Daniela Micozzi

Effects of Lyophilization and Use of Probiotics on Donkey's Milk Nutritional Characteristics

Cow milk protein allergy (CMPA) is an abnormal IgE-mediated reaction to cow milk proteins. Donkey’s milk could be considered suitable for feeding young children affected by severe IgE-mediated CMPA because its nutritional properties and composition are very close to human milk. Since donkey’s milk is available during a limited range of months during the year, it may be useful to find better storage conditions for this product. This study investigated the effects of the lyophilization treatment on donkey’s milk nutritional characteristics, and the results were compared with those obtained on fresh and frozen milk. Nutritional properties of lyophilized donkey’s milk remained basically unchanged compared with fresh milk. Two different probiotic strains were added to lyophilized donkey’s milk, and their viability was evaluated after milk reconstitution. The results obtained confirmed the possibility of producing a probiotic infant formula with beneficial properties using donkey’s milk as raw material.

Spermidine and spermine are enriched in whole blood of nona/centenarians.

Polyamines (putrescine, spermidine, and spermine) are a family of molecules that derive from ornithine through a decarboxylation process. They are essential for cell growth and proliferation, stabilization of negative charges of DNA, RNA transcription, translation, and apoptosis. Recently, it has been demonstrated that exogenously administered spermidine promotes longevity in yeasts, flies, worms, and human cultured immune cells. Here, using a cross-sectional observational study, we determined whole-blood polyamines levels from 78 sex-matched unrelated individuals divided into three age groups: Group 1 (31-56 years, n=26, mean age 44.6±6.07), group 2 (60-80 years, n=26, mean age 68.7±6.07), and group 3 (90-106 years, n=26, mean age 96.5±4.59). The total content of polyamines is significantly lower in groups 2 and 3 compared to group 1 (p=3.6×10(-12)). Interestingly, this reduction is mainly attributable to the lower putrescine content. Group 2 displays the lowest levels of spermidine and spermine. On the other hand, nona/centenarians (group 3) display a significantly higher median relative percentage content of spermine with respect to total polyamines, compared to the other groups (13.2% vs. 14.1% vs. 30.6%, p=6.0×10(-4)). For the first time, we report profiles of polyamines from the whole blood of healthy nona/centenarians, and our results confirm and extend previous findings on the role of polyamines in determining human longevity. However, although we found an important correlation between polyamines levels and age groups, further studies are warranted to fully understand the role of polyamines in determining life span. Also, longitudinal and nutritional studies might suggest potential therapeutic approaches to sustain healthy aging and to increase human life span.

A Proteomic Study on Donkey Milk

In children with Cow Milk Protein Allergy (CMPA), when it is not possible to breast feed or to use cow milk, the clinical use of donkey milk is considered since several studies have demonstrated the high similarity of donkey milk compared to human milk. An analysis was performed on donkey milk protein profile by two-dimensional electrophoresis (2-DE) followed by N-terminal sequencing in order to give a panoramic view of the proteins that are present in donkey milk. Furthermore, the interest was focused on the casein fractions and on their phosphorylation degree that may influence the calcium binding ability of caseins. At this purpose experiments on donkey milk casein dephosphorylation have been performed and the dephosphorylated casein fractions have been identified after 2-DE analysis followed by N-terminal sequencing. Among caseins were found mainly αs1- and β-caseins that showed a considerable heterogeneity due to variable degree of phosphorylation and to the presence of genetic variants. Finally, a quantitative determination of some antimicrobial proteins, such as lactoferrin and lactoperoxidase, that could be able to stimulate the development of the neonatal intestine, was performed in donkey milk, with the results being 0.080±0.0035 g/L and 0.11±0.027 mg/L, respectively. From the obtained data is evinced that human and donkey milk contain considerable amounts of lysozyme and lactoferrin but lactoperoxidase is present only in small amounts, confirming the high similarity between donkey and human milk. The present study on donkey milk proteins may be useful to assess the nutritional characteristics of this milk that is used to feed children affected by CMPA, but also may open the possibility of utilizing donkey milk in the general population to benefit subjects with CMPA, such as adults and the elderly.

