Enhanced Anti-Allergic Activity of Milk Casein Phosphopeptide by Additional Phosphorylation in Ovalbumin-Sensitized Mice.

The proteolytic digest of milk casein, known as casein phosphopeptide (CPP-III), exhibits diverse biological activities, including calcium absorption and antioxidant activities. We hypothesized that the additional phosphorylation of this peptide can enhance its immunomodulatory activity such as suppression of allergy-associated cytokine and antigen-specific immune response. This study was conducted to assess whether oral intake of additionally phosphorylated CPP-III (P-CPP) attenuates ovalbumin (OVA)-induced IgE-mediated allergic reactions because of the additional phosphate groups. Female BALB/c mice were intraperitoneally sensitized with OVA twice at intervals of 14 days and then orally fed native CPP-III (N-CPP), P-CPP, and dephosphorylated CPP-III (D-CPP) for 6 weeks. Next, the mice were orally challenged with 50 mg of OVA. Oral administration of P-CPP suppressed total and specific IgE levels in the serum. Mice fed P-CPP exhibited low levels of OVA-specific IgG1 and increased OVA-specific IgG2a. P-CPP also suppressed IL-4 production, while D-CPP showed similar a level compared to that of the control. Further, P-CPP increased the population of the T follicular helper (Tfh) cell in the spleen. These results suggest that additional phosphorylation of CPP can enhance the attenuation of allergen-specific IgE-modulated allergic reactions in a murine food allergy model.

Cysteine and arginine-rich peptides as molecular carriers.

A number of linear and cyclic peptides containing alternative arginine and cysteine residues, namely linear (CR)3, linear (CR)4, linear (CR)5, cyclic [CR]4, and cyclic [CR]5, were synthesized. The peptides were evaluated for their ability to deliver two molecular cargos, fluorescence-labeled cell-impermeable negatively charged phosphopeptide (F'-GpYEEI) and fluorescence-labeled lamivudine (F'-3TC), intracellularly in human leukemia cancer (CCRF-CEM) cells. We investigated the role of cyclization and the number of amino acids in improving the transporting ability of the peptides. The flow cytometry studies suggested that the synthesized peptides were able to work efficiently as transporters for both cargos. Among all compounds, cyclic [CR]4 was found to be the most efficient peptide in transporting the cargo into cells. For instance, the cellular uptake of F'-3TC (5µM) and F'-GpYEEI (5µM) was enhanced by 16- and 20-fold, respectively, in the presence of cyclic [CR]4 compared to that of the parent compound alone. The mechanism of F'-GpYEEI uptake by cells was found to be energy-independent. The results showed that the number of amino acids and their cyclic nature can impact the efficiency of the peptide in transporting the molecular cargos.

Efficient delivery of cell impermeable phosphopeptides by a cyclic peptide amphiphile containing tryptophan and arginine.

Phosphopeptides are valuable reagent probes for studying protein-protein and protein-ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent-labeled phosphopeptides, including F'-GpYLPQTV, F'-NEpYTARQ, F'-AEEEIYGEFEAKKKK, F'-PEpYLGLD, F'-pYVNVQN-NH2, and F'-GpYEEI (F' = fluorescein), was evaluated in the presence or absence of a [WR]4, a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR]4 improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F'-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR]4 and was found to be time-dependent. Confocal microscopy of a mixture of F'-PEpYLGLD and [WR]4 in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F'-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEpYLGLD and [WR]4. TEM results showed that the mixture of PEpYLGLD and [WR]4 formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR]4 and PEpYLGLD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR]4 can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides.

Preliminary study of a novel in-office bleaching therapy modified with a casein phosphopeptide-amorphous calcium phosphate.

