Preparation, characterization, and bioavailability evaluation of antioxidant phosvitin peptide-ferrous complex.

BACKGROUND: Iron deficiency anemia (IDA) is one of the commonest global nutritional deficiency diseases, and the low bioavailability of iron is a key contributing factor. The peptide-iron complex could be used as a novel iron supplement to improve iron bioavailability. RESULTS: In this study, antioxidant low molecular weight (<3 kDa) phosvitin peptide (named PP-4) was separated to prepare a phosvitin peptide-ferrous complex (named PP-4-Fe); then the structural conformation of PP-4-Fe was characterized and its bioavailability by in vitro digestion was evaluated. The results showed that PP-4 had good ferrous-binding activity with 96.14 ± 2.86 µg Fe2+ mg-1 , and had a strong antioxidant effect with 995.61 ± 79.75 µmol TE mg-1 in 2,2'-azinobis'3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 62.3 ± 3.95 µmol FeSO4 mg-1 in ferric ion reducing antioxidant power (FRAP). After ferrous binding, the FRAP activity of PP-4-Fe, enhanced by 1.8 times, formed a more ordered structure with an increase in α-helix and decrease in γ-random coil. The ferrous binding sites of PP-4 involved were the amino, carboxyl, imidazole, and phosphate groups. The PP-4-Fe complex displayed excellent gastrointestinal stability and antioxidant effects during digestion. The iron dialysis percentage of PP-4-Fe was 74.59% ± 0.68%, and increased to 81.10% ± 0.89% with the addition of 0.25 times vitamin C (VC). This indicated that PP-4-Fe displayed excellent bioavailability and VC in sufficient quantities had a synergistic effect on improving bioavailability. CONCLUSIONS: This study demonstrated that antioxidant phosvitin peptide was an efficient delivery system to protect ferrous ions and suggested that the phosvitin peptide-ferrous complex has strong potential as a ferrous supplement. © 2023 Society of Chemical Industry.

In vitro and in vivo wound healing-promoting activities of phosvitin-derived peptide Pt5-1c.

The closure of skin wounds is indispensable for resistance against pathogens, and fibroblast plays a critical role in skin wound healing. Our previous study demonstrates that the phosvitin-derived small peptide Pt5-1c not only possesses broad-spectrum antimicrobial activity but also exhibits synergistic effect and antibiofilm activity with traditional antibiotics against bacteria, including multi-drug resistant (MDR) strains. Here we provided the first evidence that Pt5-1c promoted the wound closure of surrogate scratch "wounds" of fibroblasts in vitro, and speeded up the healing and re-epithelialization of murine dermal wounds in vivo. We also showed that Pt5-1c activated migration of fibroblasts via a combined action of inducing migratory phenotype and trans-activating epidermal growth factor receptor (EGFR). Moreover, Pt5-1c accelerated attachment and proliferation of fibroblasts in vitro. Interestingly, Pt5-1c was able to promote collagen contraction through activation/differentiation of fibroblasts into myofibroblasts. These data together suggest that Pt5-1c is a promising candidate with therapeutic potential to promote wound healing.

Phosvitin phosphopeptides produced by pressurized hea-trypsin hydrolysis promote the differentiation and mineralization of MC3T3-E1 cells via the OPG/RANKL signaling pathways.

Phosvitin (PV) from egg yolk is an excellent substrate for the production of phosphopeptides, which have a strong calcium chelating capacity and promoting calcium absorption and bone mineralization. This study investigated the effect of PV hydrolysates produced using a effective preparation method (high temperature (121°C) and mild pressure (0.1 MPa), HTMP) or HTMP pretreatment and trypsin hydrolysis combination (HTMP-PV18) on the physiology of an osteoblast MC3T3-E1 cells line. The proliferation, apoptosis, and differentiation of MC3T3-E1 cells were analyzed using the CCK-8, flow cytometry, and RT-PCR reactions, respectively. Both the HTMP-PV and HTMP-PV18 increased the proliferation, and inhibited the apoptosis of MC3T3-E1 cells significantly. The HTMP-PV increased the proliferation of MC3T3-E1 cells by 147.12 ± 2.11% and the HTMP-PV18 by 136.43 ± 4.51%. In addition, the HTMP-PV and HTMP-PV18 effectively promoted the expression of genes related to the OPG/RANKL signaling channel during cell differentiation. This indicated that both the HTMP-PV and HTMP-PV18 have the potential to promote bone mineralization by improving the proliferation and differentiation of osteoblastic cells.

