L. Ramunno

Molecular characterization of the goat CSN1S1 01 allele

Caseins (α s1 , β, α s2 , e κ) represent about 80% of the whole protein content of ruminant milk. Each of these proteins is encoded by single copy genes ( CSN1S1 , CSN2 , CSN1S2 and CSN3 , respectively) clustered on a ∼200-kb segment of chromosome 6 (Ferretti et al. 1990; Gallagher et al. 1994) in the order: CSN1S1 , CSN2 , CSN1S2 and CSN3 (Mercier & Vilotte, 1993). Furthermore, in cattle and goat CSN1S1 and CSN2 are convergently transcribed (Leroux & Martin, 1996; Rijnkles et al. 1997) and are only 20 and 12 kb apart, respectively.

Endogenous Tissue Factor Pathway Inhibitor Modulates Thrombus Formation in an In Vivo Model of Rabbit Carotid Artery Stenosis and Endothelial Injury

BACKGROUND Tissue factor pathway inhibitor (TFPI) is the sole known inhibitor of the extrinsic coagulation pathway of physiological importance; however, its role in modulating thrombosis in vivo is still unclear. METHODS AND RESULTS Intravascular thrombosis was initiated by placing an external constrictor around endothelially injured rabbit carotid arteries (n=10). Carotid blood flow velocity was measured by a Doppler flow probe. After placement of the constrictor, cyclic flow reductions (CFRs), due to recurrent thrombosis, developed at the site of stenosis. Transstenotic TFPI plasma activity was measured in blood samples before induction of CFRs and after 30, 60, and 180 minutes of CFRs. TFPI plasma activity distal to the site of thrombosis was significantly lower than the corresponding proximal values at 30, 60, and 180 minutes of CFRs. In addition, a progressive decrease in TFPI plasma activity was observed in both the proximal and the distal samples, indicating consumption of TFPI during thrombus formation. In 10 additional rabbits, CFRs were abolished by administration of aspirin (10 mg/kg). In the animals in which aspirin abolished CFRs, endogenous TFPI was depleted by a bolus of a polyclonal antibody against rabbit TFPI, and the effects on restoration of CFRs were monitored. In 5 of 6 animals in which aspirin abolished CFRs, depletion of endogenous TFPI activity caused full restoration of CFRs. CONCLUSIONS The data of the present study support the involvement of endogenous TFPI in the process of thrombus formation in vivo and its active role in modulating arterial thrombosis.

Proteomic characterization of donkey milk “caseome”

At present, compared with bovine milk, the characterization of donkey milk caseins is at a relatively early stage progress, and only limited data are related to its genetic polymorphism. In this work, the heterogeneity of donkey caseome was investigated using a proteomic approach, based on one- (PAGE, UTLIEF) and two-dimensional (PAGE-->UTLIEF) electrophoresis, stained with either Coomassie Brilliant Blue or specific polyclonal antibodies, and structural MS analysis. These combined methodologies allowed the contemporary identification of donkey alpha(s1), alpha(s2), beta and kappa-CN with their related heterogeneity due to phosphorylation (alpha(s1), alpha(s2) and beta-CN), glycosylation (kappa-CN) and incorrect splicing of RNA in mRNA (deleted forms of alpha(s1)-CN and beta-CN). The results achieved showed 11 components for kappa-CN, six phosphorylated components for beta and alpha(s1)-CN and three main phosphorylated components for alpha(s2)-CN, each accounting for 10, 11 and 12 P/mole. At this regard, for the first time, the primary structure of the expressed protein corresponding to the only available donkey alpha(s2)-CN cDNA sequence was determined. Furthermore beta-CN was found in homozygous and heterozygous state for the occurrence of a genetic beta-CN variant having a MW value 28 mass units higher than the common beta-CN phenotype.

