The use of stable isotopes to trace small-scale movements by small fish species

Valuable biological information can be obtained by monitoring the movement of organisms. However, the choice of monitoring method becomes highly restricted when following small organisms (<100 mm), especially in aquatic ecosystems. Stable isotopes are being increasingly used in this respect but rarely at the local spatial scale, i.e. 10–1000 s of metres. We sought to identify movement of small fishes between a main river channel and its tributary. Little overlap in isotope baseline was detected between the two channels despite some temporal variability in δ¹⁵N of baseline indicator organisms in the main river. The individuals of two small cyprinid fish species (Leuciscus souffia and Alburnoides bipunctatus) of all the size classes (40–100 mm) caught within the tributary showed considerable heterogeneity in δ¹⁵N values. Classification and discriminant analysis on isotope-derived data distinguished two significantly different groups. Moreover, this result was supported by further sampling of fish caught in the main river (in May and December 2006). Alternative hypotheses, such as dietary differences, biological factors, temporal shifts and spatial differences in diet, did not explain δ¹⁵N variability. This application of stable isotopes at a relatively small spatial and temporal scales further demonstrates its potential as a tool for ecologists.     

Antitumour activities of levans produced by Zymomonas mobilis strains

Levans produced by four Zymomonas mobilis strains showed antitumour activity against sarcoma 180 and Ehrlich carcinoma in Swiss albino mice. Levans from two strains (ZAP and CP4) had the highest effects. NMR analysis showed that the polymers were composed only of fructose units. The results suggested that the antineoplasic effect is associated to the polysaccharide molecular weight and that a particular molecular weight range may be responsible for this effect.     

Studies on the mechanism of stimulation of T cells by the Mycoplasma arthritidis-derived mitogen. Role of class II IE molecules

A mitogen derived from the supernatant of broth cultures of Mycoplasma arthritidis (MAS-P) stimulates a proliferative response by normal, unprimed T cells and interleukin 2 production by some, but not all, T cell hybridomas. The response requires an IE-positive accessory cell (AC). The direct participation of IE, and not IA, in this system was confirmed by two sets of experiments. First, L cells transfected with IE, but not IA, provided effective AC function for both normal T cells and the T cell hybridoma DO-11.10. Second, we have taken a more direct approach by showing that purified IE incorporated in liposomes and used to coat glass beads can support the MAS-P response of the DO-11.10 T cell hybridoma in the absence of intact AC or other AC molecules. Although the receptor for IE-MAS-P has not been identified, we have eliminated from consideration two potential T cell recognition structures. Monoclonal antibody to the antigen-major histocompatibility complex specific receptor failed to inhibit the MAS-P response of DO-11.10 or the T cell line LBRM-33. Furthermore, the L3T4 molecule did not appear to be involved since an L3T4-negative variant of DO-11.10 responded well to the mitogen. In addition, we show that both Lyt-2-positive and L3T4-positive T cells respond to this class II-restricted stimulus. Thus, we postulate the existence of a non-T cell receptor, non-L3T4 receptor that recognizes MAS-P in association with a presumed nonpolymorphic region of IE.     

Solution conformation of the branch points of N-linked glycans: synthetic model compounds for tri'-antennary and tetraantennary glycans

The solution conformation of model compounds for the tri'-antennary and tetraantennary (six-arm) branch point of N-linked glycans has been determined through the use of chemical shift, relaxation, and nuclear Overhauser enhancement data. The object was to establish the conformation about the glycosidic linkages in the N-linked substructure GlcNAc(beta 1,6) [GlcNAc(beta 1,2)] Man(alpha)- by estimation of values for the appropriate glycosidic torsional angles. The GlcNAc(beta 1,6) linkage in a trisaccharide model compound was found to be constrained to a narrow rotameric subpopulation about the substituted Man C5-C6 bond (omega = -60 degrees) and a narrow range of possible phi - psi values. Free rotation about the Man C5-C6 bond was obstructed by unfavorable steric interactions between the GlcNAc(beta 1,6) and GlcNAc(beta 1,2) residues. A phi, psi value of 55 degrees, 190 degrees was found to be consistent with the NMR data for the GlcNAc(beta 1,6) linkage. However, the value of psi appears to be "virtual" in that the molecule is in equilibrium between two different values (90 degrees and 252 degrees). For the GlcNAc(beta 1,2) linkage, complete agreement between all the observed NMR parameters and all the calculated ensemble average values could only be obtained with a set of potential energy functions which included hydrogen bonding. Other choices of potentials yielded calculated values that disagreed with at least two of the observed quantities. As a result, we infer that an interresidue hydrogen bond is formed, and we find it to be between the GlcNAc(beta 1,2) ring oxygen and the Man C3 hydroxyl.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Antigen presentation by splenic B cells: resting B cells are ineffective, whereas activated B cells are effective accessory cells for T cell responses