Phosvitin-wheat gluten complex catalyzed by transglutaminase in the presence of Na2SO3: Formation, cross-link behavior and emulsifying properties.

Phosvitin (PSV) is considered as a good emulsifier, although it has a low proportion of hydrophobic regions and steric hindrance. Wheat gluten (WG) possesses excellent hydrophobicity and macromolecular network structure. In this work, WG was subjected to a series of Na2SO3 solution, followed by cross-linking with PSV under transglutaminase (TGase) catalyzation. The results showed that Na2SO3 could break disulfide bonds of WG and increase its solubility from 7.33% to 42.82% with 1200 mg/L of Na2SO3. Correspondingly, the cross-linking degree was significantly enhanced. Compared to PSV, the cross-linked PSV-WG exhibited a higher surface hydrophobicity and thermal stability, with a lower zeta potential and apparent viscosity. The emulsifying activity of PSV-WG reached 17.42, 20.63 and 20.28 m2/g with Na2SO3 concentration of 300, 600 and 900 mg/L, which were all higher than that of PSV (15.19 m2/g). This work provided a novel strategy to elevate emulsifying properties of PSV by cross-link reaction.

How Do Actinyls Interact with Hyperphosphorylated Yolk Protein Phosvitin?

The development of the nuclear industry has raised multiple questions about its impact on the biotope and humans. Proteins are key biomolecules in cell machinery and essential in deciphering toxicological processes. Phosvitin was chosen as a relevant model for phosphorylated proteins because of its important role as an iron, calcium, and magnesium storage protein in egg yolk. A multitechnique spectroscopic investigation was performed to reveal the coordination geometry of two oxocations of the actinide family (actinyl UVI , NpV ) in speciation with phosvitin. IR spectroscopy revealed phosphoryl groups as the main functional groups interacting with UVI . This was confirmed through laser luminescence spectroscopy (U) and UV/Vis absorption spectroscopy (Np). For UVI , X-ray absorption spectroscopy at the LIII edge revealed a small contribution of bidentate binding present, along with predominantly monodentate binding of phosphoryl groups; for NpV , uniquely bidentate binding was revealed. As a perspective to this work, X-ray absorption spectroscopy speciation of UVI and NpV in the extracted yolk of living eggs of the dogfish Scyliorhinus canicula was determined; this corroborated the binding of phosphorous together with a reduction of the actinyl moiety. Such data are essential to pinpoint the mechanisms of heavy metals (actinyls) accumulation and toxicity in oviparous organisms, and therefore, contribute to a shift from descriptive approaches to predictive toxicology.

Effects of egg phosvitin on mucosal transcriptional profiles and luminal microbiota composition in murine colon.

Egg phosvitin has been traditionally considered as a nutritionally negative protein for its low digestibility in the gastrointestinal tract. Whether it could exert nutritional functions in the large intestine is not known. In this work, the influence of phosvitin on luminal microbiota composition and mucosal transcriptome was investigated with young (3-week) and adult (8-week) mouse models. In young groups, phosvitin mainly suppressed genes related to lipid metabolism, whereas the regulated genes in adult individuals encompassed various biological processes, such as carbohydrate metabolism, sigestive system and others. Phosvitin increased the proportion of Bifidobacterium in the young group, and reduced amounts of pathogenic microbes in the adult group, including Helicobacter and Mucispirillum. There was a close relationship between gene expression changes and abundance of bacteria. Finally, phosvitin reduced the ammonia concentrations in feces for both young and adult groups. These findings suggested that phosvitin modified the large intestinal ecosystem, exhibiting potentially beneficial effects on gut health.

The Golgi 'casein kinase' Fam20C is a genuine 'phosvitin kinase' and phosphorylates polyserine stretches devoid of the canonical consensus.

