Removal of Paracoccus denitrificans outer membrane material by sodium chloride

The effects of water washing and NaCl treatment on the cell surface of P. denitrificans were studied. Both treatments caused a release of material from cells. Chemical studies showed that NaCl treatment released material containing components characteristic of outer membrane. This treatment also increased the susceptibility of the organism to lysozyme. Scanning electron microscopy was used to monitor the effects of water washing and NaCl treatment on the cell surface. Both treatments were shown to alter the appearance of the cell surface. The disruptive effects of these procedures were found to be dependent upon the age of the culture.Non-Standard Abbreviations WW water wash - SE saline extract     

Occurrence,localization, and possible significance of an ornithine-containing lipid in Paracoccus denitrificans

A ninhydrin-positive, phosphorus-negative lipid from Paracoccus denitrificans ATCC 13543 has been isolated and purified by mild alkaline methanolysis followed by silicic acid column chromatography and preparative thin-layer chromatography. The lipid was identified as an ornithine-containing lipid. The major ester-linked fatty acid was cis vaccenic acid. Major amide-linked fatty acids were 3-OH-20:1 and 3-OH-18:0. Ornithine-containing lipid was a major lipid component of P. denitrificans. Phospholipids made up about 57% and ornithine-containing lipid about 14% of the weight of the total lipid of the organism. The ratios of lipid ornithine: lipid phosphorus were 0.23, 0.65 and 0.58 in cytoplasmic membrane, outer membrane, and an NaCl extract, which is thought to represent chiefly outer membrane, respectively. Thus ornithine-containing lipid appears to be present in larger amounts in outer membrane than cytoplasmic membrane. No substantial variations in lipid ornithine levels were noted in stationary phase versus exposnential phase organisms, organisms grown in complex medium versus organisms grown in minimal medium with and without amino acid supplements, or in organisms grown in low phosphate-containing medium.Non standard abbreviations TLC thin-layer chromatography - Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride - TMS trimethylsilyl - TFA triluoroacetyl - NPPN ninhydrin-positive, phosphorus-negative - ECL equivalent chain length     

The reactions of the oxidase and reductases ofParacoccus denitrificans with cytochromesc

Electron transport in theParacoccus denitrificans respiratory chain system is considerably more rapid when it includes the membrane-bound cytochromec ₅₅₂ than with either solubleParacoccus c ₅₅₀ or bovine cytochromec; a pool function for cytochromec is not necessary. Low concentrations ofParacoccus or bovine cytochromec stimulate the oxidase activity. This observation could explain the multiphasic Scatchard plots which are obtained. A negatively charged area on the back side ofParacoccus c which is not present in mitochondrialc could be a control mechanism forParacoccus reactions.Paracoccus oxidase and reductase reactions with bovinec show the same properties as mammalian systems; and this is true ofParacoccus oxidase reactions with its own soluble cytochromec if added polycation masks the negatively charged area. Evidence for different oxidase and reductase reaction sites on cytochromec include: (1) stimulation of the oxidase but not reductase by a polycation; (2) differences in the inhibition of the oxidase and reductases by monoclonal antibodies toParacoccus cytochromec; and (3) reaction of another bacterial cytochromec withParacoccus reductases but not oxidase. Rapid electron transport occurs in cytochromec-less mutants ofParacoccus, suggesting that the reactions result from collision of diffusing complexes.     

Characteristics of the energy-transducing NADH-quinone oxidoreductase ofParacoccus denitrificans as revealed by biochemical,biophysical, and molecular biological approaches

A comparison of the mitochondrial NADH-ubiquinone oxidoreductase and the energy-transducing NADH-quinone oxidoreductase (NDH-1) ofParacoccus denitrificans revealed that both systems have similar electron-transfer and energy-transduction pathways. In addition, both complexes are sensitive to the same inhibitors and contain similar electron carriers, suggesting that theParacoccus NDH-1 may serve as a useful model system for the study of the human enzyme complex. The gene cluster encoding theParacoccus NDH-1 has been cloned and sequenced. It is composed of 18,106 base pairs and contains 14 structural genes and six unidentified reading frames (URFs). The structural genes, URFs, and their polypeptides have been characterized. We also discuss nucleotide sequences which are believed to play a role in the regulation of the NDH-1 gene cluster andParacoccus NDH-1 subunits which may contain the binding sites of substrates and/or electron carriers.     

