Functional incorporation and preferred orientation of phytohemagglutinin receptor glycoproteins in phospholipid vesicles

Porcine lymphocyte Phaseolus vulgaris phytohemagglutinin (PHA) receptor glycoproteins purified by affinity chromatography have been reassembled into vesicles made of phosphatidylcholine and phosphatidylserine by detergent (dodecyltrimethylammonium bromide) dialysis. The receptor glycoproteins were incorporated into the lipid vesicles in a nonselective manner with a yield of 65-70%. Vesicles containing the glycoproteins were sealed as evidenced by their impermeability to calcium ions, using quin 2 trapped inside the vesicles. The vesicles were agglutinated by PHA, suggesting that the saccharidic moiety of the reconstituted glycoproteins was, at least in part, oriented towards the extravesicular medium. This observation was further supported by the fact that the vesicles bound 125I-labeled PHA in a specific and saturable manner. At maximum amount of lectin bound, a ratio of 1.01 +/- 0.05 microgram of PHA per microgram glycoprotein incorporated was measured. When the binding data were analyzed by Scatchard plot, a downward concave profile was observed, suggestive of a positive cooperativity at low concentrations of lectin. The orientation of the reconstituted lectin receptor glycoproteins was determined by proteolytic treatments of labeled glycoproteins. The combined action of trypsin and chymotrypsin released, in the 120,000 X g supernatant, approximately 80% of label when 125I-tagged PHA receptor glycoproteins were incorporated into the vesicles. When the oligosaccharidic moieties of the receptor glycoproteins were specifically labeled, the simultaneous action of the two enzymes released approximately 70% of tritium labeling present in the reconstituted system. Taken together, these results suggest that the reconstituted PHA receptors are preferentially oriented into the phospholipid vesicles. The reconstituted PHA receptor glycoproteins competed effectively with cellular receptors in the assay of PHA-induced porcine lymphocyte activation. A 50% inhibition of [3H]thymidine incorporation was observed when 1 microgram of glycoproteins in vesicles was added to the cultured cells, whereas vesicles alone had no effect at this (equivalent) concentration.     

IgE receptor on human lymphocytes. IV. Further analysis of its structure and of the role of N-linked carbohydrates

Previous studies have shown that the low affinity receptor for IgE (Fc epsilon R II) on human B lymphocytes was comprised of three components with apparent Mr of 45, 65 to 95, and 37 kDa. The present results indicate that the 37-kDa component is a soluble degradation fragment of the 45-kDa component and they also suggest that the 65- to 95-kDa component is probably made of aggregates of the above components that are formed after solubilization of the cells. The 45-kDa component appears to be a glycoprotein containing several sialic acid residues, O-linked carbohydrates and one N-linked carbohydrate chain that is of the complex type. Partial digestion of the purified 65- to 95-, 45-, and 37-kDa molecules with alpha-chymotrypsin or protease V8 generates several fragments with identical mobility on SDS-PAGE. The 37 kDa is not N-glycosylated but like the IgE-binding factors present in the culture supernatant of Fc epsilon R-bearing cells, it contains sialic acid and O-linked carbohydrates. On incubation with protease inhibitors, the Mr of IgE-binding factors (BF) is shifted from 25-27 to 37 kDa, indicating that IgE-BF are derived from the proteolytic cleavage of the 37-kDa molecule, previously identified as a membrane component of Fc epsilon R. On incubation with N-glycosylation inhibitors, the production of IgE-BF is significantly increased indicating that N-glycosylation inhibits the degradation of Fc epsilon R into IgE-BF. Inasmuch as the effect of glycosylation inhibitors is not prevented by monensin, it is concluded that all the IgE-BF are derived from surface Fc epsilon R and not from their intracellular precursors.     

Expedient syntheses of neoglycoproteins using phase transfer catalysis and reductive amination as key reactions

Starting from peracetylated chloro- or bromo-glycosyl donors ofN-acetylneurmainic acid,N-acetylglucosamine, glucose and lactose, the correspondingp-formylphenyl glycosides were synthesized stereospecifically under phase transfer catalysed conditions at room temperature in yields of 38–67%. After Zemplén de-O-acetylation, the formyl groups were directly and chemoselectively coupled to the lysine residues of bovine serum albumin by reductive amination using sodium cyanoborohydride. The conjugation reactions were followed as a function of time and under a series of different molar ratios of the reactants to provide glycoconjugates of varying degree of antigenicities. Thus, carbohydrate protein conjugates were made readily available using essentially two key reactions.Presented in part at the 15th International Carbohydrate Symposium, Yokohama, Japan, August 12–17, 1990.     

Interpretation of DNA vibration modes: IV--A single-helical approach to assign the phosphate-backbone contribution to the vibrational spectra in A and B conformations

A calculated approach based on the Higgs method for assigning the vibration modes of an infinite helicoidal polymeric chain has been performed on the basis of a reliable valence force field. The calculated results allowed the phosphate-backbone marker modes of the A and B forms, to be interpreted. In the dynamic models used, the bases have been omitted and no interchain interaction was considered. The calculation can also interprete quite satisfactorily the characteristic Raman peaks and infrared bands in the 1250-700 cm-1 spectral region arising from the sugar or sugar-phosphate association and reproduce their evolution upon the B----A DNA conformational transition. They clearly show that the phosphate-backbone modes in the above mentioned spectral region constitute the optical branches of the phonon dispersion curves with no detectable variation in the first Brillouin-zone.     

