BBA - General Subjects (v.1474, #2)
Diversity and functions of glycosaminoglycan sulfotransferases by Osami Habuchi (115-127).
Sulfate residues attached to the specific position of the component sugar residues of glycosaminoglycans play important roles in the formation of functional domain structures. The introduction of a sulfate group is catalyzed by various sulfotransferases with strict substrate specificities. A rapid development achieved in the cloning of various glycosaminoglycan sulfotransferases has allowed us to study the biological functions of glycosaminoglycan sulfotransferases and their products, sulfated glycosaminoglycans.
Keywords: Glycosaminoglycan; Sulfotransferase; Cloning; Substrate specificity; Function;
The myosin motor: muscle contraction and in vitro movement by Jean-Emile Morel; Nathalie D’Hahan (128-132).
The molecular mechanism of in vitro movement is assumed, by most investigators, to be identical to that of muscle contraction. We discuss this view, which raises various problems. We believe there are mechanisms for muscle contraction (in this case considerable forces are developed, with small displacements) and other mechanisms for in vitro movement (giving large displacements, without necessarily generating substantial forces). Hybrid models may explain muscle contraction. The traditional swinging-crossbridge model may explain in vitro movement. For muscle contraction, movement may result partly from the swinging-crossbridge mechanism and partly from other factors. Comparisons of different fibres at different moments of the Mg-ATPase cycle suggest that both the value of the isometric force in muscle and in vitro and that of the Mg-ATPase activity used in vitro need to be reconsidered. The recently reported dependence of the isometric active tension of smooth skinned fibres on temperature appears to be weaker than predicted by the swinging-crossbridge theory alone. This recent observation is compatible with the existence of other forces (electrostatic repulsions) decreasing with temperature as has been known for some years. From recent experimental data, we think the biochemistry of myosin and actomyosin should be reassessed, to try to find new details of the mechanisms of muscle contraction and in vitro motility.
Keywords: Force per myosin head; Mg-ATPase activity; Force and movement generation;
Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins 1 1 This work was carried out in the Swedish Center for Bioseparation. by Ulf Sivars; Folke Tjerneld (133-146).
Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C m EO n ), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.
Keywords: Membrane protein; Partitioning; Aqueous two-phase system; Phase behavior; Purification; Extraction;
Ion–protein dissociation predicts ‘windows’ in electric field-induced wound-cell proliferation by Vladimir N Binhi; Robert J Goldman (147-156).
There are many experiments showing that weak, non-thermal electric fields influence living tissues. In many cases, biological effects display ‘windows’ in biologically effective parameters of electric fields: most dramatic is the fact that relatively intense electric fields sometimes do not cause appreciable effect, while smaller fields do. Linear resonant physical processes do not explain frequency windows in this case. Both frequency and amplitude windows are evident from experiments on human dermal fibroblasts in a collagen matrix. For this in vitro model of skin, exposure to extremely low frequency (ELF) electric fields in the frequency range 10–100 Hz and the amplitude range of 0–130 μA/cm2 macroscopic current density demonstrates such unusual ‘window’ behavior. Amplitude window phenomena suggest a non-linear physical mechanism. We consider non-linear quantum-interference effects on protein-bound substrate ions: These ions experience, due to electric fields in the media or biological tissue as small as 1 mV/m, electric gradients produced by polarized binding ligand atomic shells. The electric gradients cause an interference of ion quantum states. This ion-interference mechanism predicts specific electric-field frequency and amplitude windows within which fibroblast proliferation occurs.
Keywords: Electromagnetics; Electric field; Fibroblast; Proliferation; Metal–protein; Ion interference; Quantum physics; Wound healing;
Radical mediated indirect oxidation of a PEG-coupled polycyclic aromatic hydrocarbon (PAH) model compound by fungal laccase by Andrzej Majcherczyk; Christian Johannes (157-162).
A high molecular model compound of polycyclic aromatic hydrocarbon was synthesised by coupling pyrene to PEG5000. The pyrene-PEG was used for the study of a laccase-mediator-system. To prevent direct contact between the substrate and the enzyme the two were kept in their own compartments separated by a membrane. The low molecular mediators, 1-hydroxybenzotriazole and 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid), which were oxidised by laccase to the corresponding radicals or cations permeated the membrane and reacted with the pyrene-PEG model compound. Oxidation of the model compound resulted in an α-oxidation of the alkyl-chain leading to two main oxidation products. The same oxidation products were obtained in the reaction system without a membrane.
