Hot topic: Gene duplication at the α-lactalbumin locus: finding the evidence in water buffalo (Bubalus bubalus L.).

Studies on milk proteins revealed that a qualitative and quantitative polymorphism may often be found regarding alpha-lactalbumin (alpha-LA). In mammals, a similar phenomenon was widely documented in the alpha-globin system as the result of a gene duplication. The presence of several differently expressed alpha-lactalbumin gene (LALBA) products suggests that the mechanism underlying this phenomenon may involve nonallelic genes. To check this hypothesis, an experiment was set up to investigate the LALBA gene arrangement of a water buffalo exhibiting an alpha-LA phenotype characterized by a double-band pattern on PAGE isoelectric, focusing analysis of milk protein. In particular, the relative amount of protein inferred from the different intensity of the bands was consistent with a gene duplication. Thus, leukocyte DNA was extracted from a blood sample of the buffalo and amplified with 4 primers (2 RV-IVFW for PCR and 4 FW-IRV for nested PCR). The intergenic segments of the assumed duplicated gene were then amplified with 2 different PCR protocols. First, the segment limited by the third exon in the upstream gene and the second exon in the downstream gene was amplified by simple PCR, which gave aspecific results. Second, this PCR product was subjected to nested PCR, amplifying the segment limited by the fourth exon in the upstream gene and the first exon in the downstream gene, yielding an amplified nucleotide fragment of about 6,200 bp. Blood samples from an additional 15 buffalos were then analyzed in the same manner. The results obtained from the new samples confirmed the presence of an amplified nucleotide fragment of about 6,200 bp in most of them, though they all were characterized by an alpha-LA monomorphic phenotype. A couple of 6,200-bp fragments obtained were purified, cloned in pGEM-T easy vector system (Promega, Madison, WI) and sequenced. The sequence of the large DNA segments, containing the intergenic portion, was aligned with the LALBA gene (accession number AF194373; http://www.ncbi.nlm.nih.gov/Database/index.html). They both were found to coincide with the portion containing exon 4 and the untranslated region at the 3' end of the upstream gene and with the portion containing exon 1 and the untranslated region at the 5' end of the downstream gene. These results confirm the hypothesis that a tandemly repeated copy of the LALBA gene is present in water buffalo.

Short communication: molecular genetic characterization of ovine alpha(S1)-casein allele H caused by alternative splicing.

Sequencing of ovine CSN1S1*H cDNA showed an absence of exon 8 in comparison with GenBank sequences; the absence was confirmed by protein sequencing. We demonstrated that this allelic aberration is the result of a deletion of 4 nucleotides, the last 3 of exon 8 and the first 1 of intron 8, which are replaced by an insertion of 13 nucleotides in the DNA sequence. The insertion is a precise duplication of a part of the adjacent intronic sequence of CSN1S1*C''. These sequence differences result in an inactivation of the splice donor sequence distal to exon 8, leading to upstream exon skipping during the serial splice reactions of the ovine CSN1S1*H pre-mRNA, and may affect the specific casein expression as well as protein characteristics.

Protective effect of yogurt extract on dental enamel demineralization in vitro.

BACKGROUND: Casein phosphopeptides (CPPs) are phosphorylated casein-derived peptides produced synthetically by proteolytic digestion of alpha(s1)-, alpha(s2)- and beta-casein. The anticariogenic activity of CPPs is due to their ability to stabilize high levels of amorphous calcium phosphate (ACP) on tooth surface, preventing demineralization and enhancing remineralization of enamel caries. The aim of this study was to test the in vitro ability of natural CPPs (contained in yogurt) to prevent demineralization and promote remineralization of dental enamel. METHODS: Eighty human molars were used. After standardizing an in vitro demineralization procedure for producing artificial caries (Group 1: pH 4.8; Group 2: pH 3.97), this procedure was used on teeth, but with the addition of natural CPPs (Group 3: pH 4.8; Group 4: pH 3.97). The effects of these procedures were evaluated by quantitative analysis (change in weight and calcium titration) and qualitative analysis (SEM). Statistical analysis of the results was performed using ANOVA. RESULTS: Statistical analysis showed significant differences in weight changes between the groups with and without natural CPPs. The SEM observation showed the protective effects of natural CPPs. CONCLUSIONS: The results demonstrated that CPPs contained in yogurt have an inhibitory effect on demineralization and promote the remineralization of dental enamel.