Although in-office bleaching has been proven successful for bleaching teeth, controversy exists from morphological alterations in enamel morphology due to mineral loss and tooth sensitivity. This preliminary study aimed to evaluate the efficacy of a novel in-office tooth bleaching technique modified with a casein phosphopeptide-amorphous calcium phosphate (CPP-ACP) paste (MI paste-MI) and its effect on the enamel morphology and tooth sensitivity. Three patients received a 35% hydrogen peroxide (Whiteness HP-HP) dental bleaching system. HP was prepared and applied on the teeth on one of the hemiarches, whilst teeth on the other hemiarch were bleached with a mixture of HP and MI. Tooth color, epoxy resin replicas, and sensitivity levels were evaluated in the upper incisors. The results were analyzed descriptively. Right and left incisors showed similar color change after bleaching. Incisors bleached with the mixture of HP and MI presented unaltered enamel surfaces and lower sensitivity levels. The currently tested tooth bleaching technique did not reduce the gel effectiveness while decreasing hypersensitivity levels and protecting the enamel against surface alterations caused by the high-concentration bleaching peroxide tested. The concomitant use of MI Paste and high-concentration hydrogen peroxide might be a successful method for decreasing tooth sensitivity and limiting changes in the enamel morphology during in-office bleaching.

Effective treatment of experimental ragweed-induced asthma with STAT-6-IP, a topically delivered cell-penetrating peptide.

BACKGROUND: Treatment of allergic airways disease including asthma remains primarily local immunosuppression with topical corticosteroid and symptomatic management with antihistamines and anti-leucotrienes. We have developed a novel topical therapy designed to specifically inhibit the events associated with Th2 cell activation. OBJECTIVE: We assessed the efficacy of our cell-penetrating STAT-6 inhibitory peptide (STAT-6-IP), a novel treatment for allergic airways disease, in a model of chronic ragweed-induced asthma. METHODS: Six- to eight-week-old mice were sensitized over 5 weeks with intranasal (IN) exposures to whole ragweed allergen without adjuvant. Mice were then IN challenged with Amba 1 with and without treatment IN with STAT-6-IP and allergic responses assessed. Two weeks later, some animals were rechallenged with Amba 1 with or without STAT-6-IP. RESULTS: Animals exposed to IN ragweed developed significant airway hyperresponsiveness and airways inflammation upon challenge. Cell cultures showed increases in Th2 cytokines IL-4 and IL-13. Topical STAT-6-IP treatment reduced production of Th2 cytokines, demonstrated increased expression of IL-10 and reduced frequency of cultured IL-4 positive CD4+ T cells derived from treated mice, suggesting that STAT-6-IP treatment may be immunomodulatory. Airway responsiveness to methacholine challenge in the treatment group was similarly reduced to that of the non-allergic PBS-exposed animals. Importantly, STAT-6-IP-treated mice remained hyporesponsive following second ragweed challenge 2 weeks after treatment. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that topical application of the STAT-6-IP is sufficient to inhibit allergic airways responses in animals chronically sensitized and challenged with ragweed. Data show that a single topical treatment course is sufficient to block signs of allergic responses to ragweed in the airways for at least 2 weeks. STAT-6-IP is a novel potential treatment for chronic allergic asthma.

Anti-genotoxic potential of casein phosphopeptides (CPPs): a class of fermented milk peptides against low background radiation and prevention of cancer in radiation workers.

BACKGROUND: Radiation workers are constantly exposed to low background radiation which is their occupational hazard. This continuous and prolonged exposure produces genotoxicity and cancerous condition in many workers. The authors have tested casein phosphopeptides (CPP) as a radioprotectant against low background radiation using animal models. METHODS: Fermented milk was produced by addition of a bacterial culture, Lactobacillus acidophilus to a commercially available milk brand. After the fermentation process is completed in the milk, CPP is isolated from fermented milk by enzymatic hydrolysis-based method. The radioprotective role of CPP was proved using albino mice and Catla catla fish. RESULTS: The micronucleus assay showed higher level of cell deformation and micronucleus formation in the control animal cells than the test animal cells. CPP has found to be having radioprotective activity potential. CONCLUSIONS: This radioprotective potential of CPP can be harnessed to produce formulations which can be used by radiation workers and personnel exposed to low ionization background as an occupational hazard, thus reducing the risk and preventing any type of cancer.

A newly developed snack effective for enhancing bone volume.

