Natalina Cammertoni

Hypoallergenic properties of donkey's milk: a preliminary study

Cows milk protein allergy (CMPA) is an abnormal immunological response to cow milk proteins, which results in IgE-mediated reactions. The therapeutic strategy to respond to CMPA envisages the total elimination of milk or the administration of cows milk substitutes. For this reason the use of milk from other mammalian species was tested. Among them, donkeys milk proved to be the best alternative in feeding infants affected by CMPA, since its chemical composition is comparable to human milk. In this work an in vitrostudy was performed in order to analyze the IgE reactivity to milk protein allergens from cow, donkey and goat. In particular, immunoblotting experiments using sera from milk-allergic and non-allergic adult volunteers were conducted with the aim of verifying the hypoallergenic property of donkeys milk. This study provided a preliminary evidence of the hypoallergenicity of donkeys milk when compared to bovine and goat milk. Considering the obtained results, it would be possible to develop a sensitive diagnostic method for CMPA detection, based on chromatographic and immunoblotting analysis.

Modulation of human cytidine deaminase by specific aminoacids involved in the intersubunit interactions

An investigation was made of the role exerted by some residues supposed to be involved in the intersubunit interaction and also in the catalytic site of homotetrameric human cytidine deaminase (T‐CDA). Attention was focused on Y33, Y60, R68, and F137 residues that are a part of a conserved region in most T‐CDAs. Hence, a series of site‐directed mutagenesis experiments was set up obtaining seven mutants: Y60G, Y33G, Y33F Y33S, F137A, R68G, and R68Q. Each active purified mutant protein was characterized kinetically, with a series of substrates and inhibitors, and the effect of temperature on enzyme activity and stability was also investigated. Circular dichroism (CD) experiments at different temperatures and in presence of small amounts of sodium dodecyl sulphate (SDS) were performed in all the soluble mutant CDAs. The results obtained by site‐directed mutagenesis studies were compared to the crystallographic data of B. subtilis CDA and E. coli CDA and to molecular modeling studies previously performed on human CDA. The mutation of Y60 to glycine produced an enzyme with a more compact quaternary structure with respect to the wild‐type; this mutation did not have a dramatic effect on cytidine deamination, but it slightly affected the binding with the substrate. None of the mutant CDAs in Y33 showed enzymatic activity; they existed only as monomers, indicating that this residue, located at the intersubunit interface, may be responsible for the correct folding of human CDA. The insertion of an alanine instead of phenylalanine at position 137 led to a soluble but completely inactive enzyme unable to form a tetramer, suggesting that F137 residue may be important for the assembling of the tetramer and also for the arrangement of the CDA active site. Finally, R68G and R68Q mutations revealed that the presence of the amino group seems to be important for the catalytic process but not for substrate binding, as already shown in B. subtilis CDA. The quaternary structure of R68Q was not affected by the mutation, as shown by the SDS‐induced dissociation experiments and CD studies, whereas R68G dissociated very easily in presence of small amounts of SDS. These experiments indicated that in the human CDA, the side chain of arginine 68 involved in the catalytic process in one subunit active site might come from another subunit. The data obtained from these studies confirmed the presence of a complicated set of intersubunit interactions in the active site of human CDA, as shown in other T‐CDAs. Proteins 2008.

Profile of Nucleosides and Nucleotides in Donkey’s Milk

Nucleotides play a crucial role to cellular functions; they can be obtained from the diet or through the nucleotide salvage pathway, however, in particular situations (occurring mainly in newborns) the metabolic demand of nucleotides exceeds the capacity of their synthesis. These molecules, are receiving attention from a nutraceutical point of view because of their potential direct role in regulating metabolism and infant body condition. Donkeys milk may be considered a good replacer for cows milk in feeding children with severe Ig-E mediated cows milk protein allergy, due to its high similarity with human milk. In this study, the presence of cytidine, uridine, CMP, UMP, guanosine, and adenosine, involved in numerous biochemical and physiological activities, were detected for the first time through a RP-HPLC method.

Purification and Identification of αs1- and β-Caseins from Asses Milk

Recent clinical studies have demonstrated that feeding with asses milk is a safe and valid treatment for infants affected by protein intolerance to dairy cows milk (Businco et al., 2000; Iacono et al., 1992). In fact, the composition of asses milk is similar to humans milk, especially concerning the lipid and protein fraction contents (Fantuz et al., 2001; Salimei et al., 2004). Furthermore, the protein fraction in asses milk is characterized by low casein and β-lactoglobulin content, probably responsible for the hypoallergenic characteristics of this milk, and by a quite high lysozime content (Fantuz et al., 2001; Salimei et al., 2004). In any case, there is not much information regarding the caseinic fractions of asses milk in the literature. Therefore, in the present study αS1and β-caseins were purified from asses milk by gel filtration chromatography followed by anion exchange chromatography on HPLC. The obtained caseins were characterized by SDS-PAGE and, after blotting on a PVDF membrane, were identified by N-terminal sequencing.

