The asparagine-linked sugar chains of human follicle-stimulating hormone

The asparagine-linked sugar chains of human follicle-stimulating hormone (hFSH) were liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by N-acetylation and NaB3H4 reduction. Ninety-five percent of the oligosaccharides were acidic and all were converted to a mixture of neutral oligosaccharides on sialidase treatment. The mixture of neutral oligosaccharides was subjected to sequential immobilized lectin column chromatography on E-PHA-agarose, AAL-Sepharose, and Con A-Sepharose, and six fractions were obtained. The results of sequential exoglycosidase digestion of each oligosaccharide and methylation analysis led us to propose that the asparagine-linked sugar chains of hFSH are a mixture of complex-type bi-, tri-, and tetraantennary sialylated sugar chains with and without a fucose residue linked at the C-6 position of the proximal N-acetylglucosamine. Some of these sugar chains contain bisecting N-acetylglucosamine residue.     

Relationship among function, phenotype, and specificity in primary allospecific T cell populations: identification of phenotypically identical but functionally distinct primary T cell subsets that differ in their recognition of MHC class I and class II allodeterminants

The goal of the present study was to evaluate the relationship among function, Lyt phenotype, and MHC recognition specificity in primary allospecific T cell populations. By using Lyt-2+ and L3T4+ T cells obtained from the same responder populations, we assessed the ability of T cells of each phenotype to generate cytotoxic effector cells (CTL) and IL 2-secreting helper T cells in response to either class I or class II MHC allodeterminants. It was found that a discordance between Lyt phenotype and MHC recognition specificity does exist in primary allospecific T cells, but only in one T cell subpopulation with limited functional potential: namely, Lyt-2+ T cells with cytotoxic, but not helper, function that recognize class II MHC alloantigens. Target cell lysis by these Lyt-2+ class II-allospecific CTL was inhibited by anti-Ia monoclonal antibodies (mAb), but not anti-Lyt-2 mAb, indicating that they recognized class II MHC determinants as their "restriction" specificity and not as their "nominal" specificity even though they were Lyt-2+. A second allospecific T cell subset with limited functional potential was also identified but whose Lyt phenotype and MHC restriction specificity were not discordant: namely, an L3T4+ T cell subset with helper, but not cytotoxic, function specific for class I MHC allodeterminants presented in the context of self-Ia. Thus, the present study demonstrates that primary allospecific T cell populations contain phenotypically identical subpopulations of helper and effector cells that express fundamentally different MHC recognition specificities. Because the recognition specificities expressed by mature T cells reflect the selection pressures they encountered during their differentiation into functional competence, these findings suggest that functionally distinct but phenotypically identical T cell subsets may be selected independently of one another during ontogeny. Thus, the existence of Lyt-2+ CTL specific for class II allodeterminants can be explained by the hypothesis that the association of Lyt phenotype with MHC recognition specificity results from the process of thymic selection that these Lyt-2+ effector cells avoid.     

Structures of the sugar chains of mouse immunoglobulin G

The asparagine-linked sugar chains of mouse immunoglobulin G (IgG) were quantitatively liberated as radioactive oligosaccharides from the polypeptide portions by hydrazinolysis followed by N-acetylation, and NaB3H4 reduction. After fractionation by paper electrophoresis, lectin (RCA120) affinity high-performance liquid chromatography, and gel filtration, their structures were studied by sequential exoglycosidase digestion in combination with methylation analysis. Mouse IgG was shown to contain the biantennary complex type sugar chains. Eight neutral oligosaccharide structures, viz, +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1---- 4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc, were found after the sialidase treatment. The molar ratio of the sugar chains with 2,1, and 0 galactose residues was 2:5:3. The galactose residue in the monogalactosylated sugar chains was distributed on Man alpha 1----3 and Man alpha 1----6 sides in the ratio of 1:3. The oligosaccharides were almost wholly fucosylated and contained no bisecting N-acetylglucosamine which is present in human, rabbit, and bovine IgGs.     