Egg yolk phosvitins, generated through the fragmentation of vitellogenins (VTGs), are among the most heavily phosphorylated proteins ever described. Despite the early discovery in 1900 that chicken phosvitin is a phosphoprotein and its subsequent employment as an artificial substrate for a number of protein kinases, the identity of the enzyme(s) responsible for its phosphorylation remained a matter of conjecture until present. Here, we provide evidence that phosvitin phosphorylation is catalyzed by a family with sequence similarity 20, member C (Fam20C), an atypical protein kinase recently identified as the genuine casein kinase and responsible for the phosphorylation of many other secreted proteins at residues specified by the S-x-E/pS consensus. Such a conclusion is grounded on the following observations: (a) the levels of Fam20C and phosphorylated VTG rise in parallel upon treatment of zebrafish with oestrogens; (b) zebrafish phosvitin is readily phosphorylated upon coexpression in U2OS cells with Fam20C, but not with its catalytically inactive mutant; (c) a peptide reproducing a stretch of 12 serines, which are phosphorylated in chicken phosvitin despite lacking the C-terminal priming motif S-x-E, is efficiently phosphorylated by both recombinant and native Fam20C. The last finding expands the repertoire of potential targets of Fam20C to include several proteins known to harbor (p-Ser)n clusters not specified by any known kinase consensus.

Functions of Vitellogenin in Eggs.

Our understanding of the functions of vitellogenin (Vtg) in reproduction has undergone an evolutionary transformation over the past decade. Primarily, Vtg was regarded as a female-specific reproductive protein, which is cleaved into yolk proteins including phosvitin (Pv) and lipovitellin (Lv), stored in eggs, providing the nutrients for early embryos. Recently, Vtg has been shown to be an immunocomponent factor capable of protecting the host against the attack by microbes including bacteria and viruses. Moreover, Pv and Lv that both are proteolytically cleaved products of maternal Vtg, as well as Pv-derived small peptides, all display an antibacterial role in developing embryos. In addition, both Vtg and yolk protein Pv possess antioxidant activity capable of protecting cells from damage by free radicals. Collectively, these data indicate that Vtg, in addition to being involved in yolk protein formation, also plays non-nutritional roles via functioning as immune-relevant molecules and antioxidant reagents.

Role of phosphorylation of phosvitin in the phase transformation of mineralization.

Phosvitin is a unique highly phosphorylated protein that plays a role in the regulation of mineralization. This study investigated the role of phosphorylation of phosvitin in the phase transformation of calcium phosphate in the mineralization solution. Partially dephosphorylated phosvitins (T1, T2, T3 and T4) were prepared with 2.98, 19.46, 43.39 and 71.07% of phosphate released from native phosvitin, respectively. And their effect on regulating the phase transformation was investigated, the characterization and composition analysis was performed by circular dichroism, Fourier transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy. Results showed that phosvitin in the acceleration effect of phase transformation was dose-concentration effect by pH-stat titration. With lower dephosphorylation degree of phosvitin (<20%), the acceleration effect was weaker than native phosvitin, since phosphorylation and random structure of phosvitin were reduced. However, with higher dephosphorylation degree of phosvitin (>40%), the acceleration effect was enhanced compared to native phosvitin, in which the ß-sheet structure was increased and phosvitin was partially hydrolyzed to phosphopeptides. The acceleration effect of phase transformation was as follows: T4>T3>phosvitin>T1>T2> Control. This study clearly demonstrated that phosphorylation of phosvitin played an important role in the regulation of mineralization.

From phosphoproteins to phosphoproteomes: a historical account.

The first phosphoprotein (casein) was discovered in 1883, yet the enzyme responsible for its phosphorylation was identified only 130 years later, in 2012. In the intervening time, especially in the last decades of the 1900s, it became evident that, far from being an oddity, phosphorylation affects the majority of eukaryotic proteins during their lifespan, and that this reaction is catalysed by the members of a large family of protein kinases, susceptible to a variety of stimuli controlling nearly every aspect of life and death. The aim of this review is to present a historical account of the main steps of this spectacular revolution, which transformed our conception of a biochemical reaction originally held as a sporadic curiosity into the master mechanism governing cell regulation, and, if it is perturbed, causing cell dysregulation.

Zebrafish phosvitin is an antioxidant with non-cytotoxic activity.

Antioxidants, or anti-oxidant agents, have attracted a great deal of attention in recent years because of their roles in prevention of chronic diseases and utilization as preservatives in food and cosmetics. In this study, we clearly demonstrated that zebrafish recombinant phosvitin (rPv) is an antioxidant agent capable of inhibiting the oxidation of the linoleic acid, and scavenging the 2,2-diphenyl-1-picrylhydrazyl radical. We also showed that zebrafish rPv is a cellular antioxidant capable of protecting radical-mediated oxidation of cellular biomolecules. Importantly, zebrafish rPv is non-cytotoxic to murine macrophage RAW264.7 cells. It is the first report that showed the antioxidant activities of Pv in fishes, suggesting that zebrafish Pv can be an important antioxidant, which can be used as preservatives in food and cosmetics and even as supplementary mediator in different diseased states.

