Characterization of O-glycosidically linked oligosaccharides of rat erythrocyte membrane sialoglycoproteins

The carbohydrate units of the rat erythrocyte membrane sialoglycoprotein rSGP-4 [Edge, A. S. B., & Weber, P. (1981) Arch. Biochem. Biophys. 209, 697-705] have been characterized. All of the carbohydrate of this Mr 19,000 glycoprotein occurs in O-glycosidic linkage to the peptide; following alkaline borohydride treatment and chromatography on Bio-Gel P-2, sialic acid containing oligosaccharides terminating in N-acetylgalactosaminitol were obtained. Their structures were determined by compositional analysis, exoglycosidase digestions, alkaline sulfite degradation, and periodate oxidation. The oligosaccharides were characterized for molecular weight and linkage by direct chemical ionization and gas-liquid chromatography/mass spectrometry, respectively. The structures are proposed to be NeuAc alpha 2----3Gal beta 1----3GalNAc-ol, Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)GalNAc-ol, and NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----6)GalNAc-ol. Two of the N-acetylglucosamine-containing hexasaccharides were present per molecule of rSGP-4 along with two trisaccharides and seven tetrasaccharides.     

Chemiluminescence associated with phagocytosis of foreign particles in rabbit alveolar macrophages

Rabbit alveolar macrophages exhibit a chemiluminescent response which is associated with phagocytosis of zymosan and polystyrene-butadiene particles. The chemiluminescence reaches a peak in 15 to 25 minutes and then gradually diminishes over the next 1 to 3 hours. During the time of maximal light emission there appears to be no actual uptake of particles, but the response is dependent upon the particle concentration. The metabolic inhibitor, DNP (2,4-dinitrophenol), causes a rapid inhibition of the chemiluminescent response. The addition of ATP to the medium prior to exposure of the cells to particles causes the chemiluminescent response to be greatly diminished, i.e., 0.3mM ATP virtually abolishes the response. These experiments suggest that some metabolic response of the cell to phagocytosis is responsible for the chemiluminescence.     

Fluorimetric determination of carbamoyl phosphate

A simple fluorimetric assay for the determination of carbamoyl phosphate in tissue extracts is described. In the assay, production of ATP from carbamoyl phosphate and ADP by carbamate kinase is coupled to the formation of NADPH, using glucose, hexokinase, NADP⁺, and glucose-6-phosphate dehydrogenase. Production of NADPH in this system proved to be equal to the amount of carbamoyl phosphate present.     

Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties.

Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.     

A quantitative histochemical study of NADPH-ferrihemoprotein reductase activity

Summary A quantitative histochemical assay for NADPH-ferrihemoprotein (P450) reductase had been developed. For optimal activity, it is necessary to use a relatively electropositive tetrazolium salt such as neotetrazolium chloride as the final acceptor. The apparentK _m of the reaction is 0.83 mM. Its specificity has been proven in two ways: (i) activity is increased selectively in the pericentral zone of liver from rats treated with phenobarbitone, an inducer of the reductase, though not in liver of rats injected with 3-methylcholanthrene, which induces NAD(P)H dehydrogenase; (ii) it is competitively inhibited by NADP⁺ (K _i=1.50mm) though unaffected by dicumarol, an inhibitor of NAD(P)H dehydrogenase activity. An NADP⁺ concentration ten times greater than the substrate concentration inhibits the histochemical reaction and the reaction in a microsomal fraction assayed biochemically to the same degree (70% inhibition). The amount of inhibition is independent of temperature, of the zone of the acinus and of the treatment of the animal.Continuous microdensitometric monitoring of the reaction product as it is formed has shown that the specific reaction is linear with incubation up to 10 min, thus allowing end-point measurements to be used for cytophotometric analysis.     

