Glycerylphytate crosslinker as a potential osteoinductor of chitosan-based systems for guided bone regeneration.

Chitosan-based membranes are promising systems for guided bone regeneration. In this work, we used glycerylphytate as ionic crosslinker and osteinductor compound for the fabrication of chitosan membranes as supports for human mesenchymal stem cells. Three different glycerylphytate-crosslinked membranes were developed by changing the crosslinker concentration, from 2.5-10 wt-%, respect to chitosan. Physico-chemical characterization in terms of composition, morphology, and thermal behavior was further analyzed. Swelling degree, crosslinking density, and crosslinker release showed a glycerylphytate content-dependent behavior. Glycerylphytate suggested to improve osteointegration ability of chitosan surfaces by the formation of apatite-like aggregates after incubation in body simulated fluid. Stem cells cultured on the membranes increased their viability over time, and the incorporation of glycerylphytate improved osteogenic and osteoinductivity potential of chitosan by increasing calcium deposition and alkaline phosphatase (ALP) activity on cultured stem cells. These results demonstrated a potential application of glycerylphytate-crosslinked chitosan systems for promising bone tissue regeneration.

Glycerylphytate compounds with tunable ion affinity and osteogenic properties.

Phytic acid (PA) is a natural-occurring antioxidant, which plays an important role in many biological processes. PA is recognized as a potent inhibitor of lipid peroxidation because of its high affinity to multivalent cations, and it can play a role in osteogenic processes. However, its powerful chelating capacity is controversial because it can lead to a severe reduction of mineral availability in the organism. For this reason, compounds with beneficial biological properties of PA, but a modular ion binding capacity, are of high interest. In this work, we report the synthesis and physicochemical characterization of two hydroxylic derivatives of PA, named glycerylphytates (GPhy), through a condensation reaction of PA with glycerol (G). Both derivatives present antioxidant properties, measured by ferrozine/FeCl2 method and chelating activity with calcium ions depending on the content of glyceryl groups incorporated. Besides, the hydroxylic modification not only modulates the ion binding affinity of derivatives but also improves their cytocompatibility in human bone marrow mesenchymal cells (MSCs). Furthermore, GPhy derivatives display osteogenic properties, confirmed by COL1A and ALPL expression depending on composition. These positive features convert GPhy compounds into potent alternatives for those skeletal diseases treatments where PA is tentatively applied.

Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and ß-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

Design and synthesis of lipid-coupled inositol 1,2,3,4,5,6-hexakisphosphate derivatives exhibiting high-affinity binding for the HIV-1 MA domain.

The precursor of Gag protein (Pr55(Gag)) of human immunodeficiency virus, the principal structural component required for virus assembly, is known to bind d-myo-phosphatidylinositol 4,5-bisphosphate (PIP2). The N-terminus of Pr55(Gag), the MA domain, plays a critical role in the binding of Pr55(Gag) to the plasma membrane. Herein, we designed and synthesized myo-phosphatidylinositol 2,3,4,5,6-pentakisphosphate (PIP5) derivatives comprising highly phosphorylated inositol and variously modified diacylglycerol to examine the MA-binding properties. The inositol moiety was synthesized starting with myo-inositol and assembled with a hydrophobic glycerol moiety through a phosphate linkage. The Kd value for MA-binding of the PIP5 derivative 2 (Kd = 0.25 µM) was the lowest (i.e., highest affinity) of all derivatives, i.e., 70-fold lower than the Kd for the PIP2 derivative 1 (Kd = 16.9 µM) and 100-fold lower than the Kd for IP6 (Kd = 25.7 µM), suggesting the possibility that the PIP5 derivative blocks Pr55(Gag) membrane binding by competing with PIP2 in MA-binding.

ATR-FTIR investigation on the complexation of myo-inositol hexaphosphate with aluminum hydroxide.

The adsorption isotherm of and the pH effect on the adsorption of myo-inositol hexaphosphate (myo-IP6) on amorphous aluminum hydroxide was investigated. It was found that the adsorption isotherm of myo-IP6 on aluminum hydroxide could be well fitted with the Freundlich isotherm. The amount of myo-IP6 adsorbed remained almost constant in the range of pH 4.0 to 7.0, but it decreased considerably as the initial pH was over 7. The adsorption of myo-IP6 resulted in an increase in the pH level due to the release of OH(-) ions, which suggested that the adsorption of myo-IP6 on aluminum hydroxide was caused by a ligand exchange reaction. ATR-FTIR analysis of myo-IP6 in solution and adsorbed on aluminum hydroxide at different pH were performed. The ATR-FTIR investigation indicated that myo-IP6 was adsorbed onto aluminum hydroxide by forming inner-sphere complexes and adsorption facilitated the deprotonation of phosphate groups. The asymmetric vibration of the PO bond in AlPO(-)(3) appearing at a lower frequency than that in the terminal HPO(-)(3) indicated that Al bound to the O atom not as strongly as the H atom did. The ATR-FTIR investigation and theoretical calculation (with the Gaussian 03 program) revealed that three of the six phosphate groups in myo-IP6 molecules were bound to aluminum hydroxide while the other three remained free when myo-IP6 was adsorbed on aluminum hydroxide.