Autoradiographic study of the localization and evolution of growth zones in bacterial colonies.

Incorporation of [3H]leucine in the bacteria of 18 to 48 h-old colonies of Pseudomonas aeruginosa, Pseudomonas putida, Bacillus thuringiensis, Staphylococcus aureus and Escherichia coli enabled the localization of bacterial multiplication sites by means of autoradiography of sagittal sections. In colonies where fast diameter expansion occurred, all the bacteria from the peripheral corona contributed to peripheral growth; in colonies where the expansion was slower, the growth rate of the bacteria in this region was heterogeneous. Besides this peripheral growth, a central region of bacterial multiplication was always found, but with variable localization and extension. In aerobic species, such as P. aeruginosa and P. putida, the central growth site was limited to the zone of oxygen penetration into the bacterial mass. However, in facultatively anaerobic species, bacterial multiplication dependend on nutrient supply. For 48 h-old colonies of S. aureus, a more complex localization of growth seemed to be affected simultaneously by nutrient penetration and accumulation of toxic substances.     

Involvement of phage T5 tail proteins and contact sites between the outer and inner membrane of Escherichia coli in phage T5 DNA injection.

The penetration of phage T5 DNA into the Escherichia coli envelope takes place through ion channels (Boulanger, P., and Letellier, L. (1992) J. Biol. Chem. 267, 3168-3172). To identify putative phage protein(s) involved in the formation of these channels, E. coli cells were infected at 37 degrees C with radioactively labeled phage and their envelopes were fractionated. After a flotation gradient, proteins belonging to the phage tail were recovered both in fractions containing the contact sites between the inner and outer membranes and in the outer membrane. The electrophoretic banding pattern of phage proteins indicates that the contact sites were enriched in the protein pb2. Moreover, infected cells were significantly enriched in contact sites as compared to intact cells. There was no enrichment of contact sites and very little radioactivity was found in this fraction and in the outer membrane when the cells were infected at 4 degrees C (i.e. under conditions where the phage does not inject its DNA). These results suggest that both contact sites and pb2 may play a central role in the translocation of phage T5 DNA.     

Patterns of Root Colonization in Epacridaceous Plants Collected from Different Sites

Root colonization was studied in ten species of the Epacridaceaeat three sites in Victoria by morphological and cross-inoculationexperiments. The sites and genera chosen were Cranbourne [Epacrisimpressa Labill. andLeucopogon ericoides(Smith) R. Br.] andRye [L. parviflorus(Andrews) Lindley] on the Mornington Peninsula,and the Grampians[Astroloma conostephioides(Sond.) Benth.,A.humifusum(Cav.) R. Br.,A pinifolium(R. Br.) Benth,Brachylomadaphnoides(Smith) Benth.,E. impressa, E. impressavar.grandifloraBenth.andStyphelia adscendensR. Br.] in western Victoria. For morphologicalstudies, samples of roots from each species at each site werecleared and stained and examined microscopically. For cross-inoculationstudies, cuttings from each site were struck in potting mediuminoculated with soil from the same and other sites. The ericoidmycorrhizae in the roots of plants found at or grown in Cranbourneand Rye soils were similar. Both were significantly differentfrom the internal hyphae found in the roots of plants foundat or grown in Grampians soils, which were three times largerin diameter and formed dense coils which filled the host celland invaded adjacent epidermal cells. This suggests that morethan one fungus is involved in the relationships, that the MorningtonPeninsula sites had a different fungus from the Grampians siteand that host specificity is low. Vesicular structures werealso found commonly on plants at the Grampians site, in contrastwith other sites. Epacridaceae; root; fungus; mycorrhiza; morphology; inoculation     

Threshold effect and tissue specificity. Implication for mitochondrial cytopathies

Mitochondrial cytopathies present a tissue specificity characterized by the fact that even if a mitochondrial DNA mutation is present in all tissues, only some will be affected and induce a pathology. Several mechanisms have been proposed to explain this phenomenon such as the appearance of a sporadic mutation in a given stem cell during embryogenesis or mitotic segregation, giving different degrees of heteroplasmy in tissues. However, these mechanisms cannot be the only ones involved in tissue specificity. In this paper, we propose an additional mechanism contributing to tissue specificity. It is based on the metabolic expression of the defect in oxidative phosphorylation (OXPHOS) complexes that can present a biochemical threshold. The value of this threshold for a given OXPHOS complex can vary according to the tissue; thus different tissues will display different sensitivities to a defect in an OXPHOS complex. To verify this hypothesis and to illustrate the pathological consequences of the variation in biochemical thresholds, we studied their values for seven OXPHOS complexes in mitochondria isolated from five different rat tissues. Two types of behavior in the threshold curves can be distinguished corresponding to two modes of OXPHOS response to a deficiency. We propose a classification of tissues according to their type of OXPHOS response to a complex deficiency and therefore to their threshold values.