Keywords: Polycyclic aromatic hydrocarbon; Laccase-mediator-system; Laccase; Pyrene;
Carotenoid:methyl-β-cyclodextrin formulations: an improved method for supplementation of cultured cells by Inka Pfitzner; Pal Istvan Francz; Hans Konrad Biesalski (163-168).
A physiological, water-soluble complex of carotenoids with methyl-β-cyclodextrin (MβCD) was developed for the purpose of cell supplementation. Bioavailability, cytotoxicity and stability of the formulations were compared to carotenoid solutions in organic solvents (THF/DMSO (1:1), THF and ethanol). The stability of the different carotenoid solutions (0.5 μM) under cell culture conditions was determined by measuring absorbance 1 and 7 days after treatment. To determine the availability of β-carotene (BC), human skin fibroblasts were incubated for up to 8 days with 5 μM BC in MβCD or THF/DMSO and the cellular and medium BC contents were determined by HPLC analysis. Depending on the solubilizer, different orders of stability were found. MβCD formulation: BC>zeaxanthin>lutein>lycopene. Organic solvents: zeaxanthin>lutein>lycopene>BC. Two days after supplementation with 5 μM BC in MβCD, cellular BC levels reached a maximum of 140±11 pmol/μg DNA, leveling off to 100±15 pmol/μg DNA until day 8. Incubation with BC dissolved in THF/DMSO resulted in a lower BC uptake of 105±14 pmol/μg DNA and 64±20 pmol/μg DNA respectively. No cytotoxic effects of these formulations were detected. The results show that the MβCD formulation is an improved method for investigations of carotenoids and other lipophilic compounds in in vitro test systems compared to methods using organic solvents.
Keywords: Carotenoid; Carotene; Xanthophyll; β-Cyclodextrin; Fibroblast;
Regulation of copper uptake and transport in intestinal cell monolayers by acute and chronic copper exposure by Miguel Arredondo; Ricardo Uauy; Mauricio González (169-176).
Adaptation to high and low copper intake in mammals depends on the cellular control of influx, efflux and storage mechanisms of cellular copper concentrations. In the present study, we used an intestinal cell line (Caco-2), grown in bicameral chambers to study the effect of equilibrium loading with copper. We analyzed 64Cu uptake from the apical surface, intracellular metal (Cu, Zn, Fe) content, 64Cu transport into the basal chamber, and total copper, zinc and iron in the basal chamber. We found that the 64Cu uptake is saturable, shows a linear response phase up to 1.5 μM reaching a plateau at 4–6 μM extracellular Cu. Intracellular copper increased 21.6-fold, from 1.5 to 32.4 mM (at 0.2–20.2 μM extracellular copper respectively). The time course for 64Cu uptake and transport was linear when the cells were incubated with different copper concentrations. Uptake increased 10-fold when intracellular copper concentration was raised. Fluxes were lowest at 1.5 mM and highest at 32.4 mM Cu intracellular copper (2.03 and 20.98 pmole 64Cu insert−1 h−1, respectively). The apical-to-basolateral copper transfer rate was lower at 32.4 mM as compared to 1.5 mM intracellular copper (0.55–1.95 pmole 64Cu insert−1 h−1, respectively). The total copper in the basal chamber increased 4.2-fold (from 3.04 to 12.85 pmole Cu insert−1 h−1) when the intracellular copper concentration was raised. If cells are preincubated in a low copper medium most of the newly incorporated copper (64%) is transferred to the basolateral compartment. In contrast, under preloading with high copper concentration, only 4% of the fresh copper is transferred to the basal chamber; however, the intracellular copper contribution to this chamber increases by 4.2-fold. Thus, the process results in an increase in both storage and intracellular-to-basolateral flux of copper. In summary, our results indicate that copper fluxes from apical-to-cell and apical-to-basolateral domains are affected by intracellular copper concentration suggesting that mechanisms of copper transport involved in cellular adaptation to low and high copper exposure are different.
Keywords: Intestine; Copper uptake and transport; Caco-2 cell; Zinc; Iron; Total X-ray fluorescence;
Fluorescence quenching of dipyridamole associated to peroxyl radical scavenging: a versatile probe to measure the chain breaking antioxidant activity of biomolecules by Luigi Iuliano; Cristina Piccheri; Ilaria Coppola; Domenico Praticò; Fausta Micheletta; Francesco Violi (177-182).
