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PubMed2003

Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1.

In vertebrates, the checkpoint-regulatory kinase Chk1 mediates cell-cycle arrest in response to a block in DNA replication or to DNA damaged by ultraviolet radiation. The activation of Chk1 depends on both Claspin and the upstream regulatory kinase ATR. Claspin is a large acidic protein that becomes phosphorylated and binds to Chk1 in the presence of checkpoint-inducing DNA templates in Xenopus egg extracts. Here we identify, by means of deletion analysis, a region of Claspin of 57 amino acids that is both necessary and sufficient for binding to Xenopus Chk1. This Chk1-binding domain contains two highly conserved repeats of approximately ten amino acids. A serine residue in each repeat (serine 864 and serine 895) undergoes phosphorylation during a checkpoint response. A mutant of Claspin containing non-phosphorylatable amino acids at positions 864 and 895 cannot bind to Chk1 and is unable to mediate its activation. Our results indicate that two phosphopeptide motifs in Claspin are essential for checkpoint signalling.

Adaptor Proteins, Signal Transducing↗

PubMed1989

Indirect effect of casein phosphopeptides on calcium absorption in rat ileum in vitro.

Casein phosphopeptides (CPP) are the phosphorylated fragments of bovine milk casein. They are believed to enhance intestinal absorption of calcium by their ability to form soluble complexes with calcium thereby inhibiting the precipitation of phosphate-calcium salts. In order to evaluate whether they also act in an additional direct manner on the intestinal mucosa, these peptides were added in a phosphate-free medium at a concentration of 1, 2, or 4 mg/ml on the mucosal side of rat ileum mounted in an Ussing chamber in vitro. No effect on the electrical parameters of the tissue was observed. The unidirectional mucosal-to-serosal flux of calcium was significantly reduced in the presence of the peptides, without alteration in the serosal-to-mucosal flux. Jms was 51.71 +/- 2.67 microEq/h.cm2 for control vs. 19.23 +/- 3.95 in the presence of 4 mg/ml CPP. This effect was associated with a reduction in free calcium in the mucosal reservoir of the Ussing chamber, without modification of mucosal total calcium or of serosal total and free calcium. These results indicate that CPP did not directly act on rat ileum to enhance calcium absorption. These peptides bind calcium, and the CPP--calcium complex which was not efficiently absorbed remained on the mucosal side of the tissue. In these conditions, the physiological role of CPP on intestinal calcium absorption could be only an indirect luminal inhibition of the precipitation of phosphate-calcium salts. This effect remains to be clearly established.

Animals↗

PubMed2003

Remineralization of enamel subsurface lesions in situ by sugar-free lozenges containing casein phosphopeptide-amorphous calcium phosphate.

BACKGROUND: The anticariogenic potential of casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP) has been demonstrated using laboratory, animal and human in situ caries models. The aim of this study was to determine the effect of CPP-ACP incorporation into a sugar-free lozenge (pressed mint tablet) on enamel remineralization in a human in situ model. METHODS: The study utilized a double-blind, randomized, cross-over design with four treatments: (i) a lozenge containing 56.4mg (3 per cent w/w) CPP-ACP; (ii) a lozenge containing 18.8mg (1 per cent w/w) CPP-ACP; (iii) a lozenge not containing CPP-ACP; and (iv) a no lozenge nil-treatment control. Ten subjects wore removable palatal appliances with four, human-enamel, half-slab insets containing subsurface lesions. Lozenges were consumed, without chewing, four times per day for 14 days duration. After each treatment period the enamel slabs were removed, paired with their respective demineralized control, embedded, sectioned and subjected to microradiography and computer-assisted densitometric image analysis to determine the level of remineralization. RESULTS: The incorporation of CPP-ACP into the lozenge significantly increased enamel subsurface lesion remineralization with 18.8 and 56.4mg of CPP-ACP increasing remineralization by 78 and 176 per cent respectively, relative to the control sugarfree lozenge. CONCLUSION: This study demonstrates that lozenges are a suitable vehicle for the delivery of CPP-ACP to promote enamel remineralization.

Administration, Buccal↗

PubMedDate not supplied

Binding characteristics of Streptococcus mutans for calcium and casein phosphopeptide.

