Gianfranco Liut

Isolation of a sulfobromophthalein-binding protein from hepatocyte plasma membrane

This paper deals with the isolation and partial characterization of a protein capable of high affinity sulfobromophthalein-binding from liver plasma membrane. The purification involves acetone powder of a crude preparation of rat liver plasma membrane, salt extraction and purification through two chromatographic steps. Based on sulfobromophthalein binding, the process gives a yield of approximately 40%, with a purification of about 300 times with respect to the starting homogenate. The best preparation can bind more than 100 nmol sulfobromophthalein/mg protein. The protein behaves as a single species in dodecyl sulphate polyacrylamide gel electrophoresis, with an apparent molecular weight of 1.7 . 10(5). The molecule does not contain sugars. The dissociation constant of the protein . sulfobromophthalein complex has been found to be 4. 10(-6) M, a value in agreement with that of high affinity binding sites described on isolated liver plasma membrane.

Physicochemical properties of the exocellular polysaccharide from Cyanospira capsulata

This paper reports on the chemical structure and on some physicochemical properties of a new exopolysaccharide, CC-EPS, secreted by a filamentous nitrogen-fixing cyanobacterium, Cyanospira capsulata. Solution properties of CC-EPS were studied with the aim of characterizing the conformational state of the polysaccharide in water and in aqueous salt solution, in view of the peculiar rheological properties. Because the molecule has two different uronic acid residues, a variation of pH and/or ionic strength was considered to be the best perturbation to disclose any possible ordered structures. We found no evidence for a cooperative conformational transition from the change of the chiro-optical properties or from the smooth increase of pKa as a function of the degree of ionization, alpha, of the polyacid. The same conclusion was drawn from the study of the temperature dependence and ionic strength dependence (in the range up to 0.1 M NaCl) of solution properties. The data suggest that the solution conformation of CC-EPS is a random coil with a chain flexibility comparable to that of alginate.

The ability of the mitochondrial Ca2+-binding glycoprotein to restore Ca2+ transport in glycoprotein-depleted rat liver mitochondria.

Rat liver mitochondria may be subfractionated in sediment and supernatant fractions by swelling in the presence of EDTA and oxaloacetate. The sediment is largely depleted of the Ca2+-binding glycoprotein and its Ca2+-transporting activity may be as low as 10--20% of the starting value. Both the rate of Ca2+ uptake and the capacity to maintain a high Ca2+ concentration gradient across the membrane are depressed. Addition of an osmotic supernatant to the assay mixture may partially restore the original Ca2+-transporting ability. The active component in the supernatant is the Ca2+-binding glycoprotein. This is shown by the following facts: (a) the effect is enhanced by the addition of the purified glycoprotein to the supernatant; (b) precipitation of the glycoprotein from the supernatant by affinity chromatography-purified antibodies abolishes the stimulatory effect, and (c) in the presence of 130 microM Mg2+, the glycoprotein alone may restore fully the Ca2+-transporting ability of the particles. The maximal velocity is already reached at 0.1 microgram glycoprotein/mg mitochondrial protein.

Versatility of the Burkholderia cepacia complex for the biosynthesis of exopolysaccharides: a comparative structural investigation.

The Burkholderia cepacia Complex assembles at least eighteen closely related species that are ubiquitous in nature. Some isolates show beneficial potential for biocontrol, bioremediation and plant growth promotion. On the contrary, other strains are pathogens for plants and immunocompromised individuals, like cystic fibrosis patients. In these subjects, they can cause respiratory tract infections sometimes characterised by fatal outcome. Most of the Burkholderia cepacia Complex species are mucoid when grown on a mannitol rich medium and they also form biofilms, two related characteristics, since polysaccharides are important component of biofilm matrices. Moreover, polysaccharides contribute to bacterial survival in a hostile environment by inhibiting both neutrophils chemotaxis and antimicrobial peptides activity, and by scavenging reactive oxygen species. The ability of these microorganisms to produce exopolysaccharides with different structures is testified by numerous articles in the literature. However, little is known about the type of polysaccharides produced in biofilms and their relationship with those obtained in non-biofilm conditions. The aim of this study was to define the type of exopolysaccharides produced by nine species of the Burkholderia cepacia Complex. Two isolates were then selected to compare the polysaccharides produced on agar plates with those formed in biofilms developed on cellulose membranes. The investigation was conducted using NMR spectroscopy, high performance size exclusion chromatography, and gas chromatography coupled to mass spectrometry. The results showed that the Complex is capable of producing a variety of exopolysaccharides, most often in mixture, and that the most common exopolysaccharide is always cepacian. In addition, two novel polysaccharide structures were determined: one composed of mannose and rhamnose and another containing galactose and glucuronic acid. Comparison of exopolysaccharides obtained from cultures on agar plates with those extracted from biofilms on cellulose membranes showed important differences, thus suggesting that extrapolating data from non-biofilm conditions might not always be applicable.