Reconsideration of the efficiency of energy transduction in Paracoccus denitrificans during growth under a variety of culture conditions

1. Theoretical overall P/2e^- ratios were calculated for growth of Paracoccus denitrificans under a variety of culture conditions on the basis of a growth model and recently published schemes of electron transport and associated proton translocation. 2. Experimental overall P/2e^- ratios were calculated, using the specific rate of ATP synthesis determined in cultures grown under aerobic carbon source-limited conditions. 3. The experimental P/2e^- was equal to the theoretical P/2e^- for aerobic sulphate-limited growth with gluconate or succinate as carbon source, on the assumption that site 1 phosphorylation was completely absent. 4. The experimental and the theoretical P/2e^- were similar for growth on nitrate as terminal electron acceptor and gluconate, mannitol or succinate as carbon source, on the assumption that nitrate enters the cell via the electroneutral nitrate-nitrite antiport system. 5. The experimental and theoretical P/2e^- were similar for growth on nitrite as terminal electron acceptor and mannitol or succinate as carbon source. 6. The experimental P/2e^- was substantially lower than the theoretical P/2e^- for aerobic growth with nitrate as nitrogen source and gluconate or mannitol as carbon source. The amount of energy needed for assimilative reduction of nitrate to ammonia was calculated from this difference.Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

Metabolic regulation of pyruvate kinase isolated from autotrophically and heterotrophically grown Paracoccus denitrificans

Pyruvate kinase (ATP: pyruvate phosphotransferase (EC 2.7.1.40) was partially purified from both autotrophically and heterotrophycally grown Paracoccus denitrificans. The organism grown under heterotrophic conditions contains four times more pyruvate kinase than under autotrophic conditions. The enzyme isolated from both sources exhibited sigmoidal kinetics for both phosphoenolpyruvate (PEP) and ADP. The apparent M _m for ADP and PEP in the autotrophic enzyme were 0.63 mM ADP and 0.25 mM PEP. The effect of several low molecular weight metabolites on the pyruvate kinase activity was investigated. Ribose-5-phosphate, glucose-6-phosphate and AMP stimulated the reaction at low ADP levels; this stimulation was brought about by an alteration in the apparent K _m for ADP. The pyruvate kinases differ in their response to adenine nucleotides, but both preparations seem to be under adenylate control. The results are discussed in relation to the role of pyruvate kinase as a regulatory enzyme in P. denitrificans grown under both autotrophic and heterotrophic conditions.Non-Common Abbreviations PEP phosphoenolpyruvate - R-5-P ribose-5-phosphate - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - 3-PGA 3-phosphoglycerate     

Sulfite inhibits the F1F0-ATP synthase and activates the F1F0-ATPase of Paracoccus denitrificans

The F₁F₀ complex of Paracoccus denitrificans (PdF₁F₀) is the fastest ATP synthase but the slowest ATPase. Sulfite exerts maximal activation of the PdF₁F₀-ATPase (Pacheco-Moisés, F., García, J. J., Rodríguez-Zavala, J. S., and Moreno-Sánchez, R. (2000). Eur. J. Biochem. 267, 993–1000) but its effect on the PdF₁F₀-ATP synthase activity remains unknown. Therefore, we studied the effect of sulfite on ATP synthesis and ³²Pi ATP exchange reactions of inside-out membrane vesicles of P. denitrificans. Sulfite inhibited both reactions under conditions of maximal pH and normal sensitivity to dicyclohexylcarbodiimide. Sulfite increased by 10- and 5-fold the K _0.5 for Mg²⁺-ADP and Pi during ATP synthesis, respectively, and by 4-fold the IC₅₀ of Mg²⁺-ADP for inhibition of the PdF₁F₀-ATPase activity. Thus, sulfite exerts opposite effects on the forward and reverse functioning of the PdF₁F₀ complex. These effects are not due to membrane or PdF₁F₀ uncoupling. Kinetic and structural modifications that could account for these results are discussed.     

