Marco Signorelli

The X-Ray Structure of Zebrafish (Danio rerio) Ileal Bile Acid-Binding Protein Reveals the Presence of Binding Sites on the Surface of the Protein Molecule

The ileal bile acid-binding proteins (I-BABPs), also called ileal lipid-binding proteins or gastrotropins, belong to the family of the fatty acid-binding proteins and play an important role in the solubilization and transport of bile acids in the enterocyte. This article describes the expression, purification, crystallization, and three-dimensional structure determination of zebrafish (Danio rerio) I-BABP both in its apo form and bound to cholic acid. This is the first X-ray structure of an I-BABP. The structure of the apoprotein was determined to a resolution of 1.6 A, and two different monoclinic crystal forms of the holoprotein were solved and refined to 2.2 A resolution. Three protein molecules are present in the asymmetric unit of one of the co-crystal forms and two in the other, and therefore, the results of this study refer to observations made on five different protein molecules in the crystalline state. In every case, two cholate ligands were found bound in approximately the same position in the internal cavity of the protein molecules, but an unexpected result is the presence of clear and unambiguous electron density for several cholate molecules bound on hydrophobic patches on the surface of all the five independent protein molecules examined. Isothermal titration calorimetry was used for the thermodynamic characterization of the binding mechanism and has yielded results that are consistent with the X-ray data. Ligand binding is described in detail, and the conformational changes undergone by the protein molecule in the apo-to-holo transition are examined by superposition of the apo- and holoprotein models. The structure of the holoprotein is also compared with that of the liver BABP from the same species and those of other I-BABPs determined by NMR.

Thermodynamic and structural characterization of Liposomal-Locked in-Dendrimers as drug carriers.

A new Liposomal-Locked in-Dendrimer (LLD) formed by DPPC-DPPG and PAMAM 3.5 incorporating the anticancer drug DOX was studied by means of spectroscopic and DSC investigations. Multilamellar Lipid Bilayers were also considered for the sake of comparison. The results were in line with a picture of phase separation between DPPC-DPPG lipids and dendrimer that promotes the stability of the liposome membrane and the cooperativity of the relevant gel-to-liquid-crystal transition, which is enhanced in the presence of the dendrimer and the drug. As a result, the inner core of the liposome contained large amounts of dendrimer-DOX complex and was protected by a very stable membrane. This view was given a more general validation through investigations performed with other types of dendrimers, namely PG1 and PG2. The thermodynamic interpretation of the DSC data allowed a better understanding of the physico-chemical factors that justify this behaviour that makes these LLDs very promising as a new class of Modulatory Liposomal Controlled Release System (MLCRS) that could lead to drug formulations with higher safety and efficacy profiles.

Alkalizing Reactions Streamline Cellular Metabolism in Acidogenic Microorganisms

An understanding of the integrated relationships among the principal cellular functions that govern the bioenergetic reactions of an organism is necessary to determine how cells remain viable and optimise their fitness in the environment. Urease is a complex enzyme that catalyzes the hydrolysis of urea to ammonia and carbonic acid. While the induction of urease activity by several microorganisms has been predominantly considered a stress-response that is initiated to generate a nitrogen source in response to a low environmental pH, here we demonstrate a new role of urease in the optimisation of cellular bioenergetics. We show that urea hydrolysis increases the catabolic efficiency of Streptococcus thermophilus, a lactic acid bacterium that is widely used in the industrial manufacture of dairy products. By modulating the intracellular pH and thereby increasing the activity of β-galactosidase, glycolytic enzymes and lactate dehydrogenase, urease increases the overall change in enthalpy generated by the bioenergetic reactions. A cooperative altruistic behaviour of urease-positive microorganisms on the urease-negative microorganisms within the same environment was also observed. The physiological role of a single enzymatic activity demonstrates a novel and unexpected view of the non-transcriptional regulatory mechanisms that govern the bioenergetics of a bacterial cell, highlighting a new role for cytosol-alkalizing biochemical pathways in acidogenic microorganisms.

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed.