BACKGROUND: The incidence of primary osteoporosis is higher in Japan than in USA and European countries. Recently, the importance of preventive medicine has been gradually recognized in the field of orthopaedic surgery with a concept that peak bone mass should be increased in childhood as much as possible for the prevention of osteoporosis. Under such background, we have developed a new bean snack with an aim to improve bone volume loss. In this study, we examined the effects of a newly developed snack on bone volume and density in osteoporosis model mice. METHODS: Orchiectomy (ORX) and ovariectomy (OVX) were performed for C57BL/6J mice of twelve-week-old (Jackson Laboratory, Bar Harbar, ME, USA) were used in this experiment. We prepared and given three types of powder diet e.g.: normal calcium diet (NCD, Ca: 0.9%, Clea Japan Co., Tokyo, Japan), low calcium diet (LCD, Ca: 0.63%, Clea Japan Co.,) and special diet (SCD, Ca: 0.9%). Eighteen weeks after surgery, all the animals were sacrified and prepared for histomorphometric analysis to quantify bone density and bone mineral content. RESULTS: As a result of histomorphometric examination, SCD was revealed to enhance bone volume irrespective of age and sex. The bone density was increased significantly in osteoporosis model mice fed the newly developmental snack as compared with the control mice. The bone mineral content was also enhanced significantly. These phenomena were revealed in both sexes. CONCLUSION: It is shown that the newly developed bean snack is highly effective for the improvement of bone volume loss irrespective of sex. We demonstrated that newly developmental snack supplements may be a useful preventive measure for Japanese whose bone mineral density values are less than the ideal condition.

Short-term effect of bedtime consumption of fermented milk supplemented with calcium, inulin-type fructans and caseinphosphopeptides on bone metabolism in healthy, postmenopausal women.

BACKGROUND: Milk products are good sources of calcium and their consumption may reduce bone resorption and thus contribute to prevent bone loss. AIM OF THE STUDY: We tested the hypothesis that bedtime consumption of fermented milk supplemented with calcium inhibits the nocturnally enhanced bone resorption more markedly than fermented milk alone, and postulated that this effect was most pronounced when calcium absorption enhancers were added. METHODS: In a controlled, parallel, double-blind intervention study over 2 weeks we investigated the short-term effects of two fermented milks supplemented with calcium from milk minerals (f-milk + Ca, n = 28) or calcium from milk minerals, inulin-type fructans and caseinphosphopeptides (f-milk + Ca + ITF + CPP; n = 29) on calcium and bone metabolism in healthy, postmenopausal women, and compared them with the effect of a fermented control milk without supplements (f-milk, n = 28). At bedtime 175 ml/d of either test milk was consumed. Fasting blood samples and 48 h-urine were collected at baseline and at the end of the intervention. Urine was divided into a pooled daytime and nighttime fraction. Multifactorial ANOVA was performed. RESULTS: Fermented milk independent of a supplement (n = 85) reduced the nocturnal excretion of deoxypyridinoline, a marker of bone resorption, from 11.73 +/- 0.54 before to 9.57 +/- 0.54 micromol/mol creatinine at the end of the intervention (P = 0.005). No effect was seen in the daytime fraction. Differences between the three milks (n = 28 resp. 29) were not significant. Fermented milk reduced bone alkaline phosphatase, a marker of bone formation, from 25.03 +/- 2.08 to 18.96 +/- 2.08 U/l, with no difference between these groups either. Fermented milk increased the nocturnal but not daytime urinary excretion of calcium and phosphorus. The effects on calcium and phosphorus excretion were mainly due to the group supplemented with Ca + ITF + CPP. CONCLUSION: Bedtime consumption of fermented milk reduced the nocturnal bone resorption by decelerating its turnover. Supplemented calcium from milk mineral had no additional effect unless the absorption enhancers ITF + CPP were added. A stimulated intestinal calcium absorption may be assumed, since urinary calcium excretion increased at a constant bone resorption.

Medium-sized peptides as built in carriers for biologically active compounds.