CDA gene polymorphisms and enzyme activity: genotype–phenotype relationship in an Italian–Caucasian population

AIM To assess the distribution of CDA activity from whole blood of 142 healthy subjects, determining its main predictors among genetic (six CDA SNPs) and physiological factors (age and gender). Moreover, we performed a kinetic study of the two CDA protein variants (Q27 and K27) determined by the rs2072671 SNP. MATERIALS & METHODS CDA activity was assessed by HPLC. Selected CDA SNPs were genotyped by PCR-based methods. Recombinant CDA protein variants (Q27 and K27) were expressed in an Escherichia coli strain SØ5201 and kinetic assays were performed. RESULTS The mean value of CDA activity was 0.051 ± 0.024 mU/mg and followed a normal distribution in the study population. Carriers of the CDA*2B (-451T/-92G/-31Del/79C/435C) haplotype displayed higher CDA activity compared with the others. CDA -451G>A, -92A>G and 79A>C (K27Q) SNPs displayed significant associations with CDA activity. The best predictive model of CDA activity included the variables gender and CDA 79A>C (K27Q). Cytidine is the preferential substrate for the variant Q27. CONCLUSION We suggest the analysis of both CDA activity and CDA 79A>C (K27Q) SNP in future prospective trials with cytidine analogs, alone or in combination, in order to identify the best marker to secure the administration of these anticancer therapies.

Human cytidine deaminase: A biochemical characterization of its naturally occurring variants

Human cytidine deaminase is an enzyme of the pyrimidine salvage pathways that metabolizes several cytosine nucleoside analogs used as prodrugs in chemotherapy. We carried out a characterization of the cytidine deaminase 79A>C and 208G>A Single Nucleotide Polymorphisms, in order to highlight their functional role and provide data that could help fine-tune the chemotherapic use of cytosine nucleosides in patients carrying the above mentioned SNPs. The 79A>C SNP results in a K27Q change in a protein region not involved in the catalytic event. The 208G>A SNP produces an alanine to threonine substitution (A70T) within the conserved catalytic domain. Q27 variant is endowed with a greater catalytic efficiency toward the natural substrates and the antileukemic agent cytarabine (Ara-C), when compared to K27 variant. Molecular modeling, protein stability experiments and site-directed mutagenesis suggest that K27 variant may have an increased stability with respect to Q27 due to an ionic interaction between a lysine residue at position 27 and a glutamate residue at position 24. The T70 variant has a lower catalytic efficiency toward the analyzed substrates when compared to the A70 variant, suggesting that patients carrying the 208G>A SNP may have a greater exposure to cytosine based pro drugs, with possible toxicity consequences.

Delineation of the molecular mechanisms of nucleoside recognition by cytidine deaminase through virtual screening.

Cytidine deaminase (EC 3.5.4.5, CDA), an enzyme of the pyrimidine salvage pathways, is responsible for the degradation and inactivation of several cytidine‐based antitumor drugs such as cytarabine, gemcitabine, decitabine, and azacytidine. Thus, CDA inhibitors are highly sought after as compounds to be co‐administered with said drugs to improve their effectiveness. Alternatively, the design of antitumor drugs not susceptible to the action of CDA is also regarded as an attractive solution. Herein we describe a virtual screen for CDA ligands based on chemical similarity and molecular docking. The campaign led to the identification of three novel inhibitors and one novel substrate, with a 19 % hit rate, and allowed a significant extension of the structure–activity relationships, also in light of the compounds that resulted inactive. The most active compound identified through the screen is the inhibitor pseudoisocytidine, which has the potential to serve as a lead for highly stable compounds. The study also delineated the detrimental effect of 5‐aza and 6‐aza substitutions, the incompatibility of the presence of an amino group at the 3′‐position, as well as the presence of very strict steric requirements around the 2′‐arabino position and, even more, the N4‐position. Importantly, these features can be exploited for the design of novel anti‐neoplastic agents resistant to the action of CDA.

Role of tyrosine 33 residue for the stabilization of the tetrameric structure of human cytidine deaminase

In the present work the effect of a mutation on tyrosine 33 residue (Y33G) of human cytidine deaminase (CDA) was investigated with regard to protein solubility and specific activity. Osmolytes and CDA ligands were used to increase the yield and the specific activity of the protein. The mutant enzyme was purified and subjected to a kinetic characterization and to stability studies. These investigations reinforced the hypothesis that in human CDA the side chain of Y33 is involved in intersubunit interactions with four glutamate residues (E108) forming a double latch that connects each of the two pairs of monomers of the tetrameric CDA.

