Identification of a GM1-Binding Protein on the Surface of Murine Neuroblastoma Cells

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.     

Hemoglobin Dallas (alpha 97(G4)Asn-->Lys): functional characterization of a high oxygen affinity natural mutant.

Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (p1/2 = 1 mmHg at pH 7.3 and 20 degrees C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20 degrees C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more 'relaxed' T-state.     

Binding of lipoproteins to inert materials revisited with computer-assisted analysis

A number of studies conducted in the last decade showed that saturable ('specific') binding, by itself, does not necessarily imply biological significance. That is, biological ligands were shown to bind to inert materials as well as to biological receptors in a saturable manner. In these studies specific binding was operationally defined as binding that was displaceable by excess concentrations of unlabeled ligand. This method of measuring specific binding is now no longer considered optimal. To investigate whether optimal (computer-assisted) techniques of measuring specific binding--namely, nonlinear least-squares curve fitting of total binding data, with mathematical separation of the total binding into its various components--might ensure biological significance of measured specific binding, we studied the binding of high-density lipoproteins (HDL3) to tissue culture dishes as an example of binding without biological significance. This binding closely followed the paradigm of a ligand interacting with a class of homogeneous, saturable sites and with a class of relatively unsaturable sites, just as it would have if the HDL3 were interacting with an unpurified biological receptor. This finding indicates that computer-assisted analysis, while most accurately describing binding data, nevertheless does not ensure that measured specific binding has biological significance. Saturability is such a nonselective feature of equilibrium binding data that it should probably no longer be considered one of the criteria for deciding whether or not a defined binding site is a receptor.     

Round robin investigation of methods for recovering human enteric viruses from sludge

To select a tentative standard method for detection of viruses in sludge the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group initiated round robin comparative testing of two procedures that, after initial screening of several methodologies, were found to meet the basic criteria considered essential by the task group. Eight task group member laboratories agreed to perform round robin testing of the two candidate methods, namely, The Environmental Protection Agency or low pH-AlCl3 method and the Glass or sonication-extraction method. Five different types of sludge were tested. For each particular type of sludge, a single laboratory was designated to collect the sludge in a single sampling, make samples, and ship it to the participating laboratories. In most cases, participating laboratories completed all the tests within 48 h of sample arrival. To establish the reproducibility of the methods, each laboratory tested each sludge sample in triplicate for the two candidate virus methods. Each processed sludge sample was quantitatively assayed for viruses by the procedures of each individual round robin laboratory. To attain a more uniform standard of comparison, a sample of each processed sample from all laboratories was reassayed with one cell line and passage number by a single laboratory (Environmental Protection Agency Environmental Monitoring and Support Laboratory, Cincinnati, Ohio). When the data were statistically analyzed, the Environmental Protection Agency method was found to yield slightly higher virus recoveries for all sludge types, except the dewatered sludge. The precisions of both methods were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

The study of X-rays and TCDD effects on satellite associations may suggest a simple model for application in environmental mutagenesis

Summary Satellite associations were used as parameters to test nucleolar organizer activity. Assuming that toxic and/or mutagenic agents may affect the ribosomal genes, satellite associations in human lymphocytes were analysed following exposure to X-rays and compared with the satellite association pattern of subjects exposed to TCDD. A significant decrease in the satellite association frequency in D group chromosomes was found both in irradiated lymphocytes and in subjects exposed to Dioxin. The findings seem to be in accordance with the hypothesis based on random damage of functional nucleolar organizing regions.     

Infrared spectroscopic studies on gramicidin ion-channels: relation to the mechanisms of anesthesia

Fourier transform infrared spectroscopic studies are reported on gramicidin ion-channels in phospholipid bilayers and the effects on the spectra of the anesthetics and related compounds (methoxyflurane, halothane, chloroform, carbon tetrachloride, n-pentane and n-decane) have been determined. The addition of anesthetics containing the 'acidic hydrogen' caused unique changes particularly on the amide I bands at 1639 cm-1 and 1670 cm-1. The 1639 cm-1 band became more intense while the intensity near 1670 cm-1 decreased dramatically. These effects were not observed with carbon tetrachloride, n-pentane and n-decane. The 1670 cm-1 band is interpreted as arising from the carbonyls involved in the head-to-head hydrogen-bonded dimerization where the relationship between chains is analogous to that of the antiparallel beta-pleated sheet structure and the anesthetics with 'acidic hydrogens' are considered to disrupt the hydrogen-bonded dimerization by competitive hydrogen bonding to the carbonyls at the head-to-head junction. As the dimer-monomer equilibrium is the 'on-off' mechanism for gramicidin ion-channel conductance, the results are considered in terms of the mechanism of action of anesthetics and are taken to suggest, for certain anesthetics, a hydrogen-bonding role to protein ion-channel components.     

Decreased virulence in stable,acapsular mutants ofCryptococcus neoformans

Six acapsular strains ofCryptococcus neoformans obtained by chemical mutagenesis failed to produce a capsulein vivo and were avirulent in mice following high dose intramuscular, intraperitoneal or intravenous inoculation. Peritoneal granulomas were observed in all animals inoculated with the acapsular mutants. These granulomas were characterized by a large central mass consisting of intact, degenerating and necrotic yeast cells. This was surrounded by concentric layers of a broad band of histiocytes, a narrow band of fibroblasts, and around the periphery, a mass of lymphocytes and plasma cells. These isolates did not revert to an encapsulated or virulent state after more than a year of subculturing or 18 passages through mice.     

High performance liquid chromatography,chemical laboratory practice: By Heinz Engelhardt. Springer-Verlag,New York/Berlin, 1979. 248 pp. $29.80

A gel filtration method has been developed for the complete removal of sodium dodecyl sulfate (SDS) from proteins and peptides. The protein or peptide (20 μg–10 mg) containing SDS (up to 30–60 mg) is dissolved in a mixture of propionic acid, formic acid, and water (2:1:2, $\text{v}\text{v}$). Under these conditions, protein-SDS (or peptide-SDS) complexes, as well as SDS micelles, are dissociated. Subsequently, protein and SDS can be separated on a small Sephadex G-25 superfine column. The recovery of protein is typically 90% or more.