A growing number of oligopeptides of natural and/or synthetic origin have been described and considered as targeting structures for delivery bioactive compounds into various cell types. This review will outline the discovery of peptide sequences and the corresponding mid-sized oligopeptides with membrane translocating properties and also summarize de novo designed structures possessing similar features. Conjugates and chimera constructs derived from these sequences with covalently attached bioactive peptide, epitope, oligonucleotide, PNA, drug, reporter molecule will be reviewed. A brief note will refer to the present understanding on the uptake mechanism at the end of each section.

Caseinphosphopeptides in milk and fermented milk do not affect calcium metabolism acutely in postmenopausal women.

BACKGROUND: Caseinphosphopeptides (CPPs) are formed in food processing or during digestion in the gastrointestinal tract. CPPs prevent the formation of insoluble calcium salts; thus, the hypothesis is that CPPs increase the absorption of calcium. OBJECTIVE: We examined the effect of additional caseinphosphopeptides in milk and fermented milk on acute calcium metabolism by measuring intact PTH (iPTH), ionized calcium (iCa), total calcium (Ca) and phosphate (P) from serum, and 24-hour calcium from urine (U-Ca). METHODS: The study consisted of two separate parts, both applying a double-blind randomized crossover study with two interventions, in nine postmenopausal women. The acute effect on calcium metabolism was analysed by measuring iPTH, iCa, Ca and P from serum during the first six hours after the administration of the study milks. U-Ca was analysed 24 hours prior to the study and 0, 2, 4, 6, 12 and 24 hours after the administration of the study milks. The study included two parts, both consisting of two study days with a one-week washout period in between. In the first part the effect of control milk and CPP-enriched milk was measured. The second part evaluated the effect of fermentation by giving subjects milk or fermented milk, both enriched with CPPs. RESULTS: In the first part of the study there were no statistically significant differences in iPTH, iCa, Ca, P or U-Ca between the groups receiving control milk compared to CPP-containing milk. There was no difference in the AUC((0-6)) of iCa and iPTH. In the second part, fermentation did not affect calcium metabolism, when results from the CPP-enriched milk and CPP-enriched fermented milk groups were compared. CONCLUSION: One gram of caseinphosphopeptides does not affect calcium metabolism acutely in postmenopausal women.

Effect of the ratio of casein phosphopeptides to calcium (w/w) on passive calcium transport in the distal small intestine of rats.

OBJECTIVE: We investigated the influence of different ratios between casein phosphopeptides and calcium (CPP/Ca) in intestinal lumen on passive Ca transport across the distal small intestine of rats in vitro. METHODS: We measured the amounts of passive Ca absorbed across the everted distal small intestine of rats with the use of radioactive 45Ca as tracer. Four CPP/Ca ratios (5, 10, 15, and 20 w/w) and three mineral concentrations (4, 8, and 12 mmol/L) were tested on groups of six rats. RESULTS: We found a significant effect of the CPP/Ca ratio on mineral absorption (P < 0.001). The amounts of Ca absorbed increased from a ratio of 5 to a ratio of 15 and decreased with a ratio of 20, with a similar trend for every concentration tested. The more evident effect was found with the concentration of 12 mmol/L of Ca (P < 0.001 to 0.05). CONCLUSION: The positive effect of CPPs on passive Ca absorption seems to depend on the relative amounts of both species in intestinal lumen. In this study, the ratio 15 was identified as the most efficient at increasing mineral transport. This evidence might be considered before suggesting CPP as a Ca dietary integrator, although more studies in vitro and in vivo are needed.

Multisite phosphotyping of the ErbB-2 oncoprotein in human breast cancer.