Dypiridamole is a highly efficient chain breaking antioxidant (Iuliano et al., Free Radic. Biol. Med. 18 (1995) 239–247) with an aromatic ring system responsible for an intense absorption band in the 400–480-nm region and for an intense fluorescence. Dipyridamole fluorescence is quantitatively quenched upon reaction with peroxyl radicals. In the presence of a flux of peroxyl radicals generated by thermal dissociation of azo-initiators, dipyridamole fluorescence decays linearly, showing a first-order reaction with respect to peroxyl radicals, and zero-order with respect to dipyridamole. The pH optimum for the fluorescence quenching is in the 7–8 range, from pH 7 to 6, the decay of fluorescence rapidly decreases to became negligible below pH 5.5. Dipyridamole consumption is blocked in the presence of an added chain breaking antioxidant for a time that is proportional to the antioxidant concentration. This effect is shown for ascorbic acid, trolox, vitamin E, uric acid, and N,N′-diphenyl-p-phenylenediamine. The slope of the linear correlation relative to trolox allows calculation of the bimolecular rate constant for a given molecule and peroxyl radicals. Comparison of data obtained by the dipyridamole consumption are comparable to values obtained by the oxygen consumption method.
Keywords: Antioxidant; Peroxyl radical; Fluorescence; Rate constant; Free radical;
Docosahexaenoic acid reverses cyclosporin A-induced changes in membrane structure and function by Rupin R. Thakkar; Ou-Li Wang; Mustapha Zerouga; William Stillwell; Adeel Haq; Roger Kissling; William M. Pierce; Ned B. Smith; Frederick N. Miller; William D. Ehringer (183-195).
The use of a fish oil vehicle for cyclosporin A (CsA) can decrease the toxic effects of CsA but the mechanism is unclear. Here we examine the mechanism by which docosahexaenoic acid (DHA), a fish oil-derived polyunsaturated fatty acid, can alter the toxic effects of CsA on mouse organ function, endothelial macromolecular permeability, and membrane bilayer function. Mice given CsA and fish oil showed increased liver toxicity, kidney toxicity, incorporation of DHA, and evidence of oxidized fatty acids compared to control animals. We hypothesized that the toxic effects of CsA were primarily a result of membrane perturbation, which could be decreased if DHA were not oxidized. The presence of CsA (10 mol%) alone increased dipalmitoylphosphatidylcholine membrane permeability by seven fold over control (no CsA, no DHA). However, if non-oxidized DHA (15 mol%) and CsA were added to the membrane, the permeability returned to control levels. Interestingly, if the DHA was oxidized, the antagonistic effect of DHA on CsA was completely lost. While CsA alone increased endothelial permeability to albumin, the combination of non-oxidized DHA and CsA had no effect on endothelial macromolecular permeability. However the combination of oxidized DHA and CsA was no different than the effects of CsA only. CsA increased the fluorescence anisotropy of DPH in the liquid crystalline state of DPPC, while DHA decreased fluorescence anisotropy. However the combination of CsA and DHA was no different than DHA alone. We conclude that non-oxidized DHA can reverse the membrane perturbing effects of CsA, and the increases in endothelial macromolecular permeability, which may explain how fish oil is capable of decreasing the toxicity of CsA.
Keywords: Cyclosporin A; Docosahexaenoic acid; Membrane bilayer; Endothelial cell;
Selective ‘in synthesis’ labeling of peptides with biotin and rhodamine by Alberto Chersi; Simone Giommi; Laura Rosanò (196-200).
A new method is described for the selective ‘in synthesis’ labeling of peptides by rhodamine or biotin at a single, predetermined ε-amino group of a lysine residue. The α-amino group and other lysyl residues of the peptide remain unmodified. Peptides are assembled by the Fmoc approach, which requires mild operative conditions for the final deprotection and cleavage, and ensures little damage of the reporter group. The labeling technique involves the previous preparation of a suitable Lysine derivative, easily obtained from commercially-available protected amino acids. This new derivative, where the reporter group (biotin, or rhodamine) acts now as permanent protection of lysyl side chain functions, is then inserted into the synthesis program as a conventional protected amino acid, and linked to the preceding residue by aid of carbodiimide. A simpler, alternative method is also described for the selective ‘in synthesis’ labeling of peptides with N-terminal lysyl residues. Several applications of labeled peptides are reported.
