Diphtheria toxin-receptor interaction: A polyphosphate-insensitive diphtheria toxin-binding domain

Inositol hexaphosphate, and other polyphosphates, inhibit diphtheria toxin-mediated cytotoxicity by binding to the toxin at a highly cationic site called the P site and preventing toxin binding to cell surface receptors. The binding of diphtheria toxin to a solubilized cell surface glycoprotein (150,000 daltons) is also inhibited by these polyphosphates. Treatment of this 150,000 dalton diphtheria toxin-binding cell surface glycoprotein with papain yielded an 88,000 dalton and a 74,000 dalton diphtheria toxin-binding glycoprotein whose binding to toxin was no longer inhibited by inositol hexaphosphate. This result suggests a model of diphtheria toxin-receptor interaction in which the toxin receptor possesses one binding site which interacts with the P site of the toxin in a $\text{polyphosphate-sensitive}$ fashion, and another binding site (located within the papain-derived 74,000–88,000 dalton glycoproteins) which can interact with the toxin at a site distinct from the P site (the X site) in a $\text{polyphosphate-insensitive}$ fashion. This X site-receptor interaction may be involved in the binding of CRM proteins that bind to the toxin receptor but that do not bind polyphosphates, or it may be involved in the entry process of the toxin.     

Contact analysis of a posterior cervical spine plate using a three-dimensional canine finite element model

The development of a three-dimensional finite element model of a posteriorly plated canine cervical spine (C3-C6) including contact nonlinearities is described. The model was created from axial CT scans and the material properties were derived from the literature. The model demonstrated sufficient accuracy from the results of a mesh convergence test. Significant steps were taken toward establishing model validation by comparison of plate surface strains with a posteriorly plated canine cervical spine under three-point bending. This model was developed to better characterize the contact pressures at the various interfaces under average physiologic canine loading. The analysis showed that the screw-plate interfaces had the highest values of all the mechanical parameters evaluated.     

Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase

An assay for the enzyme responsible for the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins, a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, is reported using the synthetic peptide YSDSPSTST as the acceptor substrate. The activity is linearly dependent on time, enzyme, and substrate concentration. Replacement of the proline with a glycine in the peptide renders it ineffective as a substrate, whereas changing of the aspartic acid to a glycine has no effect. Product characterization of the glycosylated peptide demonstrates that the monosaccharide covalently attached to the peptide is N-acetylglucosamine (GlcNAc) and has not been epimerized to N-acetylgalactosamine. Mild base-catalyzed beta-elimination of the in vitro glycosylated peptide quantitatively yields GlcNAcitol, indicating that the GlcNAc is attached via an O-linkage. The transferase activity is strongly inhibited by UDP but is unaffected by GlcNAc or tunicamycin. Interestingly, EDTA only slightly inhibits activity, suggesting that the enzyme may not require divalent cations. The majority of the activity is soluble, and the remainder is lost from membranes after extracting with high salt and EDTA. Consistent with the subcellular localization of most proteins bearing O-GlcNAc, the activity appears to reside in the cytosolic portion of the cell when compared to two lumenal marker enzymes, galactosyltransferase and mannose-6-phosphatase.     

Extended physical maps and a consensus physical map of the homoeologous group-6 chromosomes of wheat (Triticum aestivum L. em Thell.)

Extended physical maps of chromosomes 6A, 6B and 6D of common wheat (Triticum aestivum L. em Thell., 2n=6x=42, AABBDD) were constructed with 107 DNA clones and 45 homoeologous group-6 deletion lines. Two-hundred and ten RFLP loci were mapped, including three orthologous loci with each of 34 clones, two orthologous loci with each of 31 clones, one locus with 40 clones, two paralogous loci with one clone, and four loci, including three orthologs and one paralog, with one clone. Fifty five, 74 and 81 loci were mapped in 6A, 6B and 6D, respectively. The linear orders of the mapped orthologous loci in 6A, 6B and 6D appear to be identical and 65 loci were placed on a group-6 consensus physical map. Comparison of the consensus physical map with eight linkage maps of homoeologous group-6 chromosomes from six Triticeaespecies disclosed that the linear orders of the loci on the maps are largely, if not entirely, conserved. The relative distributions of loci on the physical and linkage maps differ markedly, however. On most of the linkage maps, the loci are either distributed relatively evenly or clustered around the centromere. In contrast, approximately 90% of the loci on the three physical maps are located either in the distal one-half or the distal two-thirds of the six chromosome arms and most of the loci are clustered in two or three segments in each chromosome. Received: 19 April 1999 / Accepted: 28 July 1999     