Casein phosphopeptides (CPP) stabilize amorphous calcium phosphate (ACP) and may be used to localize ACP in dental plaque, maintaining a state of supersaturation with respect to tooth enamel, reducing demineralization and enhancing remineralization [Reynolds, J Dent Res 1997;76:1587-1595]. The aim of this paper is to investigate these effects by measuring the affinity and capacity of Streptococcus mutans for CPP-ACP. Using the equilibrium dialysis system described by Rose and Hogg [Biochim Biophys Acta 1995;1245:94-98], assessment of calcium binding by a plaque streptococcus at a fixed CPP-ACP concentration gives a series of CPP-ACP-influenced dissociation constants for calcium. These data can then be used to derive a true dissociation constant for CPP-ACP itself. The results demonstrate that CPP-ACP binds with about twice the affinity of the bacterial cells for calcium up to a value of 0. 16 g/g wet weight cells. Application of CPP-ACP to plaque may cause a transient rise in plaque fluid free calcium which may assist remineralization. Subsequently, CPP-ACP will form a source of readily available calcium to inhibit demineralization. Hence, CPP-ACP binds well to plaque, providing a large calcium reservoir, which is likely to restrict mineral loss during a cariogenic episode and provide a potential source of calcium for subsequent remineralization. Overall, once in place, CPP-ACP will restrict the caries process.

Binding, Competitive↗

PubMedDate not supplied

Acid resistance of enamel subsurface lesions remineralized by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate.

The aim of this clinical study was to investigate the acid resistance of enamel lesions remineralized in situ by a sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP: Recaldent). The study utilized a double-blind, randomized, crossover design with two treatments: (i) sugar-free gum containing 18.8 mg of CPP-ACP, and (ii) sugar-free gum not containing CPP-ACP as control. Subjects wore removable palatal appliances with insets of human enamel containing demineralized subsurface lesions and chewed the gum for 20 min 4 times per day for 14 days. After each treatment the enamel slabs were removed and half of each lesion challenged with acid in vitro for 8 or 16 h. The level of remineralization was determined using microradiography. The gum containing CPP-ACP produced approximately twice the level of remineralization as the control sugar-free gum. The 8- and 16-hour acid challenge of the lesions remineralized with the control gum resulted in 65.4 and 88.0% reductions, respectively, of deposited mineral, while for the CPP-ACP-remineralized lesions the corresponding reductions were 30.5 and 41.8%. The acid challenge after in situ remineralization for both control and CPP-ACP-treated lesions resulted in demineralization underneath the remineralized zone, indicating that the remineralized mineral was more resistant to subsequent acid challenge. The results show that sugar-free gum containing CPP-ACP is superior to an equivalent gum not containing CPP-ACP in remineralization of enamel subsurface lesions in situ with mineral that is more resistant to subsequent acid challenge.

Acetic Acid↗

PubMed2001

Remineralization of enamel subsurface lesions by sugar-free chewing gum containing casein phosphopeptide-amorphous calcium phosphate.

Casein phosphopeptide-amorphous calcium phosphate nanocomplexes (CPP-ACP) exhibit anticariogenic potential in laboratory, animal, and human in situ experiments. The aim of this study was to determine the ability of CPP-ACP in sugar-free chewing gum to remineralize enamel subsurface lesions in a human in situ model. Thirty subjects in randomized, cross-over, double-blind studies wore removable palatal appliances with six human-enamel half-slabs inset containing sub-surface demineralized lesions. The appliances were inserted immediately before gum-chewing for 20 min and then retained for another 20 min. This was performed four times per day for 14 days. At the completion of each treatment, the enamel half-slabs were paired with their respective demineralized control half-slabs, embedded, sectioned, and subjected to microradiography and densitometric image analysis, for measurement of the level of remineralization. The addition of CPP-ACP to either sorbitol- or xylitol-based gum resulted in a dose-related increase in enamel remineralization, with 0.19, 10.0, 18.8, and 56.4 mg of CPP-ACP producing an increase in enamel remineralization of 9, 63, 102, and 152%, respectively, relative to the control gum, independent of gum weight or type.

Adult↗

PubMed2004

Phosphopeptide mapping of proteins ectopically expressed in tissue culture cell lines.