Unique precipitation and exocytosis of a calcium salt of myo-inositol hexakisphosphate in larval Echinococcus granulosus.

The ubiquitous intracellular molecule myo-inositol hexakisphosphate (IP6) is present extracellularly in the hydatid cyst wall (HCW) of the parasitic cestode Echinococcus granulosus. This study shows that extracellular IP6 is present as its solid calcium salt, in the form of deposits that are observed, at the ultrastructural level, as naturally electron dense granules some tens of nanometers in diameter. The presence of a calcium salt of IP6 in these structures was determined by two different electron microscopy techniques: (i) the analysis of the spatial distribution of phosphorus and calcium in the outer, acellular layer of the HCW (the laminated layer, LL) through electron energy loss spectroscopy, and (ii) the observation, by transmission electron microscopy, of HCW that were selectively depleted of IP6 by treatment with EGTA or phytase, an enzyme that catalyses the dephosphorylation of IP6. The deposits of the IP6-Ca(II) salt are also observed inside membrane vesicles in cells of the germinal layer (the inner, cellular layer of the HCW), indicating that IP6 precipitates with calcium within a cellular vesicular compartment and is then secreted to the LL. Thus, much as in plants (that produce vesicular IP6 deposits), the existence of transporters for IP6 or its precursors in internal membranes is needed to explain the compound's cellular localisation in E. granulosus.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate.

myo-Inositol-1,2,3,4,5,6-hexakisphosphate (Ins P(6)) was first described as an abundant form of phosphorus in plant seeds and other plant tissues and dubbed "phytic acid". Subsequently it was found to be a common constituent in eukaryotic cells, its metabolism a basic component of cellular housekeeping. In addition to phosphate, myo-inositol (Ins) and mineral storage and retrieval in plant organs and tissues, other roles for Ins P(6) include service as a major metabolic pool in Ins phosphate and pyrophosphate pathways involved in signaling and regulation; possibly as an effector or ligand in these processes; as a form of energy currency and in ATP regeneration; in RNA export and DNA repair; and as an anti-oxidant. The relatively recent demonstration that pyrophosphate-containing derivatives of Ins P(6) can function as phosphate donors in the regeneration of ATP is reminiscent of the proposal, made four decades ago in studies of seed development, that Ins P(6) itself may serve in this function. Studies of Ins P(6) in non-plant systems rarely include the consideration that this compound might represent a significant fraction of cellular P; cellular phosphate nutrition has been viewed as either not interesting or of little importance. However, there may be few fundamental differences among diverse eukaryotes in both the metabolic pathways involving Ins P(6) and the spectrum of possible roles for it and its metabolites.

Stereo- and regiospecificity of yeast phytases-chemical synthesis and enzymatic conversion of the substrate analogues neo- and L-chiro-inositol hexakisphosphate.

Phytases are enzymes that catalyze the hydrolysis of phosphate esters in myo-inositol hexakisphosphate (phytic acid). The precise routes of enzymatic dephosphorylation by phytases of the yeast strains Saccharomyces cerevisiae and Pichia rhodanensis have been investigated up to the myo-inositol trisphosphate level, including the absolute configuration of the intermediates. Stereoselective assignment of the myo-inositol pentakisphosphates (D-myo-inositol 1,2,4,5,6-pentakisphosphate and D-myo-inositol 1,2,3,4,5-pentakisphosphate) generated was accomplished by a new method based on enantiospecific enzymatic conversion and HPLC analysis. Via conduritol B or E derivatives the total syntheses of two epimers of myo-inositol hexakisphosphate, neo-inositol hexakisphosphate and L-chiro-inositol hexakisphosphate were performed to examine the specificity of the yeast phytases with these substrate analogues. A comparison of kinetic data and the degradation pathways determined gave the first hints about the molecular recognition of inositol hexakisphosphates by the enzymes. Exploitation of the high stereo- and regiospecificity observed in the dephosphorylation of neo- and L-chiro-inositol hexakisphosphate made it possible to establish enzyme-assisted steps for the synthesis of D-neo-inositol 1,2,5,6-tetrakisphosphate, L-chiro-inositol 1,2,3,5,6-pentakisphosphate and L-chiro-inositol 1,2,3,6-tetrakisphosphate.

Analysis of myo-inositol hexakisphosphate hydrolysis by Bacillus phytase: indication of a novel reaction mechanism.