Analysis of cytoplasmic membrane from wild type and mutant Paracoccus denitrificans containing excess nitrate reductase protein and cytochrome b

Cytoplasmic membranes were isolated from wild type and mutants strain M-1 of Paracoccus denitrificans grown with low aeration to promote synthesis of nitrate reductase protein and cytochrome b. The presence of 10-100-fold excess of nitrate reductase in the wild type or the corresponding enzymically inactive protein in the mutant did not significantly affect respiratory oxidase activities with NADH, succinate or TMPD-ascorbate as electron donor. A cytochrome b-nitrate reductase complex was resolved by isoelectric focussing of Triton X-100 solubilized membranes from the wild type grown with azide and from the mutant, whereas the enzyme complex from nitrate-grown wild type was not resolved from cytochrome c. Preparations from azideinduced wild type or from the mutant could be a suitable source of the cytochrome b associated with nitrate reductase for more detailed studies.Non standard abbreviations IEF isoelectric focussing - TMPD N, N, N, N-tetramethylphenylenediamine - SDS-PAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

Electron transport chain and energy transduction in Paracoccus denitrificans under autotrophic growth conditions

Cells of Paracoccus denitrificans grown autotrophically with H₂ as energy source contained a branched respiratory chain. The presence of two terminal oxidases was indicated by two cyanide sensitive sites (K _i =10^-5 M and K _i =10^-3 M). While oxidation of NADH and succinate apparently proceeded via both electron pathways as shown by the inhibition of respiration with cyanide and Antimycin A, oxidation of H₂ involved only the terminal oxidase which was less sensitive to KCN. Oxidation of H₂ was not inhibited by rotenone, and sensitive to only relatively high concentrations of Antimycin A (50 nmol/mg).Under our growth conditions, autotrophic cells contained only very small amounts of cytochrome a +a ₃ . A cytochrome b was able to bind CO (with a peak at 418 nm and a trough at 434 nm in the reduced plus CO minus reduced difference spectrum). This cytochrome b had the spectral characteristics of cytochrome o and could be the alternate oxidase. The respiratory chain contained two b cytochromes (b ₅₅₆ and b ₅₆₂ at 77°K); under steady state conditions only b ₅₅₆ was significantly reduced by NADH and succinate while both b ₅₅₆ and b ₅₆₂ were reduced by H₂.Measurement of respiration-driven proton translocation by spheroplasts showed that the oxidation of H₂ by O₂ was associated with a vectorial ejection of H⁺ (in the outward direction) with aH⁺/O value of 6 to 7.A similar result was obtained with succinate. Oxidation of endogenous substrates gave H⁺/O values corresponding to a H⁺/site ratio of 3 with 3 sites functioning in absence of inhibitors, two sites in the presence of rotenone and one site in the presence of antimycin. The H⁺/O values indicated that two energy transducing sites were involved in the oxidation of H₂ by O₂.Measurement of ATP synthesis in membrane vesicles confirmed that phosphorylation was coupled to H₂ oxidation. However, such determinations which necessitated the use of inverted vesicles, gave P/O values too low to allow any conclusions to be made on the number of coupling sites.     