A novel highly charged exopolysaccharide produced by two strains of Stenotrophomonas maltophilia recovered from patients with cystic fibrosis

Stenotrophomonas maltophilia is a non-fermenting Gram-negative microorganism capable of causing chronic pulmonary infection in cystic fibrosis patients and its ability to form biofilms on polystyrene and glass surfaces, as well as on cystic fibrosis-derived bronchial epithelial IB3-I cells was recently demonstrated. The latter evidence might explain the power of S. maltophilia to produce persistent lung infections, despite intensive antibiotic treatment. In addition to being important components of the extracellular biofilm matrix, polysaccharides are involved in virulence, as they contribute to bacterial survival in a hostile environment. With the aim of contributing to the elucidation of S. maltophilia virulence factors, the exopolysaccharides produced by two mucoid clinical isolates of S. maltophilia obtained from two cystic fibrosis patients were completely characterised, mainly by means of ESI-MS and NMR spectroscopy. The results showed that, although the two isolates were recovered from two different patients living in different countries (Italy and France), the exopolysaccharides produced have an identical primary structure, with the following repeating unit: The exopolysaccharide is highly negatively charged for the presence of three uronic acids on four residues in the repeating unit. Moreover, an ether-linked d-lactate substituent is located on C-3 and one O-acetyl group on C-4 of the galacturonic acid side chain. Another O-acetyl group substitutes C-2 of the galacturonic acid in the backbone, making this primary structure unique.

Synthesis and conformational properties of cyanoethyl-Scleroglucan

Cyanoethyl derivatives were obtained as intermediates in the chemical modification of the neutral polysaccharide Scleroglucan, aimed at attaining new ionic carbohydrate polymers. The chemical and conformational properties of the cyanoethylated polymer were investigated and compared with those exhibited by the native one. In particular, capillary viscosity and small angle X-ray scattering experiments demonstrated that in aqueous solutions cyanoethyl derivatives were not able to assume the triple helix structure typical of the parent sclecroglucan.

Specific and non-specific ion-polysaccharide interactions '

The interaction of ions with certain carbohydrate ligands in aqueous solution is examined. Simple systems are analysed to understand the role of conformational transitions and the possible specificity often advanced as an explanation for the complexity of the binding. Evidence of an extrinsic chirality of bound ionic chromophores is presented for nearly enantiomeric complexes of alginate and pectins with Pb2+ ions. Data on the thermodynamics of interaction between divalent cations and typical ionic polysaccharides are presented with a view to offering an interpretation based upon current theoretical models of polyelectrolyte solutions. In particular, reference is made to the linear correlation between thermodynamic state functions.

Biofilms from Klebsiella pneumoniae: Matrix Polysaccharide Structure and Interactions with Antimicrobial Peptides

Biofilm matrices of two Klebsiella pneumoniae clinical isolates, KpTs101 and KpTs113, were investigated for their polysaccharide composition and protective effects against antimicrobial peptides. Both strains were good biofilm producers, with KpTs113 forming flocs with very low adhesive properties to supports. Matrix exopolysaccharides were isolated and their monosaccharide composition and glycosidic linkage types were defined. KpTs101 polysaccharide is neutral and composed only of galactose, in both pyranose and furanose ring configurations. Conversely, KpTs113 polysaccharide is anionic due to glucuronic acid units, and also contains glucose and mannose residues. The susceptibility of the two strains to two bovine cathelicidin antimicrobial peptides, BMAP-27 and Bac7(1–35), was assessed using both planktonic cultures and biofilms. Biofilm matrices exerted a relevant protection against both antimicrobials, which act with quite different mechanisms. Similar protection was also detected when antimicrobial peptides were tested against planktonic bacteria in the presence of the polysaccharides extracted from KpTs101 and KpTs113 biofilms, suggesting sequestering adduct formation with antimicrobials. Circular dichroism experiments on BMAP-27 in the presence of increasing amounts of either polysaccharide confirmed their ability to interact with the peptide and induce an α-helical conformation.

Some Properties of Two Proteins Involved in Membrane Transport

One of the most intensively investigated fields of biological research nowadays is membrane structure and function.The reason for this great interest resides in the great number of biological functions connected with these microscopic structures. It is not appropriate to recall here all the concepts which have developed in this area since some decades (see for a recent review 1). The most generally accepted structure today is that illustrated by Singer (2) and referred to as fluid-mosaic model. In the model phospholipids are arranged in a bilayer system,with the hydrophobic tails in close association to each other,whereas the polar heads are facing the water phase on both sides of the membrane. Associated to the phospholipid bilayer are the protein molecules which are to be classified in peripheral and integral ones. The type of interaction of the two classes of proteins with phospholipids are totally different. In the former case protein are bound to the surface via polar groups. Changes in ionic strength and/or chelation of divalent cations is often sufficient to cause the peripheral proteins to be released. On the contrary,integral proteins are firmly bound to the lipid bilayer and hydrophobic interactions contribute greatly to the stabilization of the structure. Obviously depending on the physical properties of the surface of the protein three possibilities are open: a) a protein molecule is completely embedded in the lipid bilayer; b) a protein molecule is only partially embedded in hydrophobic core of the membrane and faces one of the two surfaces with a hydrophilic tail and c) a protein molecule may be so arranged that only the intermediate portion of it shows a hydrophobic outer surface and the protein may be visualized as a transmembrane component.