Bacteriophage-Resistant Staphylococcus aureus Mutant Confers Broad Immunity against Staphylococcal Infection in Mice

In the presence of a bacteriophage (a bacteria-attacking virus) resistance is clearly beneficial to the bacteria. As expected in such conditions, resistant bacteria emerge rapidly. However, in the absence of the phage, resistant bacteria often display reduced fitness, compared to their sensitive counterparts. The present study explored the fitness cost associated with phage-resistance as an opportunity to isolate an attenuated strain of S. aureus. The phage-resistant strain A172 was isolated from the phage-sensitive strain A170 in the presence of the MSa phage. Acquisition of phage-resistance altered several properties of A172, causing reduced growth rate, under-expression of numerous genes and production of capsular polysaccharide. In vivo, A172 modulated the transcription of the TNF-α, IFN-γ and Il-1β genes and, given intramuscularly, protected mice from a lethal dose of A170 (18/20). The heat-killed vaccine also afforded protection from heterologous methicillin-resistant S. aureus (MRSA) (8/10 mice) or vancomycin-intermediate S. aureus (VISA) (9/10 mice). The same vaccine was also effective when administered as an aerosol. Anti-A172 mouse antibodies, in the dose of 10 µl/mouse, protected the animals (10/10, in two independent experiments) from a lethal dose of A170. Consisting predominantly of the sugars glucose and galactose, the capsular polysaccharide of A172, given in the dose of 25 µg/mouse, also protected the mice (20/20) from a lethal dose of A170. The above results demonstrate that selection for phage-resistance can facilitate bacterial vaccine preparation.

Identification and Quantification of αS1, αS2, β, and κ-Caseins in Water Buffalo Milk by Reverse Phase-High Performance Liquid Chromatography and Mass Spectrometry

A method for the simultaneous quantitation of alpha(S1), alpha(S2), beta, and kappa-caseins in water buffalo (Bubalus bubalis) milk using reverse phase high-performance liquid chromatography was developed. The molecular masses of the peaks separated by the described chromatographic protocol were determined by ESI-MS. alpha(S1)- and kappa-caseins were found to be heteromorphic in several individual milk samples. In particular, alpha(S1)-casein showed two peaks with a molecular mass of 23,490 Da and 23,516 Da, and kappa-casein showed three peaks with molecular masses of 19,165 Da, 19,177 Da, and 19,247 Da. Only one form for beta-casein (24,033 Da) and alpha(S2)-casein (22,741 Da) were detected. The mean values of casein fraction concentration observed throughout the individual samples were 8.89 gL(-1) with a relative standard deviation (RSD) of 20% for alpha(S1)-casein, 5.08 gL(-1) with a RSD of 25% for alpha(S2)-casein, 20.91 gL(-1) with a RSD of 16% for beta-casein, and 4.13 gL(-1) with a RSD of 24% for kappa-casein. Linear and second-order polynomial correlations with total nitrogen were calculated for all casein fractions.

A Ssp I PCR-RFLP detecting a silent allele at the goat CSN2 locus.

The goat calcium-sensitive caseins (alphas1, beta and alphas2) represent, over many years, an excellent model for demonstrating that the major part of the variability observed in the content of these proteins in goat milk is mostly due to the presence of autosomal alleles at single structural loci (CSN1S1, CSN2 and CSN1S2 respectively) clustered on a 200 kb segment of chromosome 6; furthermore, CSN1S1 and CSN2 are convergently transcribed and are only 12 kb apart (Rijnkels, 2002).

An association analysis between OXT genotype and milk yield and flow in Italian Mediterranean river buffalo

The aim of this study was to evaluate possible associations between three SNPs at the oxytocin locus (AM234538: g.28C>T; g.204A>G and g.1627G>T) and two productive traits, milk yield and milkability, in Italian Mediterranean river buffaloes. Effects of parity, calving season and month of production were also evaluated. A total of 41 980 test-day records belonging to 219 lactations of 163 buffalo cows were investigated. The allele call rate was 98·8% and the major allele frequency for all the investigated loci was 0·76. The OXT genotype was significantly associated with milk yield (P=0·029). The TT genotype showed an average daily milk yield approximately 1·7 kg higher than GT buffaloes. Such a difference represents about 23% more milk/d. A large dominance effect (-1·17±0·43 kg) was estimated, whereas the contribution of OXT genotype (r(2)(OXT)) to the total phenotypic variance in milk yield was equal to 0·06. The TT genotype showed higher values also for the milk flow, even though the estimated difference did not reach a level of statistical significance (P=0·07). Such an association, among the first reported for the oxytocin locus in ruminants, should be tested on a population scale and possible effects on milk composition traits should be evaluated in order to supply useful indications for the application of marker-assisted selection programmes in river buffaloes.

