Detection of terminal N-linked N-acetylglucosamine residues in the Golgi apparatus using galactosyltransferase and endoglucosaminidase F/peptide N-glycosidase F: adaptation of a biochemical approach to electron microscopy

Purified human milk beta-N-acetylglucosaminide beta 1, 4 galactosyltransferase (EC 2.4.1.38) was used to galactosylate N-acetylglucosamine (GlcNAc) residues present in ultra-thin sections of Lowicryl K4M-embedded rat and pig liver. Both endogenous galactose and galactosylated transferase products could be revealed by Ricinus communis lectin I-gold complexes (RcL I-g15). Without galactosyltransferase (GT) treatment, labeling for galactose (gal) was limited to the trans region of rat and pig hepatocyte Golgi apparatus. After exposure to GT, additional labeling was found over cis Golgi apparatus cisternae. RcL I-g15 labeling was sensitive to a purified preparation of endoglucosaminidase F/peptide N-glycosidase F (at pH 9). This indicates that endogenous gal and gal transferred by GT to terminal GlcNAc residues are present N-linked oligosaccharides. The RcL I-g15 labeling produced by GT was insensitive to extensive washing with solutions containing either EDTA and urea or SDS and 2-mercaptoethanol or 0.1 M GlcNAc. Substrate inhibition studies showed that 50 mM GlcNAc specifically inhibited the additional RcL I-g15 labeling produced by GT. The use of purified glycosyltransferases therefore appears to allow specific detection of oligosaccharide substrates and their high resolution localization in thin sections by electron microscopy.     

Studies on the mechanism of T cell inhibition by the Pseudomonas aeruginosa phenazine pigment pyocyanine

Pseudomonas aeruginosa and its products have been shown to inhibit mitogen-induced human lymphocyte blastogenesis as measured by [3H]TdR uptake. The phenazine pigment pyocyanine has been identified as one of the inhibitors present in cellfree culture supernatants. To determine the mechanism of the inhibitory action of pyocyanine, we studied its effect on the early stages of T cell activation. Pyocyanine inhibited lymphocyte stimulation induced by specific antigens, the lectin concanavalin A and the calcium ionophore, ionomycin, suggesting that its inhibitory effect is not dependent on interference with the T cell antigen receptor complex itself. Using quin-2, we showed that pyocyanine did not interfere with the mitogen-induced increase in cytosolic-free Ca2+. We also showed that pyocyanine did not interfere with the function of calmodulin stimulated Ca2+-Mg2+ ATPase activity, indicating that the mechanism of action of pyocyanine differs from that of the structurally related phenothiazine compounds. Analysis of IL 2 production and IL 2 receptor expression clearly showed that pyocyanine inhibits the production of this essential lymphokine as well as the expression of IL 2 receptors on the T cell membrane. This inhibition is dose dependent and not due to cellular toxicity. There was parallel inhibition of growth in cell volume as well as [3H]TdR uptake. Thus, our results demonstrate that pyocyanine inhibits T cell proliferation by decreasing the production of the critical lymphokine IL 2 and by decreasing the expression of the IL 2 receptor. Local suppression of lymphocyte stimulation by phenazine pigments such as pyocyanine may interfere with cellular immune responses that may be necessary for eradication of chronic infection with P. aeruginosa.     

Affinity labeling of the carbohydrate binding site of the lectin discoidin I using a photoactivatable radioiodinated monosaccharide

N-(4-Azidosalicyl)galactosamine (GalNASA), a photoactivatable, radioiodinatable analogue of N-acetylgalactosamine (GalNAc), has been prepared and characterized. We have used this reagent for labeling of the carbohydrate binding site of discoidin I, an endogenous lectin produced by Dictyostelium discoideum. GalNASA behaved as a ligand for discoidin I, as judged by its ability to compete in an assay measuring the carbohydrate binding activity of discoidin I. In this assay, it exhibited a Ki,app of 800 microM, comparable to that of GalNAc. The Ki,app of GalNASA decreased to 40 microM upon prior photolysis with ultraviolet light. In contrast, N-(4-azidosalicyl)ethanolamine produced no inhibition of carbohydrate binding regardless of photolysis. Covalent labeling of discoidin I with 125I-GalNASA was entirely dependent upon ultraviolet light. A portion of the labeling, representing 40-60% of the total, was sensitive to reagents which were known to inhibit carbohydrate binding by discoidin I, including GalNAc, asialofetuin, and ethylenediaminetetraacetic acid. N-Acetylglucosamine, which is not a ligand of discoidin I, was without effect. As a control, no carbohydrate-sensitive labeling was observed upon incubation of 125I-GalNASA with bovine serum albumin. The carbohydrate-sensitive fraction of discoidin I photolabeling with 125I-GalNASA exhibited a Kd of 15-40 microM, in agreement with the Ki,app of prephotolyzed GalNASA observed in the carbohydrate binding assay. Some labeling occurred if 125I-GalNASA was photolyzed prior to incubation with discoidin I, suggesting the involvement of long-lived species in the labeling reaction. Partial proteolytic digestion of photolabeled discoidin I revealed specific fragments whose labeling was completely blocked by GalNAc.(ABSTRACT TRUNCATED AT 250 WORDS)     

