Casein phosphopeptides L L-CN(1^25)4P and K K s1 -CN(59^79)5P, from L L-and K K s1 -casein, respectively, both carrying the characteristic 'acidic motif' Ser(P)-Ser(P)-Ser(P)-GluGlu, were chemically synthesized and administered to HT-29 cells di¡erentiated in culture, which are a used model of intestinal epithelium for absorption studies. Both casein phosphopeptides caused an increase of [Ca 2+ ] i due to in£ux of extracellular Ca 2+ . The response was quantitatively higher with L L-CN(12 5)4P than K K s1 -CN(59^79)5P. The synthetic peptide corresponding to the 'acidic motif' was ine¡ective and the dephosphorylated form of L L-CN(1^25)4P almost inactive. The lack of the N-terminally located ¢ve amino acids, or sequence modi¢-cations within the N-terminal segment of L L-CN(1^25)4P, caused a total loss of activity, whereas the lack of the C-terminal segment preserved activity. In conclusion, the in£ux of calcium into HT-29 cells caused by L L-CN(1^25)4P appears to depend on the phosphorylated 'acidic motif' and the preceding N-terminal region.

We investigated the direct effects of casein phosphopeptides (CPP), which are formed by the proteolytic degradation of alpha- and beta-caseins, on calcium uptake by human HT-29 intestinal tumor cells, which undergo an enterocytically oriented differentiation in culture. A commercial preparation containing a mixture of purified CPP and an individual CPP of 25 amino acids, both containing the characteristic Ca(2+) binding motif, ser(P)-ser(P)-ser(P)-glu-glu, were employed. The study was performed at the single-cell level and on a cell population and measured the changes in cytosolic calcium concentration before and after CPP addition. In the presence of 2 mmol/L extracellular calcium, both CPP preparations induced a transient rise of free intracellular calcium ions, which did not influence ATP-induced release of calcium from intracellular stores, and which disappeared completely in the absence of extracellular calcium. Pretreatment of these cells with thapsigargin, which completely empties the intracellular calcium stores, did not abolish the cell responses to CPP. Repetitive stimulation of HT-29 cells with CPP always elicited a transient calcium rise, suggesting a lack of desensitization. The CPP-stimulated cytosolic calcium rise was dependent on CPP dose, in a seemingly nonsaturating mode, and on cell numbers. All of this is consistent with the hypothesis that CPP do not influence membrane-bound receptors or ion channels, but may act as calcium ionophores or calcium carriers across the membrane. The reported findings provide a new basis on which to assess the possibility that CPP enhance calcium absorption and bioavailability in animals.

An intestinal 70/30 Caco2/HT-29 co-culture was set up starting from the parental populations of differentiated cells to mimic the human intestinal epithelium. Co-culture was harvested at confluence 0 (T0) and at 3, 6, 10, and 14 days post confluence after plating (T3, T6, T10, and T14, respectively) for morphological and functional analysis. Transmission electron microscopy revealed different features from T0 to T14: microvilli and a complete junctional apparatus from T6, mucus granules from T3, as also confirmed by PAS/Alcian Blue staining. The specific activity of alkaline phosphatase (ALP), aminopeptidase N (APN), and dipeptidyl peptidase IV (DPPIV) progressively increased after T0, indicating the acquirement of a differentiated and digestive phenotype. Transepithelial electrical resistance (TEER), indicative of the barrier properties of the monolayer, increased from T0 up to T6 reaching values very similar to the human small intestine. The apparent permeability coefficient for Lucifer Yellow (LY), along with morphological analysis, reveals a good status of the tight junctions. At T14, HT-29 cells reduced to 18.4% and formed domes, indicative of transepithelial transport of nutrients. This Caco2/HT-29 co-culture could be considered a versatile and suitable in vitro model of human intestinal epithelium for the presence of more than one prevalent intestinal cell type, by means of a minimum of 6 to a maximum of 14 post-confluence days obtained without the need of particular inducers of subclones and growth support to reach an intestinal differentiated phenotype.

The pathways of metabolic processing of exogenously administered GM1 ganglioside in rat liver was investigated at the subcellular level. The GM1 used was 3H-labelled at the level of long-chain base ([Sph(sphingosine)-3H]GM1) or of terminal galactose ([Gal-3H]GM1). The following radioactive compounds, derived from exogenous GM1, were isolated and chemically characterized: gangliosides GM2, GM3, GD1a and GD1b (nomenclature of Svennerholm [(1964) J. Lipid Res. 5, 145-155] and IUPAC-IUB Recommendations [(1977) Lipids 12, 455-468]); lactosylceramide, glucosylceramide and ceramide; sphingomyelin. GM2, GM3, lactosylceramide, glucosylceramide and ceramide, relatively more abundant shortly after GM1 administration, were mainly present in the lysosomal fraction and reflected the occurrence of a degradation process. 3H2O was also produced in relevant amounts, indicating complete degradation of GM1, although no free long-chain bases could be detected. GD1a and GD1b, relatively more abundant later on after administration, were preponderant in the Golgi-apparatus fraction and originated from a biosynthetic process. More GD1a was produced starting from [Sph-3H]GM1 than from [Gal-3H]GM1, and radioactive GD1b was present only after [Sph-3H]GM1 injection. This indicates the use of two biosynthetic routes, one starting from a by-product of GM1 degradation, the other implicating direct sialylation of GM1. Both routes were used to produce GD1a, but only the first one for producing GD1b. Sphingomyelin was the major product of GM1 processing, especially at the longer times after injection, and arose from a by-product of GM1 degradation, most likely ceramide.

It is known that milk is an excellent source of bioavailable calcium, due to the presence of caseins, which bind calcium, keeping it in a soluble and absorbable state [1][2][3][4][5]. In bovine milk, about two-thirds of the calcium and one-half of the inorganic phosphate are bound to various species of caseins, a S1 -casein, a S2 -casein, b-casein, and k-casein, forming colloidal micelles with a calcium ⁄ phosphate ⁄ casein molar ratio of 30 : 21 : 1 [6]. The casein micelles, of about 100 nm radius, are stable structures composed of hundreds of smaller aggregates, named calcium phosphate nanoclusters, or nanocomplexes, having a core of calcium phosphate surrounded by a shell of casein molecules [7][8][9][10]. The portion of the casein molecule responsible for the ability to maintain calcium and phosphate ions in a soluble form are amino acid sequences containing the common motif Ser(P)-Ser(P)-Ser(P)-Glu-Glu (the 'cluster sequence' or 'acidic motif'). Peptides containing this sequence (casein phosphopeptides, CPPs) are produced in vivo from the digestion of a S1 -casein, a S2 -casein and b-casein by gastrointestinal proteases [11][12][13], and in vitro by tryptic and chimotryptic fragmentation of casein followed by precipitation [14]. Calcium phosphate nanoclusters (or complexes) were also prepared and physicochemically characterized using CPPs, namely b-CN(1-25)4P and b-CN(1-42)5P, corresponding to the first 25 or 42 amino acids of b-casein, respectively, and a S1 -CN(59-79)5P, ] o , extracellular free calcium concentration; CN, casein; CPP, casein phosphopeptide; CPP DMV, CPP of commercial origin; KRH, Krebs ⁄ Ringer ⁄ Hepes.