Changes in the protein secondary structure of hen's egg yolk determined by Fourier transform infrared spectroscopy during the first eight days of incubation.

In this study, incubation-induced alterations in the protein secondary structures of egg yolk and its major fractions (granules, plasma, and low-density lipoproteins [LDL]) were monitored during the first 8 d of embryogenesis using Fourier transform infrared spectroscopy (FTIR) and isoelectric focusing (IEF). Two factors potentially connected with egg yolk protein secondary structure changes were evaluated, i.e., the pH value of incubated egg yolk, and phosvitin, an important egg yolk protein assumed to play an important role in hematopoiesis as the iron carrier during early embryogenesis. However, neither the significant increase in pH value (6.07 to 6.92) of egg yolk during incubation of fertilized eggs, nor the release of iron from phosvitin were found to be directly related to the changes in protein secondary structure in egg yolk and its fractions. FTIR showed that the protein conformation in whole egg yolk, granules, and LDL was stable during incubation, but separate evaluation of the plasma fraction revealed considerable changes in secondary structure. However, it is unlikely that these changes were provoked by structure changes of the proteins originally present in plasma; instead, the physiological influx of albumen into the yolk sac was expected to play an important role in the protein modifications of egg yolk, as was shown both by FTIR and IEF of the water-soluble egg yolk proteins. Moreover, FTIR was used to determine the naturally occurring proportions (%) of the secondary structure elements in egg yolk and its 3 fractions on d 0 of incubation. The granules fraction mainly consisted of a mixture of inter- and intramolecular ß-sheets (57.04%±0.39%). The plasma fraction was found to consist mainly of α-helices (43.23%±0.27%), whereas LDL was composed almost exclusively of intramolecular ß-sheets (67.36%±0.56%) or ß-turns, or both. On the other hand, whole egg yolk was mainly composed of intermolecular ß-sheets (39.77%±0.48%), potentially indicating molecular interchanges between the individual fractions.

Phosvitin phosphorus is involved in chicken embryo bone formation through dephosphorylation.

The aim of this study was to investigate the role of phosvitin in bone formation in chicken embryos. The yolk P content, P/N ratio and secondary structure of phosvitin, alkaline phosphatase (ALP) activity of the tibia, and body length were determined during incubation. A high correlation was found between the phosphate group content of phosvitin and both secondary structure and bone metabolism (ALP activity in the tibia, body length). The ALP activity and body length growth slightly lagged behind changes in the P/N ratio and the secondary structure of phosvitin. The phosphate content of phosvitin decreased, the γ-random coil and ß-turn gradually transformed into α-helixes, and the secondary structure of protein tended to become more orderly; these changes mainly occurred on d 13 to 16. Bone formation of the chicken embryos occurred primarily on d 14 to 18, whereas ALP activity and body length growth increased substantially. The results indicate that phosvitin P is involved in chicken embryo bone formation through dephosphorylation.

Degradation of M(r) 25,000 protein by cathepsin L-like protease in Xenopus laevis oocytes.

A phosphorylated protein with a molecular mass of 25,000 (pp25) is involved in Xenopus laevis vitellogenin B1 and partially overlaps with phosvitin and lipovitellin 2. The protease responsible for pp25 degradation was studied in vitro since this occurs during embryogenesis. Initially, a protease thought to be a contaminant of the purified pp25 preparation was analyzed and an antipain-sensitive protease presumed to be involved. When commercially available proteases were examined, pp25 was not degraded by calpain I or 20S proteasome, but it was degraded by cathepsin L in vitro. A survey of the protease responsible for pp25 degradation in the cytoplasm of Xenopus oocytes found partially purified pp25 was degraded in partly antipain-sensitive manner. These results suggest that an antipain-sensitive protease or cathepsin L (or a related protease) is a candidate for pp25 degradation.

Isolation, purification and characterization of the egg-yolk proteins from the oocytes of the indian freshwater murrel, Channa punctatus (Bloch).

