Influence of Dairy Products on Bioavailability of Zinc from Other Food Products: A Review of Complementarity at a Meal Level.

In this paper, we reviewed the role of dairy products in dietary zinc absorption. Dairy products can have a reasonable contribution for dietary zinc intake in Western diets, where dairy consumption is high. However, the co-ingestion of dairy products can also improve zinc absorption from other food products. Such improvements have been observed when dairy products (e.g., milk or yoghurt) were ingested together with food such as rice, tortillas or bread products, all of which are considered to be high-phytate foods with low inherent zinc absorption. For foods low in phytate, the co-ingestion of dairy products did not improve zinc absorption. Improved zinc absorption of zinc from high-phytate foods following co-ingestion with dairy products may be related to the beneficial effects of the citrate and phosphopeptides present in dairy products. Considering that the main dietary zinc sources in areas in the world where zinc deficiency is most prevalent are typically high in phytate, the inclusion of dairy products in meals may be a viable dietary strategy to improve zinc absorption.

Vasorelaxing cell permeant phosphopeptide mimetics for subarachnoid hemorrhage.

Subarachnoid hemorrhage (SAH) due to rupture of an intracranial aneurysm leads to vasospasm resulting in delayed cerebral ischemia. Therapeutic options are currently limited to hemodynamic optimization and nimodipine, which have marginal clinical efficacy. Nitric oxide (NO) modulates cerebral blood flow through activation of the cGMP-Protein Kinase G (PKG) pathway. Our hypothesis is that SAH results in downregulation of signaling components in the NO-PKG pathway which could explain why treatments for vasospasm targeting this pathway lack efficacy and that treatment with a cell permeant phosphopeptide mimetic of downstream effector prevents delayed vasospasm after SAH. Using a rat endovascular perforation model, reduced levels of NO-PKG pathway molecules were confirmed. Additionally, it was determined that expression and phosphorylation of a PKG substrate: Vasodilator-stimulated phosphoprotein (VASP) was downregulated. A family of cell permeant phosphomimetic of VASP (VP) was wasdesigned and shown to have vasorelaxing property that is synergistic with nimodipine in intact vascular tissuesex vivo. Hence, treatment targeting the downstream effector of the NO signaling pathway, VASP, may bypass receptors and signaling elements leading to vasorelaxation and that treatment with VP can be explored as a therapeutic strategy for SAH induced vasospasm and ameliorate neurological deficits.

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.

Enhanced skin penetration of P20 phosphopeptide using protein transduction domains.

Protein transduction domains (PTDs) were recently demonstrated to increase the penetration of the model peptide P20 when the PTD and P20 were covalently attached. Here, we evaluated whether non-covalently linked PTDs were capable of increasing the skin penetration of P20. Two different PTDs were studied: YARA and WLR. Porcine ear skin mounted in a Franz diffusion cell was used to assess the penetration of P20 in the stratum corneum (SC) and viable skin (VS); VS consists of dermis and epidermis without SC. The transdermal delivery of P20 was also assessed. At 1mM, YARA promoted a 2.33-fold increase in the retention of P20 in the SC but did not significantly increase the amount of P20 that reached VS. WLR significantly increased (2.88-fold) the penetration of P20 in VS. Compared to the non-attached form, the covalently linked WLR fragment was two times more effective in promoting the penetration of P20 into VS. None of the PTDs promoted transdermal delivery of P20 at 4h post-application. It was concluded that selected non-covalently linked PTDs can be used as a penetration enhancer, but greater skin penetration efficiency can be achieved by covalently binding the PTD to the therapeutic agent.

Fish-bone peptide increases calcium solubility and bioavailability in ovariectomised rats.

Fish-bone peptides (FBP) with a high affinity to Ca were isolated using hydroxyapatite affinity chromatography, and FBP II with a high ratio of phosphopeptide was fractionated in the range of molecular weight 5.0-1.0 kDa by ultramembrane filtration. In vitro study elucidated that FBP II could inhibit the formation of insoluble Ca salts in neutral pH. In vivo effects of FBP II on Ca bioavailability were further examined in the ovariectomised rat. During the experimental period, Ca retention was increased and loss of bone mineral was decreased by FBP II supplementation in ovariectomised rats. After the low-Ca diet, the FBP II diet, including both normal level of Ca and vitamin D, significantly decreased Ca loss in faeces and increased Ca retention compared with the control diet. The levels of femoral total Ca, bone mineral density, and strength were also significantly increased by the FBP II diet to levels similar to those of the casein phosphopeptide diet group (no difference; P>0.05). In the present study, the results proved the beneficial effects of fish-meal in preventing Ca deficiency due to increased Ca bioavailability by FBP intake.

A cell-permeable phospholipase Cgamma1-binding peptide transduces neurons and impairs long-term spatial memory.

Growth factor-mediated signaling has emerged as an essential component of memory formation. In this study, we used a phospholipase C gamma 1 (PLCgamma1) binding, cell-penetrating peptide to sequester PLCgamma1 away from its target, the phosphotyrosine residues within the activated growth factor receptor. Peptides appear to transduce neurons but not astrocytes or oligodendrocytes. The presence of the peptides in the hippocampus during training in the Morris water maze significantly impaired long-term memory, but not memory acquisition. These results, along with previous studies on extracellular signal-regulated kinase (ERK) and phosphoinositide-3 kinase (PI3K), implicate all three key growth factor receptor-activated intracellular signaling pathways in memory storage.

