Anna Maria Caroli

Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors

Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of β-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor.

Direct effects of casein phosphopeptides on growth and differentiation of in vitro cultured osteoblastic cells (MC3T3-E1).

Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation. The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function. The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE- and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs.

Repeatability of milk clotting aptitude evaluated by lactodynamographic analysis

Lactodynamographic analysis is commonly employed to evaluate the clotting aptitude of milk (Frentz, 1965; Tarodo de la Fuente et al. 1969). The individual lactodynamographic response can therefore be used in selection as a quantitative trait. The aim of this work was to estimate either the biological repeatability of the lactodynamographic response or the repeatability of the analytical method itself.

The hairless (hr) gene is involved in the congenital hypotrichosis of Valle del Belice sheep

Congenital hypotrichosis in mammalian species consists of partial or complete absence of hair at birth. The hairless gene is often responsible for this disorder in men, mice and rats. Recent experimental data on Valle del Belice sheep reared in Sicily for milk production, support the genetic control of the ovine hypotrichosis as a Mendelian recessive trait. The ovine hairless gene was chosen as the candidate gene involved in this disorder. Blood samples were collected from Valle del Belice sheep with the normal and hypotrichotic phenotypes. Almost the entire hairless gene was successfully amplified using the long PCR technique. Unrelated sheep with differing phenotypes were randomly chosen for sequencing the amplified products. Different mutations related to the hypotrichotic phenotype were found in exon 3. In fact, sequencing revealed an A/T transversion at position 739, a G/A transition at position 823, and a C/T transition at position 1312. From these nucleotide exchanges, three substitutions of the processed mature protein were deduced at the amino acid positions 247 (Thr/Ser), 275 (Ala/Thr), and 438 (Gln/Stop). A PCR-SSCP based test was developed in order to detect the last mutation, which is responsible for the hypotrichotic phenotype.

Short communication: Monitoring nutritional quality of Amiata donkey milk: Effects of lactation and productive season

Milk nutritional characteristics are especially interesting when donkey milk is aimed at consumption by children and the elderly. The aim of this study was to monitor the nutritional quality of Amiata donkey milk during lactation and productive season to provide information on the milk characteristics and to study action plans to improve milk yield and quality. Thirty-one pluriparous jennies belonging to the same farm were selected. Individual samples of milk from the morning milking were taken once per month starting from the d 30 of lactation until d 300. Milk yield and dry matter, fat, and ash content were constant throughout the experimental period. Milk total protein content showed a progressive decrease during the first 6 mo of lactation; after this period, the protein percentages remained constant (1.50%). Caseins and lactose were lower until d 60 of lactation and remained constant thereafter. During summer and autumn, milk yield and casein and lactose contents were higher, whereas during the spring season, higher protein and ash contents were found. The percentages of fat and dry matter were stable as were most of the minerals in the milk, except for calcium, which was higher in the spring. In conclusion, Amiata donkey milk was found to be relatively stable during lactation. This is an advantage in terms of the production and trade of a food product with consistent characteristics. The different milk yield and quality during the productive seasons were probably related to better adaptability of the animals to warm and temperate periods.

Characterization and genetic analysis of bovine αs1-casein I variant

The aim of this study was to identify the molecular genetic origin underlying the I variant of alpha(s1)-casein and to develop a DNA-based test for this polymorphism as a tool for genetic analyses independent of milk sample testing. All coding exons and flanking regions of the alpha(s1)-casein gene were sequenced in DNA samples from cattle of known alpha(s1)-casein genotypes (BI, CI, II, CC), determined by isoelectric focusing of milk samples. A nucleotide substitution (A>T) in exon 11 (g.19836A>T) leads to the exchange of Glu with Asp at amino acid position 84 of the mature protein (p.Glu84Asp) and perfectly co-segregated with the presence of the alpha(s1)-casein I variant in the milk of the analysed animals. Genotyping of a total of 680 DNA samples from 31 Bos taurus and Bos indicus cattle breeds and from Bos grunniens, Bison bison and Bison bonasus by restriction fragment length polymorphism analysis revealed the occurrence of Asp at position 84 at low frequencies in Bos taurus and Bos indicus breeds and established its origin from the alpha(s1)-casein C variant (p.Glu192Gly). Ten different intragenic haplotypes in the gene region from intron 8 to intron 12 were observed by sequencing, of which two occurred in Bison bison and one in Bison bonasus only. Using available casein gene complex information, an association of Asp at position 84 to beta-casein A(2) and kappa-casein B was shown in the Bos indicus breed Banyo Gudali. Taken together, we can postulate that the alpha(s1)-casein variant I is caused by a non-synonymous nucleotide substitution in exon 11 of the gene and that it originated within Bos indicus and spread to Bos taurus subsequently.

