bisphosphorylated compound (Compound 506). The compound without the phosphate group showed no or only very weak activities. The structural requirements for each activity (i.e. binding position and composition of fatty acids and presence of phosphate groups) are discussed taking into account the results of previous investigations.     

Stereochemistry of biopterin cofactor and facile methods for the determination of the stereochemistry of a biologically active 5,6,7,8-tetrahydropterin

The structure of (-)-tetrahydrobiopterin, the cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases, was determined as (6R, 1'R, 2'S)-6-(1',2'-dihydroxypropyl)-5,6,7,8-tetrahydropterin by X-ray crystallographic analysis. The CD spectra of (-)-tetrahydrobiopterin exhibits a negative Cotton effect at 290-240 nm in pH 3.2 solution. The relationship of the negative Cotton effect and the 6R configuration was supported by the application of CD analysis with 2-methyltetrahydronaphthalene as a model compound. The stereochemistries at the C(6) position of various biologically active 6-substituted tetrahydropterins were determined from the CD spectra. The relative configuration of two asymmetric centers, C(6), and C(1'), was determined by HPLC analysis on a strong cation exchange column.     

Poor physical fitness is independently associated with mild cognitive impairment in elderly Koreans

The purpose of this study was to investigate the association between physical fitness and mild cognitive impairment (MCI) in elderly Koreans. This was a cross-sectional study that involved 134 men and 299 women aged 65 to 88 years. Six senior fitness tests were used as independent variables: 30 s chair stand for lower body strength, arm curl for upper body strength, chair-sit-and-reach for lower body flexibility, back scratch for upper body flexibility, 8-ft up-and-go for agility/dynamic balance, and 2-min walk for aerobic endurance. Global cognitive function was assessed using the Korean version of the Mini-Mental State Examination (MMSE). Potential covariates such as age, education levels, blood lipids, and insulin resistance (IR) markers were also assessed. Compared to individuals without MMSE-based MCI, individuals with MMSE-based MCI had poor physical fitness based on the senior fitness test (SFT). There were significant positive trends observed for education level (p=0.001) and MMSE score (p<0.001) across incremental levels of physical fitness in this study population. Individuals with moderate (OR=0.341, p=0.006) and high (OR=0.271, p=0.007) physical fitness based on a composite score of the SFT measures were less likely to have MMSE-based MCI than individuals with low physical fitness (referent, OR=1). The strength of the association between moderate (OR=0.377, p=0.038) or high (OR=0.282, p=0.050) physical fitness and MMSE-based MCI was somewhat attenuated but remained statistically significant even after adjustment for the measured compounding factors. We found that poor physical fitness was independently associated with MMSE-based MCI in elderly Koreans.     

Owstonia kamoharai (Perciformes: Cepolidae), a new bandfish from Japan

A new owstoniine bandfish, Owstonia kamoharai, is described based on six specimens (147–402 mm in standard length) collected from off southern Japan: Shikoku Island, Kii Peninsula, and Suruga Bay. The new species differs from its congeners by the following combination of characters: dorsal-fin rays III, 21; anal-fin rays I, 14; pectoral-fin rays 21–23; total caudal-fin rays 17 (branched 13); vertebrae 28–29; gill rakers on first arch 39–40; cheek with 88–123 scales in 8–10 rows; lateral line without branch, not forming loop in front of dorsal fin, ending below 8th–10th dorsal-fin soft rays; scales in longitudinal rows 53–56; lower margin of preopercle smooth; pelvic fin short, not reaching to anus; posterior margin of caudal fin rounded; uniformly reddish coloration with black markings on membranes of both jaws; and white margins of vertical fins.