Keywords: Biotin; Fluorescence; Peptide; Selective labeling;
Molecular cloning and functional expression of human ST6GalNAc II. Molecular expression in various human cultured cells by Bénédicte Samyn-Petit; Marie-Ange Krzewinski-Recchi; Wim F.A Steelant; Philippe Delannoy; Anne Harduin-Lepers (201-211).
A cDNA clone encoding a human Galβ1-3GalNAc α2,6-sialyltransferase (designated hST6GalNAc II) was identified employing the PCR with degenerated primers to the sialylmotifs, followed by BLAST analysis of databanks. This sialyltransferase sequence is similar to that of previously cloned ST6GalNAc II (chicken and mouse) and shows the sialylmotifs that are present in all eukaryotic members of the sialyltransferase gene family. The predicted amino acid sequence encodes a putative type II transmembrane protein as found for other eukaryotic sialyltransferases and shows significant similarity to chicken (56.8% identity) and mouse (74.6% identity) enzymes. Expression of a secreted form of hST6GalNAc II in COS-7 cells showed that the gene product had Galβ1-3GalNAc (sialyl to GalNAc) α2,6-sialyltransferase activity. In vitro analysis of substrate specificity revealed that the enzyme required a peptide aglycone fraction to be active and used both Galβ1-3GalNAc and Neu5Acα2-3Galβ1-3GalNAc as acceptor substrates. Northern analysis revealed a restricted expression pattern of two hST6GalNAc II transcripts, a 2.0 kb mRNA found mainly in skeletal muscle, heart and kidney and a 1.8 kb mRNA found in placenta, lung and leukocytes. No transcriptional expression was detected in brain, thymus or spleen. Transcriptional expression of the ST6GalNAc II gene was followed in various human cell lines and found to be expressed in almost all cell types with notable exceptions for several myeloid and lymphoid cell lines.
Keywords: Human; Sialyltransferase; Molecular cloning; O-Glycosylation;
Glycine prevents the phenotypic expression of streptococcal glucan-binding lectin by Jirapon Luengpailin; R.J Doyle (212-218).
Glycine has been used extensively in bacterial cell surface research. Some researchers employ glycine in growth media so as to increase the transformability of streptococci during electroporation. Others have found that glycine, similar to wall antibiotics, ‘weakens’ peptidoglycan. It is now shown that when glycine is incorporated into the growth medium, Streptococcus sobrinus exhibits a diminished ability to aggregate with high molecular weight α-1,6-glucan. Growth of the bacteria in either a rich or a chemically defined medium results in a cell population with full lectin (glucan-binding) fidelity. Incorporation of glycine, but not serine or other amino acids, at concentrations of 100–200 mM gives rise to bacteria with lowered lectin activities. Bacteriolytic enzymes were able to lyse bacteria from glycine-grown cultures more readily than from cultures without the glycine supplement. The bacteria produce glucan-binding proteins, including glucosyltransferases, but they do not readily aggregate with added dextran. Furthermore, SDS-PAGE gels of supernatants of growth media (±glycine) are similar, suggesting the bacteria do not produce a unique set of proteins. Western blotting with a fluorescein isothiocyanate-labeled dextran probe reveals normal amounts of glucan-binding proteins in glycine-grown streptococci. Glycine may be acting as a type of antibiotic, reducing wall integrity upon which glucan promoted cellular aggregation depends.
Keywords: Glucan; Lectin; Glycine; Streptococcus;
In vitro inhibition of the activity of phosphorylase kinase, protein kinase C and protein kinase A by caffeic acid and a procyanidin-rich pine bark (Pinus marittima) extract by Mirella Nardini; Cristina Scaccini; Lester Packer; Fabio Virgili (219-225).
Caffeic acid (CA) is a common constituent of human diet while pine bark extract (PBE) is utilized either as nutritional supplement or as phytochemical remedy for different diseases. CA and PBE, are reported as efficient antioxidants and more recently have been described to modulate cellular response to oxidative challenge and to possess many other biological activities, i.e. anti-inflammatory, antimutagenic, antitumoral effects. In order to investigate in depth the mechanism of action of these polyphenols, the effects of CA and PBE on the activity of some protein kinases involved in the regulation of fundamental cellular processes were studied in vitro: phosphorylase kinase (PhK), protein kinase A (PKA), protein kinase C (PKC). PBE at the concentration of 20 μg/ml (corresponding to 69 μM catechin equivalents) inhibited PKA, PhK and PKC by about 90, 59, 57%, respectively, while 100 μM CA inhibited by 37, 52 and 54%, respectively. Considerable inhibitions have been still observed at even lower concentrations of CA and PBE. For PhK and PKA, the inhibition follows a non-competitive mechanism. CA also inhibits PKC activity in a partially purified cellular extract. The results suggest a possible involvement of CA and PBE in modulation of cellular functions.