Characterization of cell surface carbohydrates on asexual spores of the water moldSaprolegnia ferax

Summary In asexual reproduction of the water mold,Saprolegnia ferax, four distinct and sequentially produced spores are involved in dispersal, two of which are motile and two of which are nonmotile. Composition of cell surface glycoproteins may be important in dispersal strategies for each of these stages. Binding patterns of fluorescently labelled lectins were investigated to identify differences in glycoproteins of asexually produced dispersal stages. The pattern of lectin binding to zoospores was diverse. FITC-Con A bound to surfaces of zoospores and membranes of the water expulsion vacuole system, indicating the prescence of mannosyl and glucosyl residues. In zoospores incubated for more than 30 min in FITC-WGA and FITC-GS II. which bind N-acetyl glucosamine, fluorescence was sometimes localized in peripheral, intracellular patches. In shorter incubations, secondary zoospores bound these lectins along the groove region where K-bodies were located. Surfaces of cystospores typically bound FITC-WGA, but not FITC-GS II. FITC-GS II, however, bound to empty cystospore walls, probably because reactive sugars were available at the inner surface of the wall. Germ tubes emerging from cystospores bound labelled WGA and GS II, but not Con A. The same lectin binding pattern was found along discharge papilla of primary cystospores, indicating that modifications in cystospore walls associated with direct germination and zoospore discharge were similar. Thus, glycoproteins involved in early establishment of the hyphal system differ from those forming the cell surface of cystospores. Differences in the binding pattern of lectins to zoospores and cystospores highlight differences between cell surface carbohydrates of motile and nonmotile asexual stages.Abbreviations BPA lectin fromBauhinia purpurea - C₁ primary cystospore - C₂ secondary cystospore - Con A concanavalin A, lectin fromCanavalia ensiformis - DBA lectin fromDolichos biflorus - DIC Nomarski differential interference contrast optics - DS dilute salts - FITC fluorescein isothiocyanate - FUC fucose - Gal galactose - GalNAc N-acetyl galactosamine - Glc glucose - GlcNAc N-acetyl glucosamine - GS I Griffonia simplicifolia lectin I - GS II G. simplicifolia lectin II - Man mannose - MPA lectin fromMaclura pomifera - PC phase contrast optics - PNA lectin fromArachis hypogaea - SBA soybean agglutinin, lectin fromGlycine max - UEA-1 lectin fromUlex europaeus - WGA wheat germ agglutinin fromTriticum vulgare - WV water expulsion vacuole     

Growth yields and the efficiency of oxidative phosphorylation of Paracoccus denitrificans during two- (carbon) substrate-limited growth

Paracoccus denitrificans was grown aerobically during two-(carbon)substrate-limitation on mannitol and methanol in chemostat cultures. Theoretical growth parameters were calculated based on the presence of 2 or 3 sites in the electron-transport chain of Paracoccus denitrificans. Experimental growth parameters determined during two-(carbon)substrate growth were conform to the presence of 3 sites of oxidative phosphorylation, while cells grown only on mannitol possessed 2 sites. The maximum growth yield on adenosine triphosphate (ATP), corrected for maintenance requirements, determined in chemostat experiments in which the methanol concentration is less than 2.11 times the mannitol concentration was 8.6 g of biomass. When the methanol concentration was more than 2.11 times the mannitol concentration the maximum growth yield on adenosine triphosphate decreased due to the more energy consuming process of CO₂-assimilation. Cells use methanol only as energy source to increase the amount of mannitol used for assimilation purposes. When the methanol concentration in chemostat experiments was more than 2.11 times the mannitol concentration, all mannitol was used for assimilation and excess energy derived from methanol was used for CO₂-assimilation via the ribulose-bisphosphate cycle. The synthesis of ribulosebisphosphate carboxylase was repressed when the methanol concentration in chemostat experiments was less than 2.11 times the mannitol concentration or when Paracoccus denitrificans was grown in batch culture on both methanol and mannitol. When in chemostat experiments the methanol concentration was more than 2.11 times the mannitol concentration ribulose-bisphosphate carboxylase activity could be demonstrated and CO₂-assimilation will occur. It is proposed that energy produced in excess activates or derepresses the synthesis of the necessary enzymes of the ribulose-bisphosphate cycle in Paracoccus denitrificans. Consequently growth on any substrate will be carbonas well as energy-limited. When methanol is present in the nutrient cells of Paracoccus denitrificans synthesize a CO-binding type of cytochrome c, which is essential for methanol oxidase activity.The reason for the increase in efficiency of oxidative phosphorylation from 2 to 3 sites is most probably the occurrence of this CO-binding type of cytochrome c in which presence electrons preferentially pass through the a-type cytochrome region of the electron-transport chain.Non Standard Abbreviations X prosthetic group of methanol dehydrogenase - q _substrate specific rate of consumption of substrate (mol/g biomass. h.) - Y _substrate, Y _substrate ^MAX are respectively the growth yield and the maximum growth yield corrected for maintenance requirements (g biomass/mol) - m _substrate maintenance requirement (mol substrate/g biomass) - specific growth rate (h^-1) - M [methanol]/[mannitol] ratio in the nutrient - N part of mannitol that is assimilated when M=o - R _m amount of methanol-equivalents that has the same energy content as 1 mannitol-equivalent - P/O _N , P/O _F , P/O _X is the amount of ATP produced during electron-transport of two electrons from respectively NADH+H⁺, FADH₂ and XH₂ to oxygen     