In this study, we have investigated the ability of splenic B cells to act as antigen-presenting cells. Previous data had established that lipopolysaccharide (LPS)-activated B cells were effective antigen-presenting cells; however, the relative capacity of resting B cells to carry out this function remains controversial. Splenic B cells from naive BALB/c mice were depleted of macrophages, dendritic cells, and T cells, and were fractionated on the basis of cell density by using Percoll gradient centrifugation. Fractions were collected from the 50/60, 60/65, and 65/72% interfaces and from greater than 72% (pellet). Cytofluorograph analysis of the fractionated B cells showed that the two lower density fractions (50/60 and 60/65) contained a number of cells which, by cell size determination, appeared to be activated B cells, whereas the two higher density fractions (65/72 and greater than 72) appeared to contain predominantly small resting B cells contaminated by many fewer activated B cells. Functionally, the capacity of fractionated B cells to act as accessory cells for a concanavalin A response or present the antigens chicken ovalbumin (OVA) or OVA-tryptic digest gave similar results, which indicated a striking hierarchy of accessory cell function in the different Percoll fractions. When normalized to the most active low-density fraction (50/60%), the activity of the other fractions were: 60/65 = 78%; 65/72 = 25%; and greater than 72 = 4%. The differences in the functional capacity between the various Percoll fractions did not appear to be due to differences in Ia expression. Although the expression of Ia varied approximately 12-fold within any one fraction, there was little difference in the mean amount of Ia on cells obtained from the various fractions. Kinetic studies showed that activation of B cells with LPS and dextran sulfate resulted in the expression of two stages of functional development. The first stage was an increased efficiency of accessory cell function that was abrogated by irradiation with 4000 rad followed by a second stage, which was characterized by the acquisition of resistance to treatment with 4000 rad. When nonfractionated B cells that had been stimulated with LPS and DexSO4 were sorted on the basis of cell size into a small B cell fraction and a large B cell fraction, only the large B cells were able to present antigen. Taken together, these data suggest that much of the accessory cell function associated with splenic B cells can be accounted for by the relatively small percentage of activated B cells present in the spleen.(ABSTRACT TRUNCATED AT 400 WORDS)     

The use of peptide analogs with improved stability and MHC binding capacity to inhibit antigen presentation in vitro and in vivo

The identification of a core region for OVA 323-339, which is critical in determining binding to IAd, has enabled us to generate a series of analog peptides in which this core region was extended at both the N and C termini with different amino acid residues. When assessed for binding capacity, several peptides were shown to have increased affinity for IAd compared with the parent sequence, and in addition, some peptides had acquired binding specificities for class II MHC haplotypes not present for OVA 323-339. These peptides were next examined for their ability to inhibit T cell responses in vitro and in vivo. The correlation between binding and the ability to inhibit T cell activation in vitro was good. However, when assessed in vivo, it was clear that high Ia binding was not sufficient in itself to define the inhibitory capacity of a given peptide. That this discrepancy was due to differences in degradation of the core-extended peptides was suggested by 1) results from an inhibition of Ag presentation assay, in which the pulse period with Ag and inhibitor was extended to 20 h; and 2) direct analysis of peptide stability by using reverse phase HPLC. Finally, by protecting the peptide from degradation with N- and C-terminal substitutions of D-amino acids, the inhibitory capacity of an unstable core-extended peptide in vitro could be greatly enhanced. These data indicate that the core extension approach may be one method by which antagonists for MHC class II molecules may be generated.     

Capacity of intact proteins to bind to MHC class II molecules

Here we have demonstrated that denatured, but not native protein antigens can interact with Ia molecules. Thus, the failure of native antigens to be recognized as such by T cells appears to be at least in part due to a deficient antigen/Ia interaction. These results also support previous observations that some T cells can recognize denatured antigens without a further processing requirement. Moreover, a striking correlation was observed between the in vitro binding pattern of denatured proteins and the pattern of restriction of T cell responses elicited by immunization with the native antigen, raising the possibility that an unfolding step may actually occur early during in vivo processing and influence the final outcome of Ia-restricted T cell responses.     

Variation in the ribosomal internal transcribed spacers (ITS1 and ITS2) among eight taxa of the Mimulus guttatus species complex

The internal transcribed spacer region (ITS1 and ITS2) of the 18S-25S nuclear ribosomal DNA sequence and the intervening 5.8S region were sequenced from three individuals in each of eight taxa of the Mimulus guttatus species complex. Three discrete variants, or "types," of ITS sequences were found, among which 30%-40% of sites differed, compared with 1%-2% within types. Dot plots indicate that these types were not related by conspicuous rearrangements or inversions. More than one ITS type was often found in the same taxon, and two of three ITS types span species boundaries, indicating their presence prior to speciation. These ITS sequences showed essentially no positional homology with the nearest sequenced relative, tomato. In contrast, the 5.8S region was relatively unvaried, with 8 of 162 sites varied in the sample among all eight taxa. The phylogeny inferred by the most common ITS sequence type, rooted by the two other ITS types, agreed with isozymes in showing the distinctness of M. nudatus, M. laciniatus, and M. tilingii from the other five taxa.