A single nucleotide polymorphism in the promoter region of river buffalo stearoyl CoA desaturase gene (SCD) is associated with milk yield.

An association study between the milk yield trait and the stearoyl-CoA desaturase (SCD) polymorphism (g.133A > C) in Italian Mediterranean river buffalo was carried out. A full characterization of the river buffalo SCD promoter region was presented. Genotyping information was provided and a quick method for allelic discrimination was developed. The frequency of the C allele was 0·16. Test-day (TD) records (43 510) of milk production belonging to 226 lactations of 169 buffalo cows were analysed with a mixed linear model in order to estimate the effect of g.133A > C genotype, as well as the effect of parity and calving season. The SCD genotype was significantly associated with milk yield (P = 0·02). The genotype AC showed an over-dominance effect with an average daily milk yield approximately 2 kg/d higher than CC buffaloes. Such a difference represents about 28% more milk/d. The effect of the genotype was constant across lactation stages. The contribution of SCD genotype (r(2)SCD) to the total phenotypic variance in milk yield was equal to 0·12. This report is among the first indications of genetic association between a trait of economic importance in river buffalo. Although such results need to be confirmed with large-scale studies in the same and other buffalo populations, they might offer useful indications for the application of MAS programmes in river buffalo and in the future they might be of great economic interest for the river buffalo dairy industry.

Cytogenetic characterization of alpaca (Lama pacos, fam. Camelidae) prometaphase chromosomes

The present study provides specific cytogenetic information on prometaphase chromosomes of the alpaca (Lama pacos, fam. Camelidae, 2n = 74) that forms a basis for future work on karyotype standardization and gene mapping of the species, as well as for comparative studies and future genetic improvement programs within the family Camelidae. Based on the centromeric index (CI) measurements, alpaca chromosomes have been classified into four groups: group A, subtelocentrics, from pair 1 to 10; group B, telocentrics, from pair 11 to 20; group C, submetacentrics, from pair 21 to 29; group D, metacentrics, from pair 30 to 36 plus sex chromosomes. For each chromosome pair, the following data are provided: relative chromosome length, centromeric index, conventional Giemsa staining, sequential QFQ/C-banding, GTG- and RBG-banding patterns with corresponding ideograms, RBA-banding and sequential RBA/silver staining for NOR localization. The overall number of RBG-bands revealed was 391. Nucleolus organizer-bearing chromosomes were identified as pairs 6, 28, 31, 32, 33 and 34. Comparative ZOO-FISH analysis with camel (Camelus dromedarius) X and Y painting probes was also carried out to validate X-Y chromosome identification of alpaca and to confirm close homologies between the sex chromosomes of these two species.

Short communication: Translational efficiency of casein transcripts in Mediterranean river buffalo

Buffalo milk is characterized by the presence of all 4 casein fractions (α(S1), β, α(S2), and κ) encoded by the 4 tightly linked autosomal genes (CSN1S1, CSN2, CSN1S2, and CSN3, respectively). In the present paper, we report for the first time a quantitative characterization of buffalo casein transcripts and show that the 4 genes are not transcribed and translated with the same efficiency. In particular, the analysis of individual milk samples obtained from 9 Mediterranean river buffaloes showed that the most abundant casein fractions were β (53.45%) and α(S1) (20.61%), followed by α(S2) and κ, at 14.28 and 11.66%, respectively. Quantification of the corresponding mRNA showed that the percentage of transcripts of the 4 caseins was 16.48, 23.18, 55.87, and 4.47% for α(S1), β, α(S2), and κ, respectively. Translation efficiency was 0.25 for CSN1S2, 1.31 for CSN1S1, 2.39 for CSN2, and 2.69 for the CSN3 transcripts, respectively. A comparison of nucleotide sequences with the Kozak consensus sequence was also carried out to investigate if the mRNA sequences might be responsible for the observed differences.