Risk-Based Approaches to Managing Contaminants in Catchments

Risk-based methods promise improved decision-making for managing of contaminants, such as salinity, sediments, nutrients, and toxicants, that can adversely affect the ecological condition of aquatic ecosystems. Two aspects of ecological risk assessment (ERA) and management—stakeholder involvement and more quantitative approaches to risk analysis—are particularly challenging. Stakeholder involvement is crucial both in the risk assessment process and the development, acceptance, and implementation of a risk management plan. Additionally, a number of quantitative approaches (particularly Bayesian approaches and multi-criteria decision-making) have been identified as having the potential to include expert-based inputs into risk-based decision-making. These offer promise for better inclusion of stakeholder knowledge and preferences into the decision-making process, and for improving the links between stakeholder inputs and potential risks to the ecological condition of the system. A major challenge for ecologists and natural resource managers is to make the ERA process more quantitative. Most ERAs conducted to date have been qualitative assessments that suffer from a number of deficiencies, the most serious being the lack of transparency and a reliance on subjective judgments. This article argues that the most productive way forward may be to use Bayesian methods to couple existing process-based models, empirical relationships based on good data, and expert opinion, to make the analysis of ecological risks more robust, consistent, and repeatable.     

Regional Amino Acid Concentration in the Brains of Rats Exposed to High Pressures

Regional amino acid concentrations were measured in rat brain fixed by microwave irradiation at three levels of elevated atmospheric pressure corresponding to different phases of the high-pressure neurological syndrome [20 atmospheres absolute (ATA), no clinical signs; 60 ATA, tremor; 85 ATA, severe tremor and myoclonic jerks]. No changes in amino acid content occurred at 20 or 60 ATA. At 85 ATA glutamine content increased in hippocampus, striatum, cerebellum, and substantia nigra, and gamma-aminobutyric acid content increased in hippocampus. It is suggested that enhanced glutamate release in various subcortical structures contributes to the myoclonic activity observed at 85 ATA.     

Differences in control by UV radiation of inflammatory airways disease in na?ve and allergen pre-sensitised mice

Exposure of skin to UV radiation (UVR) prior to allergen exposure can inhibit inflammatory airways disease in mice by reducing effector CD4+ T cells in both the trachea and the airway draining lymph nodes. This study analysed the immunomodulatory properties of UVR delivered to na?ve versus allergen pre-sensitised mice. In a model of inflammatory airways disease, BALB/c mice were sensitised by peritoneal injection of the allergen, ovalbumin (OVA) (20 μg/mouse), in the adjuvant, alum (4 mg/mouse), on days 0 and 14. On day 21, the mice were exposed to aerosolised OVA and 24 h later, proliferative responses by the cells in the airway draining lymph nodes were examined. UVR (8 kJ m(-2)) was administered 3 days prior to first OVA sensitisation (day -3), or OVA aerosol challenge (day 18). UVR before sensitisation reduced immune responses associated with expression of allergic airways disease; seven days after first OVA sensitisation, regulation of OVA-induced proliferation in vitro but not in vivo by CD4+CD25+ cells from UV-irradiated mice was detected. UVR administered to pre-sensitised mice regulated allergen responsiveness by cells from the airway draining lymph nodes only with a sensitisation protocol involving allergen and adjuvant at 5% strength of the original dose (1 μg OVA in 0.2 mg alum/mouse). These results suggest that UVR may modulate allergic airways disease by two mechanisms. The first, and more potent, is by reducing effector cells in respiratory tissues and requires UV delivery prior to sensitisation. The second, associated with administration to pre-sensitised mice, is weaker and is detected when the mice are sensitised with lower levels of allergen and adjuvant.     

Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.

Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migrating with an apparent molecular weight of 340,000 on molecular sieve chromatography. Analysis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two protein species migrating at 110 (alpha subunit) and 78 (beta subunit) kDa in approximately a two-to-one ratio. Thus, the enzyme likely exists as a heterotrimer complex with two subunits of 110 kDa and one of 78 kDa (alpha 2 beta). The alpha subunit appears to contain the enzyme's active site since it is selectively radiolabeled by a specific photoaffinity probe (4-[beta-32P]thiouridine diphosphate). Photoinactivation and photolabeling of the enzyme are dependent on time and long wavelength ultraviolet light. Photolabeling of the alpha subunit is specifically blocked by UDP. The enzyme has an extremely high affinity for UDP-GlcNAc (Km = 545 nM). This unusually high affinity for the sugar nucleotide donor probably provides the enzyme an advantage over the nucleotide transporters in the endoplasmic reticulum and Golgi apparatus which compete for available cytoplasmic UDP-GlcNAc. The multimeric state and large size of the O-GlcNAc transferase imply that its activity may be highly regulated within the cell.