New strategies in dental caries prevention: experimental study on casein phosphopeptides.

AIM: Casein phosphopeptides (CPPs) are phosphorylated casein-derived peptides produced by proteolytic digestion of alphas 1-, betas 2- and beta-casein in vitro or in the digestive tract. CPPs exhibit anti-caries activity relates to their capability to localise high levels of amorphous Ca2+ phosphate on tooth surface. Aim of this study is in vitro testing of the capability of CPPs to prevent demineralisation and promote remineralization of early enamel lesions. MATERIALS AND METHODS: 159 samples of dental enamel were divided into 3 groups, which subsequently underwent 3 different chemical treatments: the samples from group I (control group) were preserved in distilled water; the samples from group II were treated with a demineralizing solution for producing artificial caries; the samples from group III underwent the same treatment as group II, but with the addition of CPPs. The effects of these procedures were evaluated by quantitative analysis (change in weight and calcium titration) and qualitative analysis (SEM). STATISTICS: Statistical analysis of the results was performed using ANOVA. RESULTS In presence of CPPs, acid dissolution of human enamel is reduced by over 50% in vitro. CONCLUSION Our results demonstrate that CPPs could be a valid preventive system against demineralisation of early enamel lesions.

Mass spectrometry-based procedure for the identification of ovine casein heterogeneity.

The efficiency of reversed-phase HPLC, capillary electrophoresis (CE), PAGE and isoelectric focusing with immunoblotting in separating ovine caseins has been evaluated. The assessment was carried out by employing electrospray ionization-mass spectrometry (ESI-MS) and matrix-assisted laser desorption ionization-time of flight as reference tools for identifying protein components. Ovine casein was fractionated by HTPC into four major peaks. With ESI-MS, each peak contained components belonging to only one of the four casein families. On-line liquid chromatography-ESI-MS allowed us to determine each fraction's composition by detecting thirteen alphas1-, eleven alphas2-, seven beta-, and three kappa-casein (CN) components. The alphas1-CN and alphas2-CN consisted of eight and two protein chains respectively of lengths differing through the deletion of one or more peptide sequences; they were also discretely phosphorylated as kappa-CN and beta-CN. By CE at pH 2.5, each casein fraction was as heterogeneous as that resulting from ESI-MS for the single HPLC-derived fractions. The separation of alphas1-CN and alphas2-CN proved to be excellent, with the exception of a co-migration of kappa0-CN with a minor alphas1-CN component and of a glycosylated kappa-CN for with low-phosphorylated = alphas1-CN and beta-CN components. Dephosphorylation of whole casein was used to reduce the heterogeneity of the native fractions and by applying currently used analytical techniques it was possible to visualize the protein moiety difference along the CE profile. CE, HPLC, and immunoblotting were all equally capable of effecting an accurate separation of the four dephosphorylated casein families. The spectra obtained by ESI-MS directly on dephosphorylated whole ovine casein samples contained the signals of the four casein families and the relative alphas1-CN variants, the non-allelic alphas1-CN and alphas2-CN forms, dimeric kappa-CN and other newly formed peptides. We suggest using this procedure for rapid characterization of whole casein.

Qualitative and quantitative analysis of wheat gluten proteins by liquid chromatography and electrospray mass spectrometry.