Phytic acid (myo-inositol hexakisphosphate, InsP(6)) hydrolysis by Bacillus phytase (PhyC) was studied. The enzyme hydrolyses only three phosphates from phytic acid. Moreover, the enzyme seems to prefer the hydrolysis of every second phosphate over that of adjacent ones. Furthermore, it is very likely that the enzyme has two alternative pathways for the hydrolysis of phytic acid, resulting in two different myo-inositol trisphosphate end products: Ins(2,4,6)P(3) and Ins(1,3,5)P(3). These results, together with inhibition studies with fluoride, vanadate, substrate and a substrate analogue, indicate a reaction mechanism different from that of other phytases. By combining the data presented in this study with (1) structural information obtained from the crystal structure of Bacillus amyloliquefaciens phytase [Ha, Oh, Shin, Kim, Oh, Kim, Choi and Oh (2000) Nat. Struct. Biol. 7, 147-153], and (2) computer-modelling analyses of enzyme-substrate complexes, a novel mode of phytic acid hydrolysis is proposed.

Dietary factors influencing zinc absorption.

Marginal zinc deficiency and suboptimal zinc status have been recognized in many groups of the population in both less developed and industrialized countries. Although the cause in some cases may be inadequate dietary intake of zinc, inhibitors of zinc absorption are most likely the most common causative factor. Phytate, which is present in staple foods like cereals, corn and rice, has a strong negative effect on zinc absorption from composite meals. Inositol hexaphosphates and pentaphosphates are the phytate forms that exert these negative effects, whereas the lower phosphates have no or little effect on zinc absorption. The removal or reduction of phytate by enzyme (phytase) treatment, precipitation methods, germination, fermentation or plant breeding/genetic engineering markedly improves zinc absorption. Iron can have a negative effect on zinc absorption, if given together in a supplement, whereas no effect is observed when the same amounts are present in a meal as fortificants. Cadmium, which is increasing in the environment, also inhibits zinc absorption. The amount of protein in a meal has a positive effect on zinc absorption, but individual proteins may act differently; e.g., casein has a modest inhibitory effect of zinc absorption compared with other protein sources. Amino acids, such as histidine and methionine, and other low-molecular-weight ions, such as EDTA and organic acids (e.g., citrate), are known to have a positive effect on zinc absorption and have been used for zinc supplements. Knowledge about dietary factors that inhibit zinc absorption and about ways to overcome or remove these factors is essential when designing strategies to improve the zinc nutrition of vulnerable groups.

Studies on avian erythrocyte metabolism. 5. Relationship between the major phosphorylated metabolic intermediates and while blood oxygen affinity in embryols and poults of the domestic turkey (Meleagris gallopavo).

The changes in organic phosphates of turkey erythrocytes (RBC) have been determined in relation to the changes in oxygen affinity of whole blood during growth of the embryo and poult. On a molar basis, 2,3 diphosphoglyceric acid (2,3-DPG) is the predominant organic phosphate of erythrocytes from turkey embryos during the last week of incubation. However, on the basis of relative % phosphate, 2,3-DPG is the major organic phosphate of the erythrocytes from turkey embryos on day 23 and 25 of incubation only. With the exception of day 23 and 25 incubation, adenosine triphosphate (ATP) represents the major organic phosphate of the erythrocytes of the turkey embryo and poult during the last week of embryonic development and through the first 29 days after hatching. The whole blood P50 during the last week of incubation and the first 8 days after hatching correlates best with the amount of ATP in the erythrocytes. The effects of inositol pentaphosphate on P50 of the whole blood is much more gradual and appears to become of major influence after 2-3 weeks post-hatching.

Isolation of monoferric phytate from wheat bran and its biological value as an iron source to the rat.

The objectives of the study were to isolate and chemically characterize the iron in wheat and to determine the biological availability to the rat of the iron as the purified complex(es). Hard wheat bran contained no butanol extractable or water extractable iron, but approximately 60% of the iron was extracted by 1 to 1.2 M NaCl or ammonium acetate solution. This salt extractable iron complex was purified and identified as monoferric phytate. The purified monoferric phytate was soluble in water. Synthetic monoferric phytate was prepared from sodium phytate and ferric chloride and determined to have spectral characteristics and gel filtration chromatography behavior identical to the complex isolated from wheat bran. The butanol-water-salt extracted bran residue contained no detectable phytate and an as yet uncharacterized form of iron. The biological availability of the iron to the rat was determined by a hemoglobin depletion-repletion bioassay. The relative biological value of the iron as monoferric phytate, either isolated from wheat bran or the synthetic product, was equal to the reference compound, ferrous ammonium sulfate. In contrast, the biological availability of the iron in the bran residue was significantly lower and the low biological availability of an insoluble form of ferric phytate was confirmed. It is concluded that the major portion of the iron in wheat is monoferric phytate and has a high biological availability to the rat. Monoferric phytate in bran may be bound to cationic sites of proteins or other cellular components and utilization of the iron may be through solubilization of the monoferric phytate by ion exchange type mechanism rather than by hydrolysis of the phytate as has been postulated.