Interaction study between maltose-modified PPI dendrimers and lipidic model membranes.

The influence of maltose-modified poly(propylene imine) (PPI) dendrimers on dimyristoylphosphatidylcholine (DMPC) or dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) (3%) liposomes was studied. Fourth generation (G4) PPI dendrimers with primary amino surface groups were partially (open shell glycodendrimers - OS) or completely (dense shell glycodendrimers - DS) modified with maltose residues. As a model membrane, two types of 100nm diameter liposomes were used to observe differences in the interactions between neutral DMPC and negatively charged DMPC/DMPG bilayers. Interactions were studied using fluorescence spectroscopy to evaluate the membrane fluidity of both the hydrophobic and hydrophilic parts of the lipid bilayer and using differential scanning calorimetry to investigate thermodynamic parameter changes. Pulsed-filed gradient NMR experiments were carried out to evaluate common diffusion coefficient of DMPG and DS PPI in D2O when using below critical micelle concentration of DMPG. Both OS and DS PPI G4 dendrimers show interactions with liposomes. Neutral DS dendrimers exhibit stronger changes in membrane fluidity compared to OS dendrimers. The bilayer structure seems more rigid in the case of anionic DMPC/DMPG liposomes in comparison to pure and neutral DMPC liposomes. Generally, interactions of dendrimers with anionic DMPC/DMPG and neutral DMPC liposomes were at the same level. Higher concentrations of positively charged OS dendrimers induced the aggregation process with negatively charged liposomes. For all types of experiments, the presence of NaCl decreased the strength of the interactions between glycodendrimers and liposomes. Based on NMR diffusion experiments we suggest that apart from electrostatic interactions for OS PPI hydrogen bonds play a major role in maltose-modified PPI dendrimer interactions with anionic and neutral model membranes where a contact surface is needed for undergoing multiple H-bond interactions between maltose shell of glycodendrimers and surface membrane of liposome.

Microbial biosensors to monitor the encapsulation effectiveness of Doxorubicin in chimeric advanced Drug Delivery Nano Systems: A calorimetric approach

The release of the anticancer drug doxorubicin (DOX) incorporated in a new drug carrier, namely a chimeric nanosystem formed by liposomes and dendrimers, was studied following the influence of the drug on the growth kinetics of the Lactobacillus helveticus bacterium, that would mimic the intestinal microflora. The bacterial growth was followed at 37°C by means of Isothermal Titration Calorimetry (ITC) and the method was assessed to monitor the overall effect of the delivered drug obtaining simple objective parameters to define the encapsulation effectiveness of the system, discriminating dose effects even in cases of very low release. Traditional microbiological investigations and in vitro release tests were also performed in parallel for validation. The achieved results suggest that L. helveticus is an excellent candidate as biosensor to assess the sealing effectiveness of these DOX drug carriers through ITC investigations. This approach can be extended for quantitative comparison of drug delivery systems with the same drug inserted in other supramolecular bodies for quantitative comparison. The peculiar results for the DOX drug carrier system investigated, indicate also that, the use of hydrophilic dendrimers in this case, produce a high sealing effect that seems promising in terms of the intestinal flora protection.

Shelf life extension of whole-wheat breadsticks: Formulation and packaging strategies

The aim of this study was the shelf life extension of whole-wheat breadsticks through the addition of a rosemary extract and packaging under nitrogen. Shelf life was studied at four temperatures (20, 27, 35, 48°C) for up to 200 storage days. The minimal changes observed in moisture, water activity and texture of the samples, coupled with the high peroxide values (13-539meqO2/kgfat) measured at the end of storage, and the exponential increase of hexanal concentrations (up to 13-34mg/kg) confirmed that quality decay of whole-wheat breadsticks is mainly associated to lipid oxidation. The kinetic study of oxidation development and the consumer sensory acceptance determined by the survival analysis demonstrated that the rosemary extract addition yields a 42% shelf life extension, higher than that observed using nitrogen in the package (24-29%). The combination of the formulation and packaging strategies gave the best result (83% shelf life extension at 25°C).