Based on analysis by liquid chromatography/electrospray ionisation mass spectrometry, we have developed a new method for fast and sensitive fingerprinting of gliadins and glutenins in wheat flour. Using this procedure the two protein fractions from seven durum wheat varieties have been analysed by high resolution high performance liquid chromatographic separation coupled to accurate determination of molecular mass. In this way, the molecular mass of the single components from both gliadin and glutenin fractions were measured and more than forty components were detected for each fraction indicating a high heterogeneity. Although the chromatographic profiles were similar, the molecular masses of protein components with similar retention times among the varieties were often different. The difference ranged from a few mass units corresponding to single amino acid substitution(s) up to thousands implying peptide deletion or insertion along the protein chain. Two components representing about a half of the gliadin fraction, e.g. gamma(2)- and gamma(3)-gliadin, were identified through the N-terminal sequence and molecular mass determination. We suggest the use of the high level and the molecular mass of these gliadin components as markers to detect traces of wheat in gluten-free food preparations for celiac patients.

Alternative nonallelic deletion is constitutive of ruminant alpha(s1)-casein.

Multiple forms of alpha(s1)-casein were identified in the four major ruminant species by structural characterization of the protein fraction. While alpha(s1)-casein phenotypes were constituted by a mixture of at least seven molecular forms in ovine and caprine species, there were only two forms in bovine and water buffalo species. In ovine and caprine forms the main component corresponded to the 199-residue-long form, and the deleted proteins differed from the complete one by the absence of peptides 141-148, 110-117, or Gln78, or a combination of such deletions. The deleted segments corresponded to the sequence regions encoded by exons 13 and 16, and by the first triplet of exon 11 (CAG), suggesting that the occurrence of the short protein forms is due to alternative skipping, as previously demonstrated for some caprine and ovine phenotypes. The alternative deletion of Gln78 in alpha(s1)-casein, the only form common to the milk of all the species examined and located in a sequence region joining the polar phosphorylation cluster and the hydrophobic C-terminal domain of the protein, may play a functional role in the stabilization of the milk micelle structure.

Effects of sheep alpha s1-casein CC, CD and DD genotypes on milk composition and cheesemaking properties.

The effects of sheep alpha s1-casein CC, CD and DD genotypes on milk composition and cheese yield were studied. Processed bulk milk was collected from three groups of 15 ewes, carrying alpha s1-casein CC, CD and DD genotypes. CC milk was higher in casein content than CD or DD milk (+3.5 and +8.6% respectively), and had a higher protein: fat ratio and a smaller casein micelle diameter. In addition, DD milk had a significantly lower alpha s1-casein content. The main differences were in curd formation: CC milk had better renneting properties. Cheesemaking trials, carried out in a pilot plant, showed that CC milk had better cheesemaking characteristics than DD milk, while CD milk was intermediate. Both 1 d old and fully ripened cheeses had different fat: dry matter ratios and alpha s1-I-casein electrophoretic mobilities: these were lower for DD cheese. As a consequence, these genotypes could be considered as markers of milk and/or cheese quality.

The primary structure of water buffalo alpha(s1)- and beta-casein identification of phosphorylation sites and characterization of a novel beta-casein variant.

The primary structure of water buffalo alpha(s1)-casein and of beta-casein A and B variants has been determined using a combination of mass spectrometry and Edman degradation procedures. The phosphorylated residues were localized on the tryptic phosphopeptides after performing a beta-elimination/thiol derivatization. Water buffalo alpha(s1)-casein, resolved in three discrete bands by isoelectric focusing, was found to consist of a single protein containing eight, seven, or six phosphate groups. Compared to bovine alpha(s1)-casein C variant, the water buffalo alpha(s1)-casein presented ten amino acid substitutions, seven of which involved charged amino acid residues. With respect to bovine betaA2-casein variant, the two water buffalo beta-casein variants A and B presented four and five amino acid substitutions, respectively. In addition to the phosphoserines, a phosphothreonine residue was identified in variant A. From the phylogenetic point of view, both water buffalo beta-casein variants seem to be homologous to bovine betaA2-casein.