Effect of CaCl2 on growth performance, photosynthetic efficiency and nitrogen assimilation of Cichorium intybus L. grown under NaCl stress

Pot culture experiments were conducted to assess the extent of growth, photosynthetic efficiency and nitrogen assimilation of chicory (Cichorium intybus L.) as affected by NaCl and CaCl₂ alone as well as in combination. Six treatments, i.e., 80 mM and 160 mM NaCl, 5 mM and 10 mM CaCl₂ and 80 mM + 10 mM and 160 mM + 10 mM of NaCl + CaCl₂ were given to the growing plants separately at three developmental stages, viz., the pre-flowering (30 DAS), flowering (120 DAS) and post-flowering (150 DAS) stages. Each NaCl treatment caused a significant reduction in total plant biomass, photosynthetic rate, stomatal conductance, total chlorophyll content, soluble protein content, NR activity and nitrogen content, although nitrate content increased. On the contrary CaCl₂ treatment gave a favorable effect, compared to the control. The effect of combined treatments was similar to that of NaCl but less in magnitude. Thus, the application of CaCl₂ may mitigate the adverse effect caused by NaCl.     

Quantitative response of cell growth and polysaccharide biosynthesis by the medicinal mushroom Phellinus linteus to NaCl in the medium

The effect of NaCl on cell growth and polysaccharide biosynthesis in the medicinal mushroom Phellinus linteus was studied. With the increase of NaCl concentration between 1 g/l and 7 g/l in the culture medium, the cell growth and intracellular polysaccharide (IPS) accumulation were decreased; extracellular polysaccharide (EPS) concentration was enhanced, with an increase of NaCl concentration from 1 g/l to 3 g/l. Under the optimum NaCl concentration of 3 g/l, the maximum EPS and IPS production reached 2.2±0.15 g/l and 53.6±2.45 mg/g DW on day 12, which improved 32.27% and decreased 16.89% compared to the control, respectively. Both EPS and IPS showed new polysaccharide components by fractionation with DEAE-cellulose ion exchange chromatography compared to the control. The results presented in this study are considered helpful for further investigation on the diversity of polysaccharide biosynthesis of this medicinal fungus under NaCl environments.     