In oviparous organisms, yolk accumulation in the oocytes is critical and indispensable for the development of the newly hatched young ones. In fish and many other oviparous vertebrates, the major constituents of the egg-yolk are synthesized as a precursor in the liver. The precursor is transported to the oocyte for uptake and cleaved into major yolk proteins lipovitellin, phosvitin and beta'-components. The eggs of Channa punctatus are pelagic, have large oil globule and exceptionally high lipid content. Lipovitellin was isolated by single step gel filtration chromatography on Sepharose 6B. Purified native lipovitellin showed immunological reactivity with vitellogenin antiserum. Phosvitin isolated by phenol extraction method could not be visualized with routine protein staining methods, whereas incorporation of trivalent ions in the coomassie brilliant blue stained phosvitin. It was characterized by in vivo labeling of egg-yolk proteins with 32P. The molecular mass of murrel phosvitin was less than 14,000 kDa.

Novel bioactivity of phosvitin in connective tissue and bone organogenesis revealed by live calvarial bone organ culture models.

Egg yolk phosvitin is one of the most highly phosphorylated extracellular matrix proteins known in nature with unique physico-chemical properties deemed to be critical during ex-vivo egg embryo development. We have utilized our unique live mouse calvarial bone organ culture models under conditions which dissociates the two bone remodeling stages, viz., resorption by osteoclasts and formation by osteoblasts, to highlight important and to date unknown critical biological functions of egg phosvitin. In our resorption model live bone cultures were grown in the absence of ascorbate and were stimulated by parathyroid hormone (PTH) to undergo rapid osteoclast formation/differentiation with bone resorption. In this resorption model native phosvitin potently inhibited PTH-induced osteoclastic bone resorption with simultaneous new osteoid/bone formation in the absence of ascorbate (vitamin C). These surprising and critical observations were extended using the bone formation model in the absence of ascorbate and in the presence of phosvitin which supported the above results. The results were corroborated by analyses for calcium release or uptake, tartrate-resistant acid phosphatase activity (marker for osteoclasts), alkaline phosphatase activity (marker for osteoblasts), collagen and hydroxyproline composition, and histological and quantitative histomorphometric evaluations. The data revealed that the discovered bioactivity of phosvitin mirrors that of ascorbate during collagen synthesis and the formation of new osteoid/bone. Complementing those studies use of the synthetic collagen peptide analog and cultured calvarial osteoblasts in conjunction with mass spectrometric analysis provided results that augmented the bone organ culture work and confirmed the capacity of phosvitin to stimulate differentiation of osteoblasts, collagen synthesis, hydroxyproline formation, and biomineralization. There are striking implications and interrelationships of this affect that relates to the evolutionary inactivation of the gene of an enzyme L-gulono-γ-lactone oxidase, which is involved in the final step of ascorbate biosynthesis, in many vertebrate species including passeriform birds, reptiles and teleost fish whose egg yolk contain phosvitin. These represent examples of how developing ex-vivo embryos of such species can achieve connective tissue and skeletal system formation in the absence of ascorbate.

Topographical distribution of phosphorylation sites of phosvitins by mass spectrometry.

Phosvitin, derived from the vitellogenin II gene protein, is a highly phosphorylated protein found in egg yolk. A second hypothetical protein has been predicted based on the vitellogenin I gene, but has not been defined at the protein level. Mass spectrometric analysis was used to identify the phosphopeptide sequences and the precise sites of phosphorylation of two phosvitins, phosvitin 1 and phosvitin 2 derived from vitellogenins I and II, respectively. Samples of native phosvitin were subjected to tryptic digestion followed by mass spectrometric analysis: (i) native phosvitin peptides, (ii) after treatment with NaOH, and (iii) after chemical derivatization of P-Ser/P-Thr residues by dithiothreitol under base-catalyzed conditions. A combination of these approaches led to the identification of 68 and 35 phosphopeptides with 89 (81 P-Ser and 8 P-Thr residues) and 62 (57 P-Ser and 5 P-Thr residues) phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. These data for the first time documented on a large scale the major states and sites of phosphorylation of phosvitins with a total of 151 phosphorylation sites. Importantly, the present work also provided the first direct de novo protein amino-acid sequence data for phosvitin 1 protein and evidence for the full expression of vitellogenin I gene. BIOLOGICAL SIGNIFICANCE: We have for the first time generated a large number of phosphopeptides (~100) and identified 151 phosphorylation sites of phosvitin 1 and phosvitin 2, respectively. Importantly, this study also led to the discovery of a novel phosvitin 1 and provided the first direct de novo protein amino-acid sequence data for the full expression of vitellogenin I gene. There is considerable interest in naturally occurring phosphopeptides/phosphoproteins and their application in biomedical fields and in the food industry because of their molecular characteristics and non-toxic nature, hence, our work opens new avenues to pursue such endeavors. In addition, the results provide important fundamental biologic information relevant to evolutionary developments of vertebrate animals beginning with the earliest fish, reptiles, birds and more contemporary mammals. For instance, the abundance of phosvitins with a unique degree of phosphorylation in the egg yolk of fish, reptiles, and birds suggests potential biological functions of phosvitins which are critical to the development of embryos of these distant vertebrates.