Carbohydrates of lysosomal enzymes secreted by Tetrahymena pyriformis

The carbohydrate structures of acid phosphatase and alpha-glucosidase secreted into culture medium by Tetrahymena pyriformis strain W were studied. Their asparagine-linked sugar chains were quantitatively liberated as radioactive oligosaccharides from their polypeptide moieties by controlled hydrazinolysis followed by N-acetylation and NaB3H4 reduction. The approximate amounts of total sugar chains liberated from 1 mol each of acid phosphatase and alpha-glucosidase were 6 and 4 mol, respectively. Paper electrophoresis revealed that only neutral oligosaccharides were obtained from both enzymes. The oligosaccharide fraction from acid phosphatase was separated into seven components by Bio-Gel P-4 column chromatography while that from alpha-glucosidase was resolved into three components. The structures of these oligosaccharides were determined by sequential glycosidase digestion in combination with methylation analysis. The sugar chains of the two enzymes can be primarily classified as high mannose-type oligosaccharides. However, they have the following characteristic features: 1) their common core is not the usual Man5 . GlcNAc2 structure, it is Man3 . GlcNAc2; 2) some of the sugar chains of acid phosphatase have 1 approximately 3 glucose residues linked to the nonreducing terminal Man alpha 1----2 residue. The structural characteristics of the sugar moieties of the two enzymes indicate that they might be produced by the so-called "alternate pathway," in which lipid-linked Glc3 . Man5 . GlcNAc2 functions as an oligosaccharide donor.     

Ammonia fungi of iriomote Island in the southern Ryukyus,Japan and a new ammonia fungus,Hebeloma luchuense

Species composition and fruiting season of ammonia fungi were investigated in Iriomote Island of the southern Ryukyus, Okinawa Prefecture, Japan.Castanopsis andPinus forests were surveyed and 10 species of ammonia fungi were collected, including one new agaric species,Hebeloma luchuense sp. nov. This new fungus is characterized by having a rooting, squamulose-scaly stipe and cortinate veil and forms ectomycorrhizae withCastanopsis cuspidata var.sieboldii. Although the general mushroom season in theCastanopsis forest in Iriomote island was very short and restricted to summer, ammonia fungi were observed to fruit throughout the year in urea-treated plots.     

Purification and Characterization of Membrane Protein (90 kDa) from Mycoplasma salivarium,Which Binds Immunoglobulin (Ig) G Fc Fragment

A 90 kDa protein of Mycoplasma salivarium was released from cell membranes of the organism with Triton X-100 and purified by ion-exchange chromatography and chromatofocusing. The protein was eluted at pH 5.5 by chromatofocusing. The protein was shown to react with the Fc fragments of IgG from human and nine different animal species and did not distinguish between IgG from different species, while protein A, tested for comparative purposes, displayed a strong specificity for human and swine IgG. Furthermore, the protein reacted with antigen specific goat IgG (specific for gamma chains of human IgG), sheep red blood cells (SRBC) sensitized with rabbit antiserum to SRBC, that is, the Fc part of rabbit IgG, and concanavalin A as well. These findings may suggest that the protein is a lectin which binds the carbohydrate moiety of the Fc part of IgG.     

Embryonic Development and Postnatal Changes in Free d-Aspartate and d-Serine in the Human Prefrontal Cortex

Abstract: We have analyzed free chiral amino acids (aspartate and serine) in the human frontal cortex at different ontogenic stages (from 14 weeks of gestation to 101 years of age) by HPLC with fluorometric detection after derivatization with N-tert -butyl-oxycarbonyl- l -cysteine and o -phthaldialdehyde. Exceptionally high levels of free d -aspartate and d -serine were demonstrated in the fetal cortex at gestational week 14. The ratios of d -aspartate and of d -serine to the total corresponding amino acids were also high, at 0.63 and 0.27, respectively. The concentration of d -aspartate dramatically decreased to a trace level by gestational week 41 and then remained very low during all postnatal stages. In contrast, the frontal tip contained persistently high levels of d -serine throughout embryonic and postnatal life, whereas the d -amino acid content in adolescents and aged individuals was about half of that in the fetuses. Because d -aspartate and d -serine are known to have selective actions at the NMDA-type excitatory amino acid receptor, the present data suggest that these d -amino acids might play a pivotal role in cerebral development and functions that are related to the NMDA receptor.     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Requirement of ceramide for adhesion of Helicobacter pylori to glycosphingolipids

The endogenous metabolites of the coelomic fluid of the earthworm Eisenia veneta were characterised using high-resolution one-dimensional and two-dimensional 1H nuclear magnetic resonance spectroscopy. Signals from common organic acids, such as acetate, fumarate, malonate, malate, formate, and succinate, were identified together with adenosine and nicotinamide mononucleotide. The potential use of this information as a baseline dataset for future toxicological or physiological studies was demonstrated by a metabonomic analysis: a series of earthworms were dosed with the model compound 3-fluoro-4-nitrophenol, and toxic effects followed by multivariate analysis of the spectral data of the coelomic fluid. Relative concentrations of acetate and malonate were decreased in the dosed worms compared to the controls.