The induction of chromosomal damage in rat hepatocytes and lymphocytes II. Alkylation damage and repair of rat-liver DNA after diethylnitrosamine,dimethylnitrosamine and ethyl methanesulphonate in relation to clastogenic effects

Rat-liver DNA alkylation by diethylnitrosamine (DEN), dimethylnitrosamine (DMN) and ethyl methanesulphonate (EMS) was studied in an attempt to relate chromosome-damaging effects of these agents (the formation of micronuclei in hepatocytes; see preceding paper) to specific alkylation patterns. No correlation was observed between the induction of micronuclei and liver DNA N-alkylation, measured as 3- and 7-alkyl-purines. O⁶-Alkylguanine is probably not involved in micronucleus induction because it is lost from DNA too rapidly to explain the much more persistent clastogenic effects. In contrast, both the initial amounts of alkylphosphotriesters and the persistencies of these products roughly paralleled the respective effects on micronucleus induction. The possible involvement of alkylphosphotriesters or other O-alkylation products of comparable stabilities is discussed. Results with DMN suggest that part of the primary DNA methylation damage is converted into a secondary (DNA) lesion and that both the primary and secondary lesion(s) contribute to the process of micronucleus formation.     

Failure to condition evaluative and electrodermal responses to neutral stimuli by means of prepared cues

To test Gray's proposal (1979, 1982) that prepared cues (snakes, spiders) are unconditioned, innate fear stimuli, a classical conditioning procedure was carried out in which a neutral, conditioned stimulus (CS; slides of flowers, mushrooms or apples) was repeatedly followed by a prepared, unconditioned stimulus (US; slides of snakes or spiders). Dependent variables were subjective evaluation of, and skin conductance responses to the CS slides, while respiration was used as a control variable. In one group of subjects (n = 12), skin conductance was recorded from the left hand, whereas in a second group (n = 11) it was recorded from the right hand. No evidence of skin conductance response - or of evaluative conditioning to the neutral CS as a result of its being paired with the prepared US was found. Thus, the results lend no support to Gray's proposal. No differences in skin conductance responses as a function of left versus right hand recording were found.     

Pistil Exudate Production and Pollen Tube Growth in Lilium longiflorum Thunb.

Exudate production in the pistil of Lilium longiflorum was studiedin relation to pollen tube growth, using scanning electron microscopy(SEM), transmission electron microscopy and light microscopy.In contrast with conventional fixation for SEM, during whichthe exudate of L. longiflorum largely washes away, the exudateremains present through freezing in case of cryo-SEM. Usingthe latter method we observed that exudate production on thestigma and in the style started before anthesis. Just underneaththe stigma the exudate was first accumulated at the top of eachsecretory cell, followed by a merging of those accumulationsas exudate production proceeded. Exudate is also produced bythe placenta. It was however not possible to determine whetherany of this fluid originated from the micropyle. Apart fromthe cell shape and the cuticle present in between the secretorycells, the ultrastructure of the secretory cells covering theplacenta was comparable to those of the stylar canal. The transferwall of the secretory cells of the placenta originated fromfusing Golgi vesicles but the endoplasmic reticulum seemed tohave an important role as well. After pollination the pollen tubes grew across the stigma andentered the style through one of the slits in the three stigmalobes. The pollen tubes grew straight downward through the styleand were covered by exudate. As the pollen tubes approachedthe ovary their growth was restricted to the areas with secretorycells. In the cavity the pollen tubes formed a bundle and theybent from this bundle in between the ovules towards the micropylarside. There they bent again to stay close to the secretory cells.After bud pollination the pollen tube growth was retarded. Laterarriving pollen tubes had a tendency to grow close to the secretorycells of the style, which resulted in a growth between thesecells and preceding pollen tubes. If there was still a littleexudate produced, it resulted in a lifting up of the pollentubes, out of the exudate. The relationship between exudateproduction and pollen tube growth is discussed. Both the speedand the guidance of the pollen tube seemed determined by theproperties of the exudate.Copyright 1994, 1999 Academic Press Cryo-scanning electron microscopy, exudate, Lilium longiflorum, lily, ovary, pollination, pollen tube growth, secretory cell, stigma, style