Mycorrhizal colonisation improves fruit yield and water use efficiency in watermelon (Citrullus lanatus Thunb.) grown under well-watered and water-stressed conditions

The effect of arbuscular mycorrhizal (AM) colonisation by Glomus clarum on fruit yield and water use efficiency (WUE) was evaluated in watermelon (Citrullus lanatus) cv. Crimson Sweet F1 under field conditions. Treatments were: (1) well-watered plants without mycorrhizae (WW-M), (2) well-watered plants with mycorrhizae (WW+M), (3) water- stressed plants without mycorrhizae (WS-M) and (4) water-stressed plants with mycorrhizae (WS+M). When soil water tension readings reached –20 and –50 kPa for well-watered (WW) and water-stressed (WS) treatments, respectively, irrigation was initiated to restore the top soil to near field capacity. Water stress reduced watermelon shoot and root dry matter, fruit yield, water use efficiency but not total soluble solids (TSS) in the fruit, compared with the non-stressed treatments. Mycorrhizal plants had significantly higher biomass and fruit yield compared to nonmycorrhizal plants, whether plants were water stressed or not. AM colonisation increased WUE in both WW and WS plants. Macro- (N, P, K, Ca and Mg) and micro- (Zn, Fe and Mn) nutrient concentrations in the leaves were significantly reduced by water stress. Mycorrhizal colonisation of WS plants restored leaf nutrient concentrations to levels in WW plants in most cases. This is the first report of the mitigation of the adverse effect of water stress on yield and quality of a fruit crop.     

Inhibition of nitrogenase by nitrite and nitric oxide in Rhodopseudomonas sphaeroides f. sp. denitrificans

In cells of Rhodopseudomonas sphaeroides f. sp. denitrificans nitrite and nitric oxide, the products of denitrification, inhibit activity of nitrogenase enzyme.Ferredoxin-linked CO₂ fixation, with H₂ as a reductant, was also inhibited by nitrite and NO in denitrifying cells.EPR spectroscopy of cell preparations treated with NO showed that it reacts with non-haem iron-sulphur proteins to form iron-nitrosyl complexes. Nitrite also reacts with these iron-sulphur proteins, but the formation of ironnitrosyl complexes was dependent on the presence of dithionite. Since nitrite is reduced to NO by dithionite it is likely that nitrogenase and CO₂ fixation reactions are inhibited not only by nitrite itself, but also by nitric oxide.Abbreviation DPPH 1,1-diphenyl-2-picrylhydrazyl     

The effect of NaCl on water relations,chlorophyll, and protein and proline contents of two cultivars of blackgram (Vigna mungo L.)

The physiological basis of salt tolerance of two cultivars of blackgram, cv Candhari Mash (relatively salt tolerant) and cv Mash 654 (salt sensitive), was assessed in salinized sand culture at the flowering stage. Increasing NaCl concentration in the rooting medium significantly reduced the chlorophyll a, chlorophyll b, and total chlorophyll, leaf water potential (Ψ_w), leaf solute potential (Ψ_s), and leaf turgor potential (Ψ_p) in both the cultivars. Leaf protein and proline content was increased as a result of increasing salt concentration in both cultivars. High salt concentrations had no significant effect on the seed protein content of both cultivars. At high salinities, cv Candhari Mash had significantly greater chlorophyll a, chlorophyll b and total chlorophyll, leaf water potential, solute potential, and turgor potential than cv Mash 654, but the latter had greater leaf proline content than cv Candhari Mash. Cultivars did not differ significantly for both leaf and seed protein contents. The relatively salt tolerant cv Candhari Mash maintained high leaf water potential and turgor potential to resist salt injury. Leaf proline content had negative correlation with salt tolerance in blackgram.     

Functional and spectral characterization of the respiratory chain of Chloroflexus aurantiacus grown in the dark under oxygen-saturated conditions