Correlation of conformational changes in the acrosome stabilizing factor (ASF) with its biological activity

The rabbit Acrosome Stabilizing Factor (ASF) is a glycoprotein synthesized in the corpus epididymis that reversibly decapacitates sperm. The effects of altering the conformation of ASF were evaluated by using a competitive enzyme-linked immunoabsorption assay (ELISA) with monoclonal antibodies that recognized either sequential or conformational determinants and/or an in vivo decapacitation assay. Heat denaturation (80 degrees C for 30 min) of affinity-purified ASF resulted in destruction of its native conformation concurrent with its loss of biological activity. Acid pH treatment of ASF also resulted in a conformational change in ASF, which caused a shift from the dimeric form (MW = 260,000) to the monomeric form (MW = 130,000). This manipulation allowed the biological activity of the monomeric form of ASF to be assayed separately from the dimer. The monomer was found to be biologically inactive. Proteolysis with trypsin or Staphylococcus-V8 treatment resulted in loss of the native conformation of the molecule, but Staphylococcus-V8 did not destroy the sequential determinant recognized in this analysis. This work indicates that conformation of the ASF macromolecule is important for its biological activity, and also provides a rapid means to evaluate potential decapacitation activity of purified ASF.     

Expression of HLA-DR antigens on tumour cells does not contribute to skin reactivity to autologous cholesteryl hemisuccinate (CHS)-treated tumour cells in patients with metastatic melanoma

Summary Skin tests with autologous cholesteryl hemisuccinate (CHS)-treated and untreated cells were performed in ten metastatic melanoma patients. In the majority of cases evident reaction was noted with CHS-treated cells (9/10) while the reaction with untreated cells was mostly negative (7/10). Tumour cell suspensions used for skin tests were characterized for reactivity with monoclonal antibody TAL 1B5 detecting the HLA-DR alpha chain. There were no differences between CHS-treated and untreated cells with respect to HLA-DR expression and no correlation was found between grade of skin reaction to CHS-treated cells and the proportion of HLA-DR positive cells in the injected cell sample.     

Crossovers in two German cystic fibrosis families determine probe order for MET, 7C22 and XV-2c/CS.7

Summary We have followed the segregation of the probes pJ3.11, 7C22, pB79a, and MET through cystic fibrosis families in the German Democratic Republic with two affected sibs. Two families with a crossover between MET and the CF phenotype were detected. In one of these families recombination was also observed between the DNA probe 7C22 and CF, and between the markers XV-2c and CF, which suggests that XV-2c, MET and 7C22 are all on the same side of CF. The other MET recombinant family is informative with XV-2c and does not recombine, which excludes the genetic order XC-2c-MET-CF if multiple recombinant events are disregarded. These two families together demonstrate that recombinations may occur in a very small genetic interval, which has important implications for prenatal diagnosis based on data from linked markers.     

Localization of the human progesterone receptor gene to chromosome 11q22–q23

Summary The human progesterone receptor gene was mapped by in situ hybridization using two cDNA probes corresponding to the 5′ and 3′ part of the coding sequence. This gene was localized to 11q22-q23.     

Accumulation of cadmium and copper by the terrestrial snail Arianta arbustorum L.: kinetics and budgets

Summary Specimens of the terrestrial gastropod Arianta arbustorum were fed on cadmium- or copper-enriched agar plates with the aim of performing an input/output analysis and of studying the distribution of these metals in several organs of the snails. After a feeding period of 20 days about 45% of cadmium were lost. 36% accumulated in the hepatopancreas, where a cadmium concentration of more than 500 g/g was measured. The efficiency of cadmium assimilation decreased from about 90% at the beginning to about 55% after 20 days. Copper was distributed more evenly than cadmium, but the main site of copper storage seemed to be the foot/mantle tissues, where 49% of the ingested copper were found. The efficiency of copper assimilation always exceeded 95%. The patterns of distribution and assimilation of copper and cadmium are discussed in relation to differences in the cytological and biochemical detoxification mechanisms which exist for these metals in molluscs.