Intersubunit Interactions in Human Cytidine Deaminase

Abstract In order to design new efficient cytidine based drugs, an intersubunit interactions study related to the active site has been performed on the wild-type cytidine deaminase (CDA) and on the mutant enzyme F137W/W113F. F137 is the homologous to the Bacillus subtilis CDA F125 involved in the subunit interactions. In presence of the dissociating agent SDS, wild-type human CDA dissociate into enzymatically inactive monomers without intermediate forms via a non-cooperative transition. Extensive dialysis or dilution of the inactivated monomers restores completely the activity. The presence of the strong human CDA competitive inhibitor 5-fluorozebularine disfavour dissociation of the tetramer into subunits in the wild-type CDA but not in mutant enzyme F137W/W113F.

Evidence of Anti-Gliadin and Transglutaminase Antibodies in Sera of Dogs Affected by Lymphoplasmacytic Enteritis

Celiac disease is a disorder that affects the small intestine of genetically predisposed individuals and is developed by the presence of the gliadin fraction of wheat gluten. This disease is characterized by a high seric titre of antibodies against cereal components (anti-gliadin antibodies) and anti-endomysium antibodies (Fasano and Catassi, 2001). It has recently been demonstrated that the modification of gliadin by tissue transglutaminase (tTG) and the formation of gliadin-tTG complexes is important in the immunophatogenesis of the disorder (Dieterich et al., 1998). In human medicine the diagnosis of celiac disease takes advantage of the presence of specific antibodies in the serum of patients (anti-gliadin antibodies, AAG; anti-endomysium antibodies, EmA; anti-transglutaminase antibodies, anti-tTG). The evaluation of such antibodies is useful in selection of patients that have a high probability to be affected by celiac disease, in order to perform an intestinal biopsy. In the dog lymphoplasmacytic enteritis is a significant problem, because it is correlated to weight loss, decrease in performance, diarrhoea and vomiting. In many cases strong plasmacytic infiltration can induce lymphagiectasis, and consequently hypoproteinemia. The aim of this work was to set up a method to evaluate the presence of AAG and anti-tTG antibodies in symptomatic dogs with a clinical diagnosis of lymphoplasmacytic enteritis. For this purpose, one commercial preparation of gliadin and one of transglutaminase were loaded on polyacrylamide gel electrophoresis in the presence of sodium-dodecyl sulphate (SDS-PAGE), blotted on a nitrocellulose membrane and utilized as antigens to search for AAG and anti-tTG antibodies. The sera of clinically and histologically healthy dogs were used as negative controls, while a preparation of anti-gliadin and anti-transglutaminase antibodies was used as a positive control. The obtained data indicated the presence of a high prevalence of AAG and anti-tTG antibodies in the sera of dogs with lymphoplasmacytic enteritis. Therefore, this test, once it has been tested on greater numbers of animals, may be used to evaluate the presence of an intestinal pathology and to establish an appropriate diet for the dog.

Purification and Identification of αs1

Recent clinical studies have demonstrated that feeding with asses milk is a safe and valid treatment for infants affected by protein intolerance to dairy cows milk (Businco et al., 2000; Iacono et al., 1992). In fact, the composition of asses milk is similar to humans milk, especially concerning the lipid and protein fraction contents (Fantuz et al., 2001; Salimei et al., 2004). Furthermore, the protein fraction in asses milk is characterized by low casein and β-lactoglobulin content, probably responsible for the hypoallergenic characteristics of this milk, and by a quite high lysozime content (Fantuz et al., 2001; Salimei et al., 2004). In any case, there is not much information regarding the caseinic fractions of asses milk in the literature. Therefore, in the present study αS1and β-caseins were purified from asses milk by gel filtration chromatography followed by anion exchange chromatography on HPLC. The obtained caseins were characterized by SDS-PAGE and, after blotting on a PVDF membrane, were identified by N-terminal sequencing.

Purification and identification of alpha(s-1) and beta-caseins from asses milk

Recent clinical studies have demonstrated that feeding with asses milk is a safe and valid treatment for infants affected by protein intolerance to dairy cows milk (Businco et al., 2000; Iacono et al., 1992). In fact, the composition of asses milk is similar to humans milk, especially concerning the lipid and protein fraction contents (Fantuz et al., 2001; Salimei et al., 2004). Furthermore, the protein fraction in asses milk is characterized by low casein and β-lactoglobulin content, probably responsible for the hypoallergenic characteristics of this milk, and by a quite high lysozime content (Fantuz et al., 2001; Salimei et al., 2004). In any case, there is not much information regarding the caseinic fractions of asses milk in the literature. Therefore, in the present study αS1and β-caseins were purified from asses milk by gel filtration chromatography followed by anion exchange chromatography on HPLC. The obtained caseins were characterized by SDS-PAGE and, after blotting on a PVDF membrane, were identified by N-terminal sequencing.