Variability of reduced glutathione levels in Massese ewes and its effect on daily milk production.

The variability of the erythrocyte concentration of the tripeptide glutathione was studied in four flocks of Massese sheep. A relationship was found between glutathione level and stage of lactation in which the second month showed the lowest content. Genotype at transferrin and haemoglobin loci was associated with glutathione levels. Homozygotes at the transferrin locus yielded higher levels, while the D allele at the same locus was associated with lower concentrations. Animals with haemoglobin AA showed higher contents of glutathione. Also, a negative correlation was found between daily milk production and glutathione level.

Detecting β-Casein Variation in Bovine Milk

In bovine species, β-casein (β-CN) is characterized by genetic polymorphism. The two most common protein variants are β-CN A2 (the original one) and A1, differing from A2 for one amino acid substitution (Pro67 to His67). Several bioactive peptides affecting milk nutritional properties can originate from β-CN. Among them, β-casomorphin-7 (BCM7) ranging from amino acid 60 to 66 can be released more easily from β-CN variants carrying His67 (A1 type) instead of Pro67 (A2 type). Nowadays, “A2 milk” is produced in different countries claiming its potential benefits in human health. The aim of this study was to further develop and apply an isoelectric focusing electrophoresis (IEF) method to bulk and individual milk samples in order to improve its use for β-CN studies. We succeeded in identifying A2 milk samples correctly and quantifying the percentage of A2, A1, and B variants in bulk samples not derived from A2 milk as well as in individual milk samples. The method allows us to quantify the relative proportion of β-CN variants in whole milk without eliminating whey protein by acid or enzymatic precipitation of caseins. The aim of this study was also to study the different behavior of β-CN and β-lactoglobulin (β-LG) in the presence of trichloroacetic acid (TCA). The higher sensitivity of β-CN to TCA allows quantifying β-CN variants after TCA fixation because β-LG is not visible. Monitoring β-CN variation in cattle breeds is important in order to maintain a certain balance between Pro67 and His67 in dairy products. Overall, the debate between A1 and A2 milk needs further investigation.

The casein genes in goat breeds from different Continents: analysis by Polymerase Chain Reaction - Single Strand Conformation Polymorphism (PCR-SSCP)

Abstract A screening of casein gene variability was carried out by Polymerase Chain Reaction – Single Strand Conformation Polymorphism in 8 goat breeds from Sudan (Nubian goat), Turkey (Angora Goat Lalahan Tiftic, Angora Goat Yerkoy, Hair goat) and India (Jammu, Maharashtra, Rajasthan, South Goat). A total of 16 different alleles or groups of alleles were found, showing conspicuous differences among breeds. The allele frequencies were submitted to cluster analysis in order to highlight differences between breeds, also including data from Red Sokoto, West African Dwarf Nigeria, West African Dwarf Cameroon, and Borno Goat. The tree obtained from the cluster analysis showed two main lineages. The West African goat clustered together, the Indian and Turkish breeds were in the other group. Nubian goat was found in an intermediate position.

Assessment of 29 candidate genes for milk traits in Italian dairy cattle

The Italian Bracco is one of the oldest pointing dog breed, used for hunting ever since the Renaissance time; paintings of the 14th century show hunting sceneries with dogs similar to the present day Bracco. The breed has been officially registered by ENCI (the Italian cynological club) in 1949, when the definitive standard was established. In this work, we report the first results of a study aimed at measuring morphological traits in this breed, as a part of a more comprehensive study whose objective is to identify the characters that have the potential of being genetically improved. R. Ciampolini1, F. Cecchi1, A. Bramante1, F. Casetti2, S. Presciuttini1