Population variability in CD38 activity: Correlation with age and significant effect of TNF-α -308G>A and CD38 184C>G SNPs

CD38 (EC 3.2.2.6, NAD(+)-glycohydrolase) is a multifunctional enzyme catalyzing the synthesis and hydrolysis of cyclic ADP-ribose from NAD(+) to ADP-ribose. The loss of CD38 function is associated with impaired immune responses, metabolic disturbances, and behavioral modifications. Notably, it has been linked to HIV infection, leukemias, myelomas, solid tumors, Type II Diabetes mellitus, bone metabolism, as well as Autism Spectrum Disorder. Taking into account the crucial role played by CD38 in many diseases and in clinical practice, here we assessed the distribution of CD38 NADase activity in a healthy population (104 sex-matched unrelated individuals, 12-98 years) and determined its main predictors among genetic and physiological factors (age and sex). The mean value of CD38 NADase activity was 0.051±0.023 mU/mg (0.010-0.099 mU/mg), following a normal distribution in the study population (Kolmogorov-Smirnov test P=0.200). The TNF-α -308G>A (rs1800629) resulted the main predictor (β=0.364, P=0.00008), followed by Age (β=0.280, P=0.002) and the CD38 184C>G (rs6449182) (β=0.193, P=0.033). Our study contributes to understanding CD38 enzyme physiological functions, by reporting, for the first time, its activity distribution in healthy individuals and demonstrating a significant positive correlation with age. Moreover, the possible use of TNF-α -308G>A (rs1800629) and the CD38 184C>G (rs6449182) SNPs as predictive genetic markers of CD38 activity, clearly point toward possible pharmacogenomic applications and to a more refined use of CD38 in clinical settings.

Enzymology of Pyrimidine Metabolism and Neurodegeneration

It is well known that disorders of pyrimidine pathways may lead to neurological, hematological, immunological diseases, renal impairments, and association with malignancies. Nucleotide homeostasis depends on the three stages of pyrimidine metabolism: de novo synthesis, catabolism and recycling of these metabolites. Cytidine and uridine, in addition to be used as substrates for pyrimidine nucleotide salvaging, also act as the precursors of cytidine triphosphate used in the biosynthetic pathway of both brains phosphatidylcholine and phosphatidylethanolamine via the Kennedy cycle. The synthesis in the brain of phosphatidylcholine and other membrane phosphatides can utilize, in addition to glucose, three compounds present in the blood stream: choline, uridine, and a polyunsaturated fatty acids like docosahexaenoic acid. Some authors, using rat models, found that oral administration of two phospholipid precursors such as uridine and omega-3 fatty acids, along with choline from the diet, can increase the amount of synaptic membrane generated by surviving striatal neurons in rats with induced Parkinsons disease. Other authors found that in hypertensive rat fed with uridine and choline, cognitive deficit resulted improved. Uridine has also been recently considered as a neuroactive molecule, because of its involvement in important neurological functions by improving memory, sleep disorders, anti-epileptic effects, as well as neuronal plasticity. Cytidine and uridine are uptaken by the brain via specific receptors and successively salvaged to the corresponding nucleotides. The present review is devoted to the enzymology of pyrimidine pathways whose importance has attracted the attention of several researchers investigating on the mechanisms underlying the physiopathology of brain.

Site Directed Mutagenesis as a Tool to Understand the Catalytic Mechanism of Human Cytidine Deaminase

Cytidine deaminase (CDA), is one of the enzymes involved in the pyrimidine salvage pathways, which catalyzes the formation of uridine and deoxyuridine by the hydrolytic deamination of cytidine and deoxycytidine, respectively. Human CDA is a tetrameric enzyme of identical 15 kDa subunits, each containing an essential zinc atom in the active site. The substrate binds to each active site independently and the cooperativity between subunits has not been reported. CDA is able to recognize as substrates some antitumor and antiviral cytidine analogs rendering them pharmacologically inactive. In light of the role played by this enzyme, a deep knowledge of CDA active site and mechanism of catalysis is required. Site-directed mutagenesis, associated with molecular modeling studies, may be an important tool to discover the active site structure of an enzyme and consequently its mechanism of action. In this review are summarized the site-directed mutagenesis experiments performed on human CDA: through these studies it was possible to understand the role exerted by specific amino acid residues in CDA active site and in the contacts between subunits. The obtained results may open a way for designing new cytidine based drugs or more potent CDA inhibitors.