Antiviral activity of phosvitin from zebrafish Danio rerio.

Lymphocystis disease virus (LCDV), a virus of Iridoviridae, can infect numerous teleost species, causing serious losses of aquaculture industry, and thus effective ways of prophylaxis and treatment are demanded. Previous studies have shown that phosvitin (Pv) is an antimicrobial agent in zebrafish, and vitellogenin, the precursor of yolk proteins including Pv, is able to neutralize virus, we thus hypothesize that Pv may have an antiviral activity. Here we clearly demonstrated that recombinant Pv (rPv) purified was capable of inhibiting the cytopathic effect in LCDV-infected cells and reducing the virus quantities in the infected cells as well as in the infected zebrafish. These data indicate that Pv possesses an antiviral activity and participates in immune defense of host against the infection by viruses like LCDV.

Effects of high-pressure processing and enzymatic dephosphorylation on phosvitin properties.

BACKGROUND: Egg phosvitin could be a good source of functional peptides. Enzymatic dephosphorylation and high-pressure processing combined with thermal treatment applied before proteolysis could produce phosvitin hydrolysates with different properties compared to its native form. RESULTS: Phosvitin structure was maintained overall during high-pressure treatment of 600 MPa applied at an initial temperature of 65 °C regardless of the pH and duration of treatment, confirming the high structural stability of this phosphoprotein. Treatment of phosvitin with phosphatase increased the degree of dephosphorylation from 24% to 63%, after 2 and 18 h, respectively. Moderate dephosphorylation of phosvitin prior to proteolytic digestion improved its hydrolysis, allowing formation of peptides with a molecular weight lower than 17,000 kDa as determined by size exclusion chromatography. Angiotensin-converting enzyme (ACE) inhibition and antioxidant activity of dephosphorylated and protease-treated phosvitin was increased by 52% and 39%, respectively, as compared to protease-digested native phosvitin. CONCLUSION: Enzymatic dephosphorylation before proteolysis mimicking in vivo gut conditions improved ACE inhibition and antioxidant activity of phosvitin hydrolysates.

Effects of calcium salts of acidic monomers on mineral induction of phosphoprotein immobilized to agarose beads.

The aim of this study is to evaluate the mineralizing potential of acidic monomers and their calcium salts for mineralization, using an in vitro mineral induction model. Phosvitin (PV) was used as a model phosphoprotein in this study. PV was immobilized on agarose beads with divinyl sulfone. Five aliquots of agarose-immobilized PV, acidic monomers, and their calcium salts were incubated in mineralizing solution at various concentrations. The PV beads and acidic monomers were incubated at 37°C. Samples were taken at several time points during the incubation. Then, the agarose beads were analyzed for bound calcium by atomic absorption spectrometry. The mineral formed on the agarose beads was identified as an apatite by microarea X-ray diffraction. Additionally, the specimens were observed using scanning electron microscopy (SEM). Mineral induction time decreased with increasing solution saturation. 4-METCa salt [calcium salt of 4-methacryloxyethyl trimellitate (CMET)] significantly reduced the mineral induction time. Using these data, the interfacial tension for mineral induction of PV and CMET was determined to be 90.1 and 92.7 ergs/cm(2), respectively. The mineral induced in each specimen after incubation for 24 h was identified by its X-ray diffraction pattern as apatite. SEM observation showed that lath-shaped crystals were formed on the surfaces of the CMET. We conclude that CMET could play a role in dentin remineralization.