Detection of caseinophosphopeptides in the distal ileostomy fluid of human subjects.

Caseinophosphopeptides (CPP) were detected for the first time in ileostomy fluid, collected at 2 h intervals for 10 h post milk and CPP ingestion, from human volunteers with an ileostomy. The level of CPP present in ileostomy fluid obtained from milk-fed volunteers was markedly higher than that from volunteers fed with selected CPP preparations. The findings are based on HPLC analysis in combination with peptide-bound P determination, thin-layer electrophoresis and amino acid analysis, together with ELISA studies using polyclonal antibodies raised against a set of CPP to detect immunoreactive CPP in ileostomy fluid. These procedures allowed the detection of nm concentrations of CPP. CPP, which can be released during intestinal digestion, may function as bioactive constituents and carriers for different minerals, especially Ca, and may be used as ingredients in functional foods or pharmaceutical preparations.

Understanding the cellular uptake of phosphopeptides.

Phosphopeptide-cellular uptake has been studied with a unique combination of tools designed to quantitate this phenomena and to understand properties that contribute to transmembrane penetration. High-affinity src-homology domain (SH2) hexapeptides for the phosphatidyl inositol 3-kinase system were used to judge cell penetration using red blood cells--a model system for the study of transmembrane cellular uptake. Hexapeptides without phosphate groups and devoid of charged residues poorly entered cells. N-terminal modification with bulky hydrophobic groups enhanced partitioning into octanol, an index of hydrophobicity, and allowed certain non-phosphorylated peptides to pass into red cells. However, tyrosine phosphorylation of hexapeptides markedly decreased octanol-water partitioning and completely eliminated cellular uptake. Inclusion of ion-pairing agents that masked the phosphate hydrophilic character enabled partitioning of phosphopeptides into octanol and achieved cellular uptake. This effect was demonstrated using fluorescent derivatives of phosphopeptides and CV1 cells in culture. The results validate the concept of facilitating cell entry by charge masking and open the way to future refinements of this principle. Various penetration techniques are compared and discussed in the context of maximizing cellular viability.

Bioavailability of milk micellar calcium phosphate-phosphopeptide complex in rats.

We evaluated the bioavailability of two types of calcium from milk in two experiments. One was a micellar calcium phosphate-phosphopeptide (MCP-PP) complex in which the chemical form was similar to the original form of milk, and the other was a commercial whey calcium in which the chemical form was different from that of milk. In experiment 1, the calcium absorption, bone mineral density, and bone strength were examined when growing female rats were fed either MCP-PP complex or whey calcium as the sole source of calcium for 46 d. In experiment 2, the calcium solubility in the small intestine was measured when female rats were meal-fed either MCP-PP complex or whey calcium. The apparent calcium absorption rate in both groups decreased time-dependently during the experimental period, but the time-dependent change in the apparent calcium absorption rate was statistically different. It decreased more slowly in rats fed the MCP-PP diet than in rats fed the whey calcium diet. The bone mineral density of the femur in rats fed the MCP-PP diet was significantly higher than that of the rats fed the whey calcium diet. The bone strength (breaking force and energy) of the femur in rats fed the MCP-PP diet was higher than in the rats fed the whey calcium diet. The amount of soluble calcium in the small intestinal contents in rats at 2.5 h after ingestion of the MCP-PP diet was approximately three times higher than that found in rats fed the whey calcium diet. These results indicate that the calcium bioavailability of MCP-PP complex is higher than that of whey calcium, and this difference is due in part to the solubility in the intestine.

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)

N-Phosphonomethyl dipeptides and their phosphonate prodrugs, a new generation of neutral endopeptidase (NEP, EC 3.4.24.11) inhibitors.

Inhibitors of the zinc protease neutral endopeptidase (NEP, EC 3.4.24.11) offer significant therapeutic interest as antihypertensives due to their ability to potentiate the biological action of the circulating natriuretic hormone ANF (atrial natriuretic factor). N-Phosphonomethyl dipeptides bearing a central (4-phenyl)phenylalanine residue have been designed to exert potent and selective NEP inhibition. In particular, (S)-3-[N-[2- [(phosphonomethyl)amino]-3-(4-biphenylyl)propionyl]amino]propionic acid (10a) (CGS 24592) displayed high inhibitory potency in vitro (IC50 = 1.9 +/- 0.1 nM) and a long plasma half-life in rats but lacked oral bioavailability. This drawback was overcome by using esterase-sensitive (acyloxy)alkyl phosphonates. More remarkable, several diaryl phosphonate derivatives of 10a also performed as effective prodrugs. Specifically, the structurally simple diphenyl phosphonate 18 (CGS 25462) induced potent inhibition of NEP ex vivo for at least 8 h after oral administration to rats (30 mg/kg). Its antihypertensive effect was demonstrated in DOCA-salt rats. At 30 mg/kg orally, 18 caused a significant reduction in mean arterial pressure measuring -35 +/- 7 mmHg at 5-h postdosing. The alpha-aminomethyl phosphonate 18 represents a new generation of selective NEP inhibitors that combine high potency, long duration of action, and oral bioavailability. Therefore, it holds promise as a novel therapeutic agent for the treatment of human hypertension and congestive heart failure.