Copresence of Deleted Protein Species Generates Structural Heterogeneity of Ovine alpha(s1)-Casein.

Multiple forms of mature alpha(s1)-casein have been characterized in ovine variants A and D using a combination of mass spectrometry and automated Edman degradation. Mature ovine alpha(s1)-casein was found to be a heterogeneous mixture of at least seven molecular species. The main component, representing about 50% total alpha(s1)-casein, corresponded to the full-length (199 residues long) protein. The other components were alpha(s1)-casein of different lengths: 198 (less Gln78), 191 (less peptide 110-117), 191 residues (less peptide 140-148), 190 (less peptide 110-117 and Gln78), 190 (less peptide 140-148 and Gln78), and 183 (less peptides 110-117 and 140-148) residues long alpha(s1)-casein. Each of the alpha(s1)-casein multiple forms occurred at three different phosphorylation levels, due to the partial phosphorylation of both Ser115 (at about 50%) and Ser41 (at about 20%). In the case of deleted peptide 110-117, the protein heterogeneity linked to the partially phosphorylated Ser115 was abolished, and only two levels of phosphorylation were observed. These multiple forms differing in molecular weight and degree of phosphorylation may have been developed from an exon skipping during mRNA splicing in ovine alpha(s1)-casein, similar to that recently described in the case of its caprine counterpart.

Phosphopeptides from Grana Padano cheese: nature, origin and changes during ripening.

Casein phosphopeptides (CPP) which develop in Grana Padano cheese at different ages were isolated by precipitation with Ba2+ and analysed by HPLC. Profiles were complex throughout the period between 4 and 38 months. CPP in a cheese sample 14 months old were identified by a combination of fast atom bombardment-mass spectrometry and Edman degradation. They were found to consist of a mixture of components derived from three parent peptides, beta-CNf(7-28)4P, alpha s1-CNf(61-79)4P and alpha s2-CNf(7-21)4P. In total, 45 phosphopeptides were identified: 24 from beta-CN, 16 from alpha s1-CN and 5 from alpha s2-CN. The presence of aminopeptidase activity during cheese ripening was deduced from the presence of a number of CPP of different lengths with the loss of one or more residues from the N-terminus. The longest had C-terminal lysine and seemed to be progressively hydrolysed by carboxypeptidases A and B to shorter peptides. CPP in cheese appeared to be shortened plasmin-mediated products. Moreover, those most resistant to further hydrolysis contained at least three closely located phosphoserine residues. The anticariogenic activity of CPP is also discussed.

Differential splicing of pre-messenger RNA produces multiple forms of mature caprine alpha(s1)-casein.

The identity of multiple forms of caprine alpha(s1)-casein in variants A, B, and C has been determined by structural characterisation using mass spectrometry, automated Edman degradation and peptide mapping. Mature goat alpha(s1)-casein exists as a mixture of at least four molecular species which differ in peptide chain length. The main component corresponds to the 199-residues form already described. The other three, in lesser amounts, were shorter forms of alpha(s1)-casein and differed for the deleted peptides 141-148, as shown previously for ovine alpha(s1)-casein, peptide 110-117, or Gln78. Analysis of alpha(s1)-casein mRNA from milk somatic cells demonstrated that these forms originated from skipping events at the level of exon 13 (codifying for peptide 110-117) and 16 (codifying for peptide 141-148) and from the presence of a cryptic splice site within exon 11 (whose first CAG triplet encodes Gln78) during primary transcript processing. The finding of these splicing abnormalities in the three common variants A, B, and C suggests that this is a general feature of alpha(s1)-casein in goat. A further source of heterogeneity of caprine alpha(s1)-casein was identified in the discrete phosphorylation of seryl residues. Eight serine residues (at positions 44, 46, 64 to 68 and 75) are fully phosphorylated (except in variant A because of the replacement Glu77-->Gln which prevents phosphorylation of Ser75). Conversely, Ser115 and Ser41 are phosphorylated only to about 50% and 20%, respectively. Ser12, although located in a consensus triplet, is never phosphorylated, similarly to the ovine alpha(s1)-casein variants. These results confirm that there are stabilised mechanisms of simultaneous synthesis of alpha(s1)-casein at different length and of post-translational modification in both caprine and ovine species.