Keywords: Caffeic acid; Pine bark extract; Polyphenol; Protein kinase; Phosphorylase kinase;
Primary structures of two hemagglutinins from the marine red alga, Hypnea japonica 1 1 The sequence data of this paper have been submitted to the sequence data bank and are available under the accession numbers of JC5773 for hypnin A-1 and JC5774 for hypnin A-2. by Kanji Hori; Kiminori Matsubara; Keisuke Miyazawa (226-236).
As the first examples among marine algal hemagglutinins, the primary structures of two hemagglutinins, named hypnin A-1 and A-2, from the red alga Hypnea japonica, were determined by Edman degradation. Both hemagglutinins were single-chain polypeptides composed of 90 amino acid residues including four half-cystines, all of which were involved in two intrachain disulfide bonds, Cys5-Cys62 and Cys12-Cys89. Hypnin A-1 and A-2 had calculated molecular masses of 9146.7 and 9109.7 Da which coincided with determined values, 9148 and 9109 Da, by electrospray ionization-mass spectrometry, respectively. Both hemagglutinins only differed from each other at three positions; Pro19, Arg31 and Phe52 of hypnin A-1 as compared with Leu19, Ser31, and Tyr52 of hypnin A-2. Approximately 43% of total residual numbers consisted of three kinds of amino acids: serine, glycine and proline. The hemagglutination activities were lost by reduction and alkylation of the disulfide bonds. The nature of the small-sized polypeptides, including disulfide bonds, may contribute to the extreme thermostability of the hemagglutinins. Sequence having overall similarity to hypnin A-1 or A-2 was not detected in databases. Unexpectedly, however, hypnins contained a motif similar to the alignment of the C-terminal conserved amino acids within carbohydrate-recognition domains of C-type animal lectins. Furthermore, interestingly, the hemagglutination activities were inhibited by a protein, phospholipase A-2 besides some glycoproteins, suggesting that hypnins may possess both a protein-recognition site(s) and a carbohydrate-recognition site(s).
Keywords: Marine red alga; Hemagglutinin; Lectin; Primary structure; C-type animal lectin; Phospholipase A2;
A versatile assay to study cellular uptake of gene transfer complexes by flow cytometry by M. Ogris; E. Wagner; P. Steinlein (237-243).
In this study, we present a simple and reliable method to analyse the first steps of DNA-based gene delivery into eucaryotic cells, i.e. binding and internalisation of transfection complexes. Taking advantage of flow cytometry, it is possible to discriminate quantitatively between total and internal DNA on a single-cell level. Here, we use two fluorescent dyes with high specificity and affinity to double-stranded DNA that cannot penetrate the extracellular membrane of living cells. Total DNA is stained prior to complexation with the first dye and complexes are added to cells. After the incubation, only extracellular DNA remains accessible to the second dye. Cell associated fluorescence is measured simultaneously using a flow cytometer and data are analysed using a computer program capable of calculating the ratio of fluorescence intensities on a single-cell level. These ratios are indicative of the binding and internalisation kinetics of gene transfer complexes.
Keywords: Gene transfer; Internalization; Flow cytometry; YOYO-1; TOTO-3;
Transfection using synthetic peptides: comparison of three DNA-compacting peptides and effect of centrifugation by Laurence Vaysse; Benoı̂t Arveiler (244-250).
Positively charged peptides have been shown to allow efficient transfection in vitro, especially when mixed with lipids. We have compared the ability of three positively charged peptides both to compact DNA and to increase the transfection efficiency of the cationic lipid DOTAP. The peptides are: a polymer of 17 lysines (pK17), YKAWK8WK (peptide K8) and SPKRSPKRSPKR (peptide P2). Peptides pK17 and K8 compact DNA efficiently in a gel retardation assay and protect DNA efficiently against DNase I degradation. Peptide P2, on the other hand, interacts weakly with DNA and provides poor protection. In order to compare their transfection efficiency, the three peptides were mixed with DNA (plasmid pEGFP-N1) at different charge ratios (+/−) and DOTAP (at a charge ratio of 2). The transfection efficiency was measured by FACS analysis at different times post-transfection. With NIH-3T3 cells, peptide P2 provides the highest transfection efficiency (about 40%), when compared with peptides pK17 (29%) and K8 (31%) and DOTAP alone (21%) under optimal conditions. Finally, we showed that centrifugation of the complexes onto the cells increased the transfection efficiency by a factor 1.5 to 2 with the various cell lines tested (ECV, primary human keratinocyte, CFT-2, NT-1).