BACKGROUND: Overexpression of the ErbB-2 (HER2/neu) receptor tyrosine kinase is one of the most common molecular changes in human cancer, but the functional significance of this phenotype remains uncertain. METHODS AND RESULTS: Using phosphorylation-specific antibodies recognizing different ErbB-2 functional states, we assessed the phosphorylation status of ErbB-2 in 102 human breast cancer specimens. Quantitative ErbB-2 immunoblotting intensity correlated directly with that of immunohistochemistry (r = 0.84). Widely varying phosphorylation profiles were evident in 65 ErbB-2-positive carcinomas, suggesting different ErbB-2 functions in different tumors. In a subset of patients for whom clinical data were obtainable, mortality trends were strongly associated with the quantitative signal intensities of ErbB-2 phosphoantibodies (P < or =.02), but not with those of conventional antibodies to ErbB-2 (P = .147), epidermal growth factor receptor (P = .44), or phosphotyrosine (P = .94). CONCLUSION: Although requiring corroboration in larger prospective clinical studies, these findings suggest that immunophenotyping using phosphorylation-specific antibodies may enable more accurate prediction of cancer behavior than is currently obtainable using conventional reagents.

The effect of high intakes of casein and casein phosphopeptide on calcium absorption in the rat.

The effect of the level or source of dietary protein or protein-derived peptides on Ca absorption is not well understood. We determined, therefore, the influence of habitual dietary casein level, meal casein and meal casein phosphopeptide (CPP) on Ca absorption in the rat. True fractional Ca absorption was investigated in male 7-week-old rats, Wistar strain, in three separate studies using a faecal 47Sc: 47Ca ratio method. In studies A and C, rats (n 8 per group) were fed on a purified diet containing 200 g casein/kg for 2 weeks. Rats were then given a 47Ca-labelled meal (10 g) containing (per kg) either 0, 100, 200, or 300 g casein (study A) or 0, 100, 200, 350 or 500 g CPP (study C). In study B, rats (n 24 per group) were fed on a purified diet containing (per kg) either 200, 350 or 500 g casein for 2 weeks. Each group was then further randomized into three groups (n 8 per group) and given a 47Ca-labelled meal (10 g of the same diet) containing (per kg) either 200, 350 or 500 g casein. Ca absorption from a meal was unaffected by increasing meal casein concentration from 0 to 300 g/kg (study A), but was increased with a meal casein content of 500 g/kg (study B). Fractional Ca absorption decreased with increasing usual dietary casein intake in the range 200-500 g/kg (study B), suggesting intestinal adaptation. Ca absorption was unaffected by inclusion of 100 g CPP/kg in a single meal but was significantly (P < 0.001) reduced by 200, 350 and 500 g CPP/kg meal, with no evident dose-relationship. Thus, while Ca absorption was enhanced by high-casein meals, the mechanism remains unclear.

Minimum effective dose of casein phosphopeptides (CPP) for enhancement of calcium absorption in growing rats.

We examined the effect of ingested casein phosphopeptides (CPP) on intestinal calcium (Ca) absorption and determined the minimum effective dose for enhancement of Ca absorption under conditions of marginal dietary Ca levels. Male Sprague-Dawley rats, divided upon weaning into five groups, were fed a control diet (isolated soyprotein, ISP; 20%) or a CPP diet (ISP + CPP; 20%, CPP/Ca: 0.1, 0.2, 0.35 and 1.0) for 4 weeks. All diets contained the same amounts of Ca (0.35%) and phosphorus (0.70%). The apparent Ca absorption, the retention of Ca, and the luminal soluble Ca content in the small intestine as determined at 4 weeks in the rats fed CPP diet with a weight ratio of CPP/Ca of more than 0.2 were significantly (p < 0.05) higher than those in the rats fed control diet. The wet weight, length and Ca content of the femur were not significantly different among the groups. These results indicate that the minimum effective dose of CPP for enhancement of Ca absorption is 0.7 g/kg or a weight ratio of CPP/Ca of 0.2 in the ISP diet and that CPP supplementation has the effect of significantly increasing Ca absorption at least under conditions of marginal dietary Ca levels.

Casein phosphopeptides improve zinc and calcium absorption from rice-based but not from whole-grain infant cereal.