Occurrence of five alpha s1-casein variants in ovine milk.

Five ovine alpha s1-casein variants (A-E) were identified in an Italian population sample using gel electrophoresis at alkaline pH, gel isoelectric focusing, two dimensional gel electrophoresis, and immunoblotting with polyclonal antibodies against alpha s1-casein. Each casein sample produced two peaks by fast reversed-phase HPLC. Gel isoelectric focusing and electrospray mass spectrometry were used to demonstrate that the first HPLC peak contained the 191 residue alpha s1-casein molecular species and the second the 199 residue species, in proportions of approximately 20:80. Only in the case of the sample containing alpha s1-casein CE was the method for the separation of the single short and long forms of each variant unsuccessful. Both two dimensional electrophoresis followed by staining with polyclonal antibodies against alpha s1-casein and electrospray mass spectrometry showed a heterogeneity consistent with that expected from a protein chain with three levels of phosphorylation and two different lengths. However, especially for alpha s1-caseins D and E, a further uncharacterized heterogeneity was detected.

Induction of p75NGFR in human diabetic neuropathy.

In this immunohistochemical study we investigated the expression of low-affinity NGF receptor (p75NGFR) in peripheral nerves from 16 patients with type I or type II diabetes mellitus. Fourteen nerves from age- and sex-matched normal individuals and nine nerves from non-diabetic patients with ischemic neuropathy served as controls. All nerve samples were preliminarily examined by standard histology, fiber teasing and electron microscopy. Increased p75NGFR immunoreactivity was detectable within the endoneurium of cross-sections from ischemic and particularly from diabetic nerves. Immuno-teasing demonstrated that p75NGFR immunostaining was distributed along the entire length of isolated nerve fibers undergoing axonal degeneration. Quantitative assessment of p75NGFR immunoreactivity, performed by histospectrophotometry and expressed as percentage of adsorbance, was 21.20 +/- 3.50 in nerves from diabetic patients, 13.35 +/- 3.62 in nerves from non-diabetic patients with ischemic neuropathy and 9.02 +/- 2.75 in normal controls. The increased expression of p75NGFR in diabetic nerves is consistent with an axonopathic defect and further suggests involvement of NGF and other neurotrophins in the pathogenesis of human diabetic neuropathy.

Primary structure of ovine alpha s1-caseins: localization of phosphorylation sites and characterization of genetic variants A, C and D.

The primary structures of ovine alpha s1-casein variants A, C and D (formerly called Welsh variant) were determined. Separation of variants from whole casein was achieved using a fast and reliable reversed-phase HPLC method. Extended structural characterization of the purified proteins using electrospray mass spectrometry, automated Edman degradation and peptide mapping by means of HPLC-fast atom bombardment-mass spectrometry demonstrated that the mature protein was a mixture of two molecular species that differed in the deletion of residues 141-148 and were therefore 199 and 191 residues long respectively. The 199 residue peptide chain, which accounted for approximately 80% of the entire translated alpha s1-casein, was as long as its caprine and bovine counterparts, and had a 98 and 89% degree of identity with those two proteins respectively. Nine serine residues (positions 12, 44, 46, 64 to 68 and 75) were fully phosphorylated in alpha s1-casein A, whereas Ser115 and Ser41 were phosphorylated by approximately 50 and approximately 20% respectively. The differences between the three genetic variants A, C and D were simple silent substitutions, which however involved the degree to which the protein was phosphorylated. Variant C differed from variant A in the substitution Ser13-->Pro13 which determined the loss of the phosphate group on site 12 of the protein chain, SerP12-->Ser12. A further substitution, SerP68-->Asn68 caused the disappearance of both phosphate groups in the phosphorylated residues Ser64 and Ser66 in variant D; in this last casein variant there was no evidence of phosphorylation at Ser41.