Keywords: Lysine-rich peptide; Cationic liposome; DNA condensation; Green fluorescent protein; Gene delivery; Centrifugation;
End-to-end distance distribution in bradykinin observed by Förster resonance energy transfer by Eduardo Sérgio de Souza; Izaura Yoshico Hirata; Luiz Juliano; Amando Siuiti Ito (251-261).
Förster resonance energy transfer (FRET) was used to study the conformational dynamics of bradykinin related peptides. The fluorescent probe aminobenzoic acid (Abz) bound to the amino terminal of bradykinin maintained its fluorescence characteristics, like high quantum yield and excited state decay dominated by a lifetime of 8.3 ns. The binding of the acceptor group N-[2,4-dinitrophenyl]-ethylenediamine (EDDnp) to the carboxy terminal of Abz labeled bradykinin resulted in a drastic decrease of the fluorescence intensity and in a fastening of the excited state decay. The change of the decay kinetics to an heterogeneous process, precludes the use of energy transfer models based on a single fixed distance between donor and acceptor. The computational package CONTIN was employed to the analysis of time-resolved fluorescence data, allowing the recovery of a distance distribution between donor and acceptor corresponding to the end-to-end distance of the labeled peptide. The distance distribution reflects the occurrence of distinct conformations for the peptide, that coexist in equilibrium during the fluorescence lifetime. We observed three distance populations for bradykinin in water, that merged to two populations when the solvent was trifluoroethanol (TFE). The results were consistent with those obtained from circular dichroism spectroscopy, that showed structural flexibility in water and the presence of more defined secondary structure in TFE. We also studied several peptides related to bradykinin, and the results emphasized the formation of turns involving the proline residues and the decrease of conformational flexibility induced by using TFE as the solvent.
Keywords: Ortho-aminobenzoyl-peptide; Förster resonance energy transfer; Distance distribution; Conformational dynamics; Protease fluorescent substrate;
Identification of sialic acids on the cell surface of Candida albicans by Regina M.A. Soares; Rosangela M. de A. Soares; Daniela S. Alviano; Jayme Angluster; Celuta S. Alviano; Luiz R. Travassos (262-268).
The cell-surface expression of sialic acids in two isolates of Candida albicans was analyzed by thin-layer and gas chromatography, binding of lectins, colorimetry, sialidase treatment and flow cytofluorimetry with fluorescein-labeled lectins. N-acetylneuraminic acid (NANA) was the only derivative found in both strains of C. albicans grown in a chemically defined medium. Its identification was confirmed by mass spectrometry in comparison with an authentic standard. The density of sialic acid residues per cell ranged from 1.6×106 to 2.8×106. The surface distribution of sialic acids over the entire C. albicans was inferred from labeling with fluorescein-Limulus polyphemus and Limax flavus agglutinins and directly observed by optical microscopy with (FITC)-Sambucus nigra agglutinin (SNA), abrogated by previous treatment of yeasts with bacterial sialidase. Sialidase-treated yeasts generated β-galactopyranosyl terminal residues that reacted with peanut agglutinin. In C. albicans N-acetyl-neuraminic acids are α2,6- and α2,3-linked as indicated by yeast binding to SNA and Maackia amurensis agglutinin. The α2,6-linkage clearly predominated in both strains. We also investigated the contribution of sialic acids to the electronegativity of C. albicans, an important factor determining fungal interactions in vivo. Adhesion of yeast cells to a cationic solid phase substrate (poly-L-lysine) was mediated in part by sialic acids, since the number of adherent cells was significantly reduced after treatment with bacterial sialidase. The present evidence adds C. albicans to the list of pathogenic fungi that synthesize sialic acids, which contribute to the negative charge of fungal cells and have a role in their specific interaction with the host tissue.
Keywords: Candida albicans; Sialic acid; Lectin; Sialidase;