Pierluigi Mariani

Donkey milk production: state of the art

Abstract Milk is one of the most common causes of food allergies among children under one year of age. No specific therapy exists for this allergy, and thus the only feasible response is to avoid assumption of milk and derived products. Studies conducted on the serum of children with hypersensitivity to milk have shown that caseins are the proteins with the greater allergenic potential. However, in some cases, children have also shown hypersensitivity to the β-lactoglobulines and to the α-lactal-bumins. When food intolerance is diagnosed in an infant, it is often necessary to impose a period of total parenteral feeding, followed by breast feeding, considered the most correct method of re-feeding. When human milk can not be given, alternative food sources must be sought. Clinical studies have demonstrated that donkey milk could substitute breast feeding in infants affected by severe Ig-E mediated milk allergies. In these subjects, donkey milk is not only useful, but also safer than other types of milk. In fact donkey milk composition in lipids (high levels of linoleic and linolenic acid) and proteins (low caseins content) is very close to human milk. Lysozyme content in donkey milk resulted to be very high (mean value 1.0 mg/ml) if compared to bovine (traces), caprine (traces) and human milk. The high lysozyme content of donkey milk may be responsible of the low bacterial count reported in literature and also makes this milk suitable to prevent intestine infections to infants. Among seroproteins, β-lactoglobulin and α-lactalbumin content in donkey milk was respectively 3.75 and 1.80 mg/ml and remained substancially the same during the different stages of lactation.

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.

Essential trace elements in milk and blood serum of lactating donkeys as affected by lactation stage and dietary supplementation with trace elements

The aim of this trial was to study the concentration of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), selenium (Se), cobalt (Co) and iodine (I) in milk and blood serum of lactating donkeys, taking into account the effects of lactation stage and dietary supplementation with trace elements. During a 3-month period, 16 clinically healthy lactating donkeys (Martina-Franca-derived population), randomly divided into two homogeneous groups (control (CTL) and trace elements (TE)), were used to provide milk and blood samples at 2-week intervals. Donkeys in both groups had continuous access to meadow hay and were fed 2.5 kg of mixed feed daily, divided into two meals. The mixed feed for the TE group had the same ingredients as the CTL, but was supplemented with a commercial premix providing 163 mg Zn, 185 mg Fe, 36 mg Cu, 216 mg Mn, 0.67 mg Se, 2.78 mg Co and 3.20 mg I/kg mixed feed. The concentrations of Zn, Fe, Cu, Mn, Se, Co and I were measured in feeds, milk and blood serum by inductively coupled plasma-MS. Data were processed by ANOVA for repeated measures. The milk concentrations of all the investigated elements were not significantly affected by the dietary supplementation with TE. Serum concentrations of Zn, Fe, Cu Mn and Se were not affected by dietary treatment, but TE-supplemented donkeys showed significantly higher concentrations of serum Co (1.34 v. 0.69 μg/l) and I (24.42 v. 21.43 μg/l) than unsupplemented donkeys. The effect of lactation stage was significant for all the investigated elements in milk and blood serum, except for serum manganese. A clear negative trend during lactation was observed for milk Cu and Se concentrations (-38%), whereas that of Mn tended to increase. The serum Cu concentration was generally constant and that of Co tended to increase. If compared with data reported in the literature for human milk, donkey milk showed similarities for Zn, Mn, Co and I. Furthermore, this study indicated that, in the current experimental conditions, the mineral profile of donkey milk was not dependent on dietary TE supply.

Adhesion molecules and cytokine profile in ileal tissue of sheep infected with Mycobacterium avium subsp. paratuberculosis.

Sheep develop clinical diseases after 3-5 years after infection with Mycobacterium avium subsp. paratuberculosis (MAP). Clinical symptoms of paratuberculosis include persistent diarrhea and weight loss due to a chronic inflammation of the small intestine. Tissue alterations in the areas of the ileo-cecal junction are often observed. Here, we investigate the molecular processes underlying tissue damages in intestinal mucosa of 14 sheep showing either tuberculoid or lepromatous form of MAP enteritis. We found that E-cadherins, alpha-catenin and beta1-integrins were present at significant low levels in tissues of sheep affected by lepromatous form and that this pattern was associated with high expression of TGF-beta, IL-10, IL-1beta, and TNF-alpha and with a modest increase of CD4+ and CD25+ T cells. Tissues of sheep with the tuberculoid form showed high expression of IFNgamma, IL-12, and MCP-1 and a significant presence of CD4+ and CD25+ T cells. Finally, anti-transglutaminase (tTG) IgG1 antibodies were detected in sera of infected animal belonging to both groups, as already described for human inflammatory bowel diseases. Our results further stress the similarities in the clinical and histological features between ruminant paratuberculosis and human intestinal inflammatory diseases.

Protein fraction characterization of sheep milk from the Comisana breed

Protein fraction characterization of sheep milk from the Comisana breed S. Vincenzetti & P. Polidori & P. Mariani & A. Vita Published online: 7 August 2008 # Springer Science + Business Media B.V. 2008

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.

Minor and potentially toxic trace elements in milk and blood serum of dairy donkeys

The aim of this trial was to study the concentration of Ti, V, As, Rb, Sr, Mo, Cd, Cs, and Pb in donkey milk and blood serum. One hundred twelve individual milk and blood serum samples were collected from 16 lactating donkeys (Martina-Franca-derived population; 6 to 12 yr old; 3 to 7 parities; average live weight 205.4kg; 32 to 58 d after foaling at the beginning of the trial) during a 3-mo-long experiment. The samples were analyzed for the aforementioned elements by inductively coupled plasma-mass spectrometry. Feedstuff and drinking water were also analyzed for the investigated elements. Data were processed by ANOVA for repeated measures. Average milk concentrations (±SD) of Ti, Rb, Sr, Mo, Cs, and Pb were 77.3 (±7.7), 339.1 (±82.1), 881.7 (±270.4), 4.5 (±1.6), 0.49 (±0.09), and 3.2 (±2.7) μg/L, respectively. More than 80% of samples were below the limit of detection for V, As, and Cd in milk and for Cd, and Pb in blood serum. The lower bound calculated for milk V, As, and Cd was 0.03μg/L for the 3 elements, the upper bound was calculated at 0.23, 0.10, and 0.31μg/L and the maximum value was observed at 0.54, 0.15, and 0.51μg/L, respectively. The average milk concentrations of Ti, Rb, Sr, Mo, and Cs were 600, 458, 346, 16, and 294%, respectively, than those of blood serum. Yet, Cs concentrations were in the same order of magnitude in milk and serum. Moderate to strong positive and significant correlation coefficients were observed between milk and blood serum concentrations for Ti, Rb, Sr, and Cs. The effect of the stage of lactation was significant for all the investigated elements in milk and blood serum, but most of the elements showed only small changes or inconsistent trends, and only the concentrations of Rb and Sr showed decreasing trends both in milk and blood serum. The relationship between milk and blood serum element concentrations indicates that the mammary gland plays a role in determining the milk concentrations of Mo, Ti, Rb, Sr, Mo, and Cs. In the current experimental conditions, in agreement with the low levels in drinking water and feedstuff, donkey milk concentration of potentially toxic elements was very low and did not raise health concerns for human consumption.

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.