Gel electrophoresis and immunoblotting for the detection of casein proteolysis in cheese.

The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immunoblotting with polyclonal antibodies against beta- and alpha s1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4.6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as gamma 3-casein (beta-casein(f108-209)) and alpha s1-PL1 (alpha s1-casein(f80-199). In both cases, a Lys-X bond was hydrolysed, indicating the action of a trypsin-like enzyme in beta- and alpha s1-casein hydrolysis during the ripening of this variety of hard pressed cheese.

Characterization of the oligopeptides of Parmigiano-Reggiano cheese soluble in 120 g trichloroacetic acid/l.

The non-protein nitrogen (NPN) of samples of Parmigiano-Reggiano cheese ripened for 6 and 15 months was fractionated by ion-exchange chromatography on a Cu(2+)-Chelex column to separate oligopeptides from free amino acids. Peptide components were isolated by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry (FAB-MS). Only the NPN fraction of 6 month old cheese samples contained enough peptides to be further characterized. On the basis of FAB-MNS spectral results, 39 oligopeptides were identified, the main components being phosphopeptides. Two sets of both intact and partly dephosphorylated peptides, accounting for a total of 19 phosphopeptides, were formed by the hydrolysis of beta-casein and belonged to regions 1-20 and 6-28 of beta-casein. The formation and potential role of these peptides in cheese is discussed.

Increase in serum alpha-fetoprotein without recurrent disease after hepatectomy for hepatocellular carcinoma.

The case of a 58-year-old man with clinically-stable and compensated HBsAg-positive liver cirrhosis is reported. In April 1991, the patient underwent partial hepatectomy to treat a solitary 3.5 cm hepatocellular carcinoma (HCC), (Edmonson scale I), in the 5th liver segment. His serum alpha-fetoprotein (AFP) level was 24 ng/ml. After hepatectomy, the AFP level dropped to 8 ng/ml, but between the 4th and 12th month it rose gradually from 72 ng/ml to 4,520 ng/ml. Hepatic recurrence of HCC was excluded, but a 6 cm solitary metastasis (Edmonson scale III-IV) was detected on the right adrenal. Adrenalectomy was performed and two months later the patient is doing well and his AFP level is 51 ng/ml. The methodological approach to diagnosis, treatment and follow-up of HCC, and the relationship between AFP and liver and metastatic HCC, are discussed.

Discovery of an ovine alpha S2-casein variant.

A novel ovine alpha S2-casein variant has been detected using discontinuous polyacrylamide gel electrophoresis at alkaline pH, two dimensional electrophoresis and immunoblotting. It is characterized by a greater negative net charge and a lower isoelectric point compared with the most common ovine alpha S2-casein variant. The phenotypic frequency in the Manchega breed is 5.5%.

New alpha s2-casein variant from caprine milk.

A new alpha s2-casein variant was isolated from goat milk by means of covalent chromatography. With gel electrophoresis at alkaline pH, the alpha s2-casein variant showed a double band with the highest anodic mobility amongst the casein components. The mobility of the isolated alpha s2-casein variant was similar to that occurring in three individual samples of whole casein. Two dimensional electrophoresis revealed a more pronounced heterogeneity of this protein. Developing the two dimensional plate with polyclonal antibodies obtained against the four main bovine casein fractions, we demonstrated that the variant in question belongs to the alpha s2-casein family. Since the alpha s2-casein fractions, immunospecifically developed, spread along two different zones on the gel, it was concluded that a heterozygous alpha s2-casein form was present in the sample.