Serum levels of the fetuin-mineral complex correlate with artery calcification in the rat

The present studies were carried out to evaluate the possible association between the presence of the fetuin-mineral complex in serum and vitamin D-induced artery calcification. The first experiment shows that there is a fetuin-mineral complex in the blood of rats in which extensive calcification of the artery media has been induced by treatment with vitamin D for 96 h, and that there is no detectable fetuin-mineral complex in the blood of rats in which artery calcification has been inhibited by concurrent treatment with ibandronate or osteoprotegerin. The second experiment shows that the timing of vitamin D-induced artery calcification correlates with the timing of the maximal increase in serum fetuin-mineral complex levels. Whereas both results indicate that serum levels of the fetuin-mineral complex are indeed associated with vitamin D-induced artery calcification, the biochemical basis for this association is presently unclear. One possibility is that high levels of the fetuin-mineral complex cause defects in the ability of fetuin to prevent the growth of the mineral component, which then seeds artery calcification. Another possibility is that the fetuin-mineral complex is the downstream product of a pathway that begins with the true causative agent, and that the serum level of the fetuin-mineral complex is a marker for the activity of this agent in blood. An unexpected finding of the present studies is that vitamin D-induced artery calcification is also correlated with a 65 to 75% reduction in serum fetuin, a reduction that appears to be caused by the clearance of the fetuin-mineral complex from serum.     

Biochemical characterization of the serum fetuin-mineral complex

The present study was carried out to characterize the fetuin-mineral complex (FMC), a high molecular mass complex of calcium phosphate mineral and the proteins fetuin and matrix Gla protein (MGP) that was initially discovered in serum of rats treated with etidronate and appears to play a critical role in inhibiting calcification in vivo. Fetuin purified from the FMC contains 3.3 mol of protein-bound phosphate. There is 1.3 mg of FMC/ml of serum 6 h after etidronate injection, and the FMC is 46% fetuin and 53% mineral by mass. Formation of the FMC in the first 6 h after etidronate injection does not increase serum fetuin despite the fact that 50% of serum fetuin is associated with the FMC, and clearance of the FMC in the 9-24-h interval lowers total serum fetuin by 50%. These observations suggest that the fetuin component of the FMC is derived from fetuin initially in serum and that clearance of the FMC removes the associated fetuin from circulation. One additional protein was consistently present in all preparations of the FMC, spp24 (secreted phosphoprotein 24). This 24-kDa protein is similar in domain structure to fetuin and, like fetuin and MGP, contains several residues of phosphoserine and accumulates in bone. Exogenous spp24 associated strongly with the FMC when added to serum containing it. These observations suggest that spp24 may, like fetuin and MGP, play a role in inhibiting calcification.     

Discovery of a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats.

In the present study we report the discovery of a novel protein-mineral complex in the serum of rats treated with doses of the bone-active bisphosphonate etidronate that inhibit normal bone mineralization. The composition of this high molecular mass protein-mineral complex consists of about 18% mineral, 80% fetuin, and 2% matrix Gla protein (MGP) by weight, and the presence of the complex in serum after an injection of 8 mg etidronate/100 g of body weight elevates calcium by 1.8-fold (to 4.3 mm), phosphate by 1.6-fold (to 5.6 mm), and MGP by 25-fold (to 12 microg/ml). The serum mineral complex reaches maximal levels at 6 h after subcutaneous injection of etidronate and is subsequently cleared from serum by 24 h. This highly specific complex of fetuin, MGP, and mineral prevents the growth, aggregation, and precipitation of the mineral component, which indicates that the previously reported calcification inhibitory activities of fetuin and MGP may be related to their ability to form stable complexes with nascent mineral nuclei. Treatment with the vitamin K-antagonist warfarin prevents the increase in serum MGP after etidronate injection, which shows that the increase in serum MGP is due to new synthesis and that the gamma-carboxylation of MGP is necessary for its binding to the serum mineral complex.     

Separation of the epithelial and mesenchymal components of the newt limb regenerate with salt.

After incubation of isolated forelimb regenerates of Notophthalmus (Triturus) viridescens at all developmental stages for 60 minutes at 37 degrees C in a salt medium containing 111 mM sodium chloride, 5.6 mM potassium chloride and 100 mM sodium phosphate buffer at pH 7.5, the wound epithelium of each regenerate was removed intact from its underlying mesenchymal component. The suggestion is made that the salt medium is an effective epithelial-mesenchymal separating agent due to a combination of its hypertonicity, high ionic strength and the fact that the medium precipitates calcium as calcium phosphate. Attempts to dissect away the epithelium from the mesenchyme after incubation of isolated regenerates in sodium phosphate containing 1% or 3% Difco 1:250 trypsin, 10 mM EDTA or 150 units collagenase/ml medium were unsuccessful. Epidermis of adult newt forelimb skin was removed only after extended incubation of the forelimbs in the salt medium for three hours at 37 degrees C or after freezing isolated forelimbs in buffer and subsequent thawing.     

Characterisation of non-activated and activated estrogen- and antiestrogen-receptor complexes by high performance ion exchange chromatography

The ionic species of cytosol estrogen receptors from mature rat uteri have been compared by HPIEC on a SynChrom AX-1000 column when complexed with either [125I]iodoestradiol, [3H]estradiol or [3H]4-hydroxy tamoxifen. Three species of receptors (isoforms) each suppressible by excess competitor were fractionated at identical salt concentrations regardless of ligand employed. One species eluted in the column void volume (10 mM) and the others at congruent to 90 mM and congruent to 155 mM phosphate. Activation of receptor complexes by increasing time of incubation with ligand from 1 to 24 h at 4 degrees C or addition of 10 mM GTP increased the proportion of the congruent to 90 mM species for all 3 ligands. The addition of 10 mM molybdate to homogenization and HPIEC buffers resulted in only two species being resolved at 10 and 110 mM phosphate. These species were again identical regardless of ligand employed. Increasing concentrations of estradiol (1-40 nM) tamoxifen (20 nM-4 microM) and 4-hydroxy tamoxifen (2-400 nM) were able to compete for binding of [125I]iodoestradiol to each of the three ionic species. Binding to each species was inhibited equally by each concentration of competitor. There was no preferential or unique association of estrogen or antiestrogen with any of the ionic species and all ligands gave identical ionic species of non-activated and activated receptor complexes.     

A simple and defined method to study calcification by isolated matrix vesicles. Effect of ATP and vesicle phosphatase.

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 microgram of protein, were incubated for 2 h in a 45Ca-labelled solution with 2.2 mM Ca2+, 1.6 mM PO 3/4-and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO4 hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca2+ or Mg2+ was found to stimulate matrix vesicle ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, beta-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, vesicles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.     

Mineralization by Inhibitor Exclusion: THE CALCIFICATION OF COLLAGEN WITH FETUIN

One of our goals is to understand the mechanisms that deposit mineral within collagen fibrils, and as a first step we recently determined the size exclusion characteristics of the fibril. This study revealed that apatite crystals up to 12 unit cells in size can access the water within the fibril, whereas molecules larger than a 40-kDa protein are excluded. Based on these observations, we proposed a novel mechanism for fibril mineralization: that macromolecular inhibitors of apatite growth favor fibril mineralization by selectively inhibiting crystal growth in the solution outside of the fibril. To test this mechanism, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentration and the only macromolecule in solution is fetuin, a 48-kDa inhibitor of apatite growth. Our experiments with this system demonstrated that fetuin determines the location of mineral growth; in the presence of fetuin mineral grows exclusively within the fibril, whereas in its absence mineral grows in solution outside the fibril. Additional experiments showed that fetuin is also able to localize calcification to the interior of synthetic matrices that have size exclusion characteristics similar to those of collagen and that it does so by selectively inhibiting mineral growth outside of these matrices. We termed this new calcification mechanism “mineralization by inhibitor exclusion,” the selective mineralization of a matrix using a macromolecular inhibitor of mineral growth that is excluded from that matrix. Future studies will be needed to evaluate the possible role of this mechanism in bone mineralization.The type I collagen fibril plays several critical roles in bone mineralization. The mineral in bone is located primarily within the fibril (1–6), and during mineralization the fibril is formed first and then water within the fibril is replaced with mineral (7, 8). The collagen fibril therefore provides the aqueous compartment in which mineral grows. We have recently shown that the physical structure of the collagen fibril plays an important additional role in mineralization, that of a gatekeeper allowing molecules smaller than a 6-kDa protein to freely access the water within the fibril while preventing molecules larger than a 40-kDa protein from entering the fibril (9).Molecules too large to enter the collagen fibril can have important effects on mineralization within the fibril. We have suggested that large inhibitors of apatite growth can paradoxically favor mineralization within the fibril by selectively preventing apatite growth in the solution outside of the fibril (9). We have also proposed that large nucleators of apatite formation may generate small crystals outside the collagen fibril and that some of these crystals can subsequently diffuse into the fibril and grow (9). Because the size exclusion characteristics of the fibril allow rapid penetration of molecules the size of a 6-kDa protein, apatite crystals up to 12 unit cells in size should in principle be able to freely access all of the water within the fibril (9).We subsequently tested these hypotheses for the role of large molecules in fibril mineralization by determining the impact of removing fetuin on the serum-driven calcification of collagen fibrils (10). Fetuin is the most abundant serum inhibitor of apatite crystal growth (11, 12), and with a molecular weight of 48 kDa fetuin is too large to penetrate the collagen fibril (9). Fetuin is also termed fetuin-A (to distinguish it from a recently discovered homologue, fetuin-B (13)) and is sometimes called α2-HS glycoprotein in humans. Our working hypothesis was that fetuin is required for the serum-driven calcification of a collagen fibril and that its role is to favor calcification within the collagen fibril by selectively preventing apatite crystal growth in the solution outside the fibril.The results of this study demonstrate that removing fetuin from serum eliminates the ability of serum to induce the calcification of a type I collagen matrix and that adding purified fetuin to fetuin-depleted serum restores this activity (14). This study further shows that a massive mineral precipitate forms during the incubation of fetuin-depleted serum but not during the incubation of serum containing fetuin (14). These observations are consistent with the hypothesis that a large serum nucleator generates apatite crystals in the solution outside of the collagen fibril, some of which penetrate into the aqueous interior of the fibril (14). Because fetuin can trap only those nuclei that it can access, the crystal nuclei that penetrate the fibril grow far more rapidly than those nuclei trapped by fetuin outside of the fibril, and the collagen fibril therefore selectively calcifies.The goal of the present experiments was to further understand the role of fetuin in the calcification of type I collagen fibrils. To accomplish this goal, we developed a system in which crystal formation is driven by homogeneous nucleation at high calcium phosphate concentrations and the only macromolecule in the solution is fetuin. This system allowed us to probe the impact of fetuin and only fetuin on the location and extent of collagen calcification. Because fetuin is the subject of this study, it is useful to review briefly its occurrence and calcification-inhibitory activity. Fetuin is a 48-kDa glycoprotein that is synthesized in the liver and is found at high concentrations in mammalian serum (15, 16) and bone (17–22). The serum fetuin concentration in adult mammals ranges from 0.5 to 1.5 mg/ml, whereas the serum fetuin concentration in the fetus and neonate is typically far higher (16). Fetuin is also one of the most abundant noncollagenous proteins found in bone (17–22), with a concentration of about 1 mg fetuin/g bone in rat (21), bovine (17), and human (19, 23) bone. Despite the abundance of fetuin in bone, however, it has not been possible to demonstrate the synthesis of fetuin in calcified tissues, and it is therefore presently thought that the fetuin found in bone arises from hepatic synthesis via serum (20, 22). This view is supported by the observation that fetuin binds strongly to apatite, the mineral phase of bone, and is selectively concentrated from serum onto apatite (18).In vitro studies have demonstrated that fetuin is an important inhibitor of apatite growth and precipitation in serum containing increased levels of calcium and phosphate (12) and that targeted deletion of the fetuin gene reduces the ability of serum to arrest apatite formation by over 70% (11). More recent studies have shown that a fetuin-mineral complex is formed in the course of the fetuin-mediated inhibition of apatite growth and precipitation in serum containing increased calcium and phosphate (24, 25). Purified fetuin also potently inhibits the growth of apatite crystals from supersaturated solutions of calcium phosphate (12, 24). In solutions in which a decline in calcium occurs within minutes because of the spontaneous formation of apatite crystals, the presence of added fetuin sustains elevated calcium levels for at least 24 h (24).     

Inhibition of hydroxyapatite formation in collagen gels by chondroitin sulphate.

Crystal growth in native collagen gels has been used to determine the role of extracellular matrix macromolecules in biological calcification phenomena. In this system, type I collagen gels containing sodium phosphate and buffered at pH 7.4 are overlayed with a solution containing CaCl2. Crystals form in the collagen gel adjacent to the gel-solution interface. Conditions were determined which permit the growth of crystals of hydroxyapatite [Ca10(PO4)6(OH)2]. At a Ca/P molar ratio of 2:1, the minimum concentrations of calcium and phosphate necessary for precipitation of hydroxyapatite are 10 mM and 5 mM, respectively. Under these conditions, precipitation is initiated at 18-24h, and is maximal between 24h and 6 days. Addition of high concentrations of chondroitin 4-sulphate inhibits the formation of hydroxyapatite in collagen gels; initiation of precipitation is delayed, and the final (equilibrium) amount of precipitation is decreased. Inhibition of hydroxyapatite formation requires concentrations of chondroitin sulphate higher than those required to inhibit calcium pyrophosphate crystal formation.     

Fusion of erythrocyte ghosts induced by calcium phosphate. Kinetic characteristics and the role of Ca2+, phosphate and calcium-phosphate complexes

Using an assay which allows continuous monitoring of the mixing of aqueous contents during membrane fusion, we have investigated the kinetics of calcium-phosphate-induced fusion of erythrocyte ghosts. In the presence of 10 mM phosphate, the threshold concentration for Ca2+-induced fusion was 1.25 mM, while the optimal concentration was approx. 1.75 mM Ca2+. Further enhancement of the cation concentration (greater than or equal to 2 mM) inhibited fusion of the ghosts. Initiation of fusion required the addition of phosphate prior to the addition of Ca2+, indicating that the combined interaction of Ca2+ and phosphate in or at the plane of the bilayer was a prerequisite for the induction of fusion. Furthermore, fusion was greatly facilitated upon transformation of calcium phosphate in the bulk medium from an amorphous to a solid, crystalline phase. It is suggested that membrane aggregation, and hence fusion, is facilitated by the formation of crystalline calcium phosphate nucleating on the ghost membrane. La3+, Mg2+ and Mn2+ did not trigger the fusion process, although aggregation of the ghosts did occur. Under conditions where calcium phosphate precipitation was inhibited, lanthanum phosphate precipitates facilitated fusion after prior treatment of ghosts with phosphate and Ca2+. These results indicated that fusion-prone conditions were induced prior to calcium phosphate precipitation. It is proposed that prior to calcium phosphate precipitation membrane changes are induced by separate interaction of Ca2+ and phosphate with the ghost membrane. Such an interaction could then render the ghosts susceptible to fusion and as soon as conditions are provided allowing close contact between adjacent membranes, fusion will be observed.     

Characterization of the parameters affecting covalent binding stoichiometry in binary and ternary complexes of thymidylate synthase

Covalent binding stoichiometries for both the enzyme:5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) binary complex and the enzyme:FdUMP:5,10-methylenetetrahydrofolate (inhibitory ternary) complex at equilibrium were measured by the trichloroacetic acid precipitation assay and shown to be a function of temperature, time, pH, salt concentration, buffer composition and thiol concentration. Incubation at 37 degrees C yielded the maximum covalent binding ratio (mol FdUMP/mol enzyme) for the latter binary (0.7) and ternary (1.7) complexes. In most buffers studied, the maximum covalent binding ratio (1.5-1.7) for the inhibitory ternary complex occurred over a broad pH range (4.5-8.0), while the optimum covalent binding ratio for binary complex was observed at a much narrower region centered between pH 5.5-6.5. In the presence of increasing concentrations of phosphate buffer, the maximum binding ratio for the covalent binary complex decreased from 0.63 in the absence of phosphate to 0.1 in the presence of 225 mM phosphate, while that for the inhibitory ternary complex was unchanged. When a ternary complex was formed with enzyme, FdUMP and (+/-)-tetrahydrofolate in the absence of phosphate, the FdUMP:enzyme covalent binding ratio was 1.8, while in the presence of 75 mM phosphate, the binding ratio was only 1.0. When exogenous thiol was removed by centrifugal column chromatography, the maximum binding stoichiometry of the resulting inhibitory ternary complex was 1.7 and was independent of added thiol over a 2 h incubation period at 37 degrees C. When extensive dialysis at 5 degrees C was used to remove the thiol, the maximum binding stoichiometry of the resulting inhibitory ternary complex was found to be dependent on both the concentration of added thiol and the time of incubation at 37 degrees C and did not exceed a value of 1.0.     

The mechanism by which tetracycline hydrochloride inhibits mineralization in vitro

1.

1. The inhibitory action of tetracycline hydrochloride on mineralization was demonstrated in vitro. Low concentrations of the drug interfered with teh precipitation of apatites from supersaturated mineralization buffers at 37° and pH 7.4. In the presence of 20 μM tetracycline, the ionic produce of calcium and phosphate required for spontaneous precipitation rose from 2.5 to