BACKGROUND: Casein phosphopeptides (CPP) are phosphorus-rich peptide fragments of casein, assumed to contribute to the high bioavailability of calcium from milk. METHODS: The effect of casein phosphopeptides on calcium and zinc absorption from infant foods was investigated. Twenty-two men and women were given single test meals extrinsically labeled with Ca and Zn. Absorption was calculated from measurements on whole-body retention of the radioisotopes. Each subject was given either rice-based cereal (n = 11) or whole-grain cereal (n = 11) on three occasions together with 250 ml water and added 0, 1, and 2 g CPP in random order. One serving of rice-based cereal contained 481 mg Ca and 1.29 mg Zn; whole-grain cereal contained 541 mg Ca and 1.77 mg Zn. One and 2 g of CPP contributed with additional 69 and 138 mg Ca, respectively. RESULTS: From rice-based cereal, fractional calcium absorption was not affected by CPP addition (mean +/- SD): 16.0 +/- 4.0% (no CPP), 17.6 +/- 4.5% (1 g CPP), and 15.8 +/- 4.3% (2 g CPP), while the total quantity of calcium absorbed was significantly improved: 7 +/- 19 mg, 97 +/- 25 mg, and 98 +/- 26 mg, respectively (p = 0.0004). Fractional zinc absorption as well as total quantity of zinc absorbed were increased with addition of CPP: 19.4 +/- 9.0% (0.25 +/- 0.12 mg), 25.2 +/- 7.5% (0.33 +/- 0.10 mg) and 23.9 +/- 5.4% (0.31 +/- 0.07 mg) at the three CPP levels (p = 0.04). From whole-grain cereal, CPP had no effect on the percentage or actual quantity of calcium absorbed: 17.0 +/- 3.2% (92 +/- 18 mg), 17.2 +/- 4.5% (105 +/- 27 mg), and 15.0 +/- 4.6% (102 +/- 31 mg), respectively. Zinc absorption was also not influenced by CPP: 16.0 +/- 5.1% (0.28 +/- 0.09 mg), 15.3 +/- 3.1% (0.27 +/- 0.06 mg) and 18.1 +/- 4.4% (0.32 +/- 0.08 mg), respectively. CONCLUSIONS: CPP addition improved calcium and zinc absorption from rice-based cereal, while no effect was seen from whole-grain cereal.

Dietary casein phosphopeptides prevent bone loss in aged ovariectomized rats.

The effect of dietary Ca-bound casein phosphopeptides (CaCPP) on the bones of aged ovariectomized (OVX) rats was studied as a model for post-menopausal bone loss. Three groups of ovariectomized rats were fed a control diet or one of two experimental diets, and one group of sham-operated rats (SHAM) was fed the control diet. The experimental diets contained 0.5% Ca and 0.4% P. In one diet, CaCPP was the sole source of calcium and provided 62.5% of dietary phosphorus (CaCPP diet). In the other, Ca-free CPP provided 100% of dietary phosphorus (Ca-free CPP diet). In the control diet, CaCO3 and KH2PO4 were used. During a 17-wk feeding period, there was little change in femoral bone mineral densities (BMD) of ovariectomized rats fed CaCPP and Ca-free CPP, or in the SHAM rats fed the control diet, whereas the bone mineral densities in the control ovariectomized rats decreased with time. Some of the segmental bone mineral densities of the excised femurs from the rats fed CaCPP were significantly higher than those from the control ovariectomized rats, but the values of the Ca-free CPP group were similar to those of the control ovariectomized rats. In the Ca-free CPP group, the discrepancy in bone mineral densities obtained between in vivo results and excised specimens might have been the result of a loss in bone mass due to their significant loss in body weight. There were no significant differences in serum inorganic phosphorus, alkaline phosphatase activity, osteocalcin or 1 alpha-25-dihydroxycholecalciferol concentrations among the ovariectomized groups. In the CaCPP and Ca-free CPP groups, urinary phosphorus excretion decreased and urinary calcium excretion increased significantly with time. The inhibitory effect on bone loss in aged ovariectomized rats could be due to the effects of dietary CaCPP on phosphorus and calcium metabolism.

Caseinphosphopeptides (CPP) in feces and contents in digestive tract of rats fed casein and CPP preparations.