Post-translational modifications such as phosphorylation play a vital role in the regulation of protein function. In our study of the basic Helix-loop-Helix (bHLH) transcription factor HAND1, it was suspected that HAND1 was being phosphorylated during trophoblast giant cell differentiation and that coexpression of a constitutively active kinase with HAND1 resulted in changes in the proteins dimerization profile. In order to accurately document HAND1 phosphorylation and identify the resides being modified, we employed metabolic cell labeling with (32)P of tissue culture cells coexpressing a Flag-epitope tagged HAND1 along with a number of active kinases and phosphatase subunits. We generated phosphopeptide maps of the phosphorylated HAND1 using the methods described below and linked these modifications to changes in HAND1 biological function.

Journal Article↗

PubMed2001

[The remineralization of enamel lesions by casein phosphopeptide-amorphous calcium fluoride phosphate in vitro].

OBJECTIVE: To evaluate the remineralizing effect on artificial enamel subsurface lesions in vitro by 2% casein phosphopeptide-amorphous calcium fluoride phosphate complexes (CPP-ACFP) solution. METHODS: Twenty-eight enamel blocks were cut from human third molars, in which 2 lesions were prepared from each block. The 28 samples were randomly assigned to the four study groups, in which each group was incubated in remineralization solution for 1, 3, 5, 10 days respectively. After remineralization the samples were sectioned, microradiographied and the mineral content were determined by microdensitometry. RESULTS: The remineralizing solution replaced 9.19%, 14.27%, 29.07%, 38.45% of mineral lost after 1, 3, 5, 10 days respectively (y = 8.9316 x0.6347, R2 = 0.9322; y: remineralizing rate, x: time). Significant differences were found in remineralizing rate between any two groups using One-way Scheffe multiple comparison. CONCLUSIONS: The 2% CPP-ACFP solution could significantly remineralize the artificial enamel subsurface lesion in vitro. The remineralizing rates were increased with the experiment time (within 10 days).

Caseins↗

PubMed1991

Two-dimensional phosphopeptide analysis of the autophosphorylation cascade of a soluble insulin receptor tyrosine kinase. The tyrosines phosphorylated are typical of those observed following phosphorylation of the heterotetrameric insulin receptor in intact cells.

A soluble derivative of the human insulin receptor cytoplasmic domain, as expressed in insect cells via a Baculovirus vector, is an active protein-tyrosine kinase. In the present study, we find that three forms of the enzyme (48, 43, and 38 kDa) can be partially purified by MonoQ fast protein liquid chromatography. Two-dimensional thin layer phosphopeptide mapping reveals that the 48-kDa enzyme undergoes a rapid autophosphorylation on the same tyrosines (residues 1158, 1162, 1163, 1328, and 1334) that have previously been shown to be major autophosphorylation sites on the native insulin receptor beta-subunit in intact cells. Furthermore, the 48- and 43-kDa proteins are phosphorylated on serine residues by a serine kinase(s) that copurifies through MonoQ fast protein liquid chromatography. Tyrosine autophosphorylation sites 1328 and 1334 and virtually all serine phosphorylation sites are absent in the 38-kDa kinase. Partial tryptic proteolysis of the 48-kDa kinase generates a core 38-kDa enzyme that undergoes autophosphorylation almost exclusively on tyrosines 1158, 1162, and 1163. Phosphorylation of these tyrosine residues occurs in a cascade manner analogous to that found in the intact insulin receptor beta-subunit.

Amino Acid Sequence↗

PubMed1987

Ascaris suum phosphofructokinase. Phosphorylation by protein kinase and sequence of the phosphopeptide.

Phosphorylation of the ascarid phosphofructokinase with the catalytic subunit of beef heart cyclic AMP-dependent protein kinase results in the incorporation of 1 mol of P/mol of subunit. Accompanying the phosphorylation there is a 3-4-fold increase in catalytic activity when measured at pH 6.8 with inhibitory levels of ATP. Studies on the effect of phosphorylation on the ATP saturation curve demonstrated that phosphorylation decreased the inhibitory action of ATP. The apparent Km of the catalytic subunit for the phosphofructokinase was 11.2 microM. Chymotryptic or subtilisin digestion of the labeled enzyme released distinct but overlapping phosphopeptides that were purified by high pressure liquid chromatography and sequenced by gas phase peptide sequencing. The sequence of the chymotryptic peptide was Ala-Lys-Gly-Arg-Ser-Asp-Ser(P)-Ile-Val-Pro-Thr. Based on these results and earlier observations, it is proposed that phosphorylation of phosphofructokinase plays an important role in the regulation of energy metabolism in the parasitic helminth.

Adenosine Triphosphate↗

PubMed1965