In this paper we attempt a functional and spectral characterization of the membrane-bound cytochromes involved in respiratory electron transport by membranes from cells of Chloroflexus aurantiacus grown in the dark under oxygen saturated conditions. We conclude that the NADH-dependent respiration is carried out by a branched respiratory chain leading to two oxidases which differ in sensitivity to CN^- and CO. The two routes also show a different sensitivity to the ubiquinone analogue, HQNO, the pathway through the cytochrome c oxidase being fully blocked by 5 M HQNO, whereas the alternative one is insensitive to this inhibitor. The cytochrome c oxidase containing branch is composed by at least two c-type haems with E _m 7.0 of +130 and +270 mV ( bands at 550/553 nm and 549 nm, respectively), plus a b-type cytochrome with E _m 7.0 of +50 mV ( band at 561 nm). From this, and previous work, we conclude that respiratory and photosynthetic electron transport components are assembled together and function on a single undifferentiated plasma membrane.Abbreviations HQNO heptylhydroxy-quinoline-N-oxide - UHDBT undecyl-hydroxydioxobenthiazole - Q/b-c ubiquinol/cytochrome c oxidoreductase complex - BChl bacteriochlorophyll     

Resistance to water flow in xylem of Picea abies (L.) Karst. trees grown under contrasting light conditions

Summary The relative hydraulic conductivity (k) of xylem and resistance (R) to water flow through trunk, primary roots and branches in Picea abies trees growing under contrasting light conditions were investigated. The xylem permeability to water was measured by forcing 10 mM water solution of KC1 through excised wood specimens. From the values of k, the sapwood transverse area and the length of conducting segments, R of the whole trunk, branches and roots was calculated. The relative conductivity of xylem in open-grown trees exceeded that of shade-grown trees by 1.4–3.1 times, while k was closely correlated with the hydraulically effective radius (R _e) of the largest tracheids (R ² was 0.85–0.94 for open- and 0.51–0.79 for shade-grown trees). Because of both a low k and a smaller sapwood area in shade-grown trees the resistance to water movement through their trunk, roots and branches was many times higher. The distribution of R between single segments of the water-conducting pathway differed considerably in trees from different sites. At high water status the largest share of the total resistance in open- as well as shade-grown trees resides in the apical part of the trunk. The contribution of the branches to total xylem resistance is supposed to increase with developing water deficit.     

The effect of tunicamycin on Leishmania brasiliensis. Glycosylation and the cell surface components

Culture conditions of Leishmania cells were developed to allow the study of the effect of tunicamycin (TM) on glycosylation and on the cell surface components. Leishmania incorporate [¹⁴C]-mannose and [³⁵S]-methionine in vitro. The incorporation of [¹⁴C]-mannose is linear for 150 min and is inhibited by TM (2 g/ml) in a time dependent effect which reaches a plateau of 45% inhibition at 36 h. Under the same experimental conditions [³⁵S]-methionine incorporation into protein is slightly affected. This is reflected by an almost identical polypeptide pattern for TM treated and non-treated cells when analyzed on SDS-PAGE. On the contrary, strong differences were detected on the labeled compounds analyzed on SDS-PAGE followed by autoradiography when the precursor used was [¹⁴C]-mannose. A shift in the electrophoretic mobility of most of the glycopeptides synthesized in the presence of TM was observed, which is also reflected in the structure of the main Leishmania cell surface components.The findings are discussed in the light of biological implications.     

Modifications of cell surface sialic acids modulate cell adhesion mediated by sialoadhesin and CD22

An increasing number of mammalian cell adhesion molecules, including sialoadhesion, CD22 and the family of selectins, have been found to bind cell surface glycoconjugates containing sialic acids. Here we describe how the structural diversity of this sugar influences cell adhesion mediated by the related molecules sialoadhesin and CD22 in murine macrophages and B-cells respectively. We show that the 9-O-acetyl group of Neu5,9Ac₂ and theN-glycoloyl residue of Neu5Gc interfere with sialoadhesin binding. In contrast, CD22 binds more strongly to Neu5Gc compared to Neu5Ac. Of two synthetic sialic acids tested, only CD22 bound theN-formyl derivative, whereas aN-trifluoroacetyl residue was accepted by sialoadhesin. The potential significance for the regulation of sialic acid dependent cell adhesion phenomena is discussed.Dedicated to Professor Dr Gerhard Uhlenbruck on the occasion of his 65th birthday.