A part of caseinphosphopeptides (CPP) formed during the digestion of casein in the small intestine of rats fed casein was not hydrolyzed in the digestive tract, but was excreted into the feces. Amino acid compositions of CPP fraction of feces for wk 1 and 2 were almost identical. The residual CPP in the feces expressed by the rate of bound phosphoserine in the CPP fraction of feces to bound phosphoserine ingested (phosphoserine-CPP/phosphoserine-ingested rate) in the ileum was the highest 4 h after the start of feeding of casein but decreased significantly after 10 h. No significant difference was observed in the rats of contents in jejunum, cecum, and colon between 4 h and 10 h after the start of feeding. No significant difference was observed in the phosphoserine-CPP/phosphoserine-ingested rate in contents of any part of the digestive tract between 50% casein and 50% CPP I (a commercial CPP product with nearly the same amino acid composition as that of casein) 4 h and 10 h after the start of feeding, except for the cecum 4 h after the start of feeding. No significant difference in phosphoserine-CPP/phosphoserine-ingested rate was observed in the contents of any part of digestive tract between the groups fed 50% casein and 5% CPP III +45% soybean protein isolate (SPI) 4 h or 10 h after the start of feeding (CPP III is a commercial CPP product containing nearly 8 times the concentration of phosphoserine as CPP I).(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase.

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.

Effect of casein, casein phosphopeptides and calcium intake on ileal 45Ca disappearance and temporal systolic blood pressure in spontaneously hypertensive rats.

Paracellular 45Ca absorption and temporal systolic blood pressure (SBP) measurements were recorded in spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats fed on casein (C) and soya-bean-protein isolate (S) diets, containing 20 (H), 5 (H) and 0.5 (L) g Ca/kg. Similar measurements were also taken in SHR rats only fed on C-M and S-M diets supplemented with 30 g caseinophosphopeptides (CPP)/kg. Absorption of 45Ca from the ileal loop was equivalent in both SHR and WKY animals and largely affected by the level of dietary Ca. In addition, animals fed on C diets exhibited significantly (P < 0.05) greater ileal absorption of 45Ca compared with S-fed animals. This result was attributed to the presence of CPP and a greater (P < 0.05) proportion of soluble 45Ca in the contents of the ileum. Animals fed on S diets supplemented with CPP confirmed this finding. The SBP of SHR rats was higher (P < 0.01) than WKY controls after 9-10 weeks of age. The temporal pattern of observed hypertension was independent of dietary influence in the SHR. The severity of hypertension in SHR rats was affected only by dietary Ca deficiency, and not by Ca supplementation or CPP enhancement of Ca bioavailability. These findings suggest that tryptic digestion products of casein in milk can enhance Ca bioavailability by increasing Ca solubility; however, this action had no effect in reducing hypertension in SHR.

Intestinal absorption of calcium in rats given diets containing casein or amino acid mixture: the role of casein phosphopeptides.

1. In an attempt to investigate calcium absorption in the rat during the postprandial period, with the least alteration of the physical environment, the undisturbed small intestine was ligated in situ 2.5 or 3.0 h after ingestion of a diet containing 200 g casein/kg or an equivalent amino acid mixture, or 925 g casein/kg. Estimation of Ca absorption was made by comparing the amount of soluble 40Ca or 45Ca in the contents of segments from the rats receiving 45Ca by intubation 30 min after withdrawal of food, ligated after a further 30 min, then killed after 0 or 30 min. 2. Under conditions such that the estimated amount of a marker, polyethylene glycol, in segments ligated in a defined position was little changed in rats killed 30 min apart, the difference in the amount of soluble 40Ca was much higher in the rats fed on the basal diet containing 200 g casein/kg than in other groups. 3. This specific effect on Ca absorption, particularly in the distal portion of the small intestine, could be seen also after 45Ca was directly injected into ligated segments in situ. The amount of 45Ca in the portal blood 15 min after injection of the label was also highest in the rats given the basal diet. 4. The results were in agreement with our previous findings that the formation and accumulation of casein phosphopeptides causes an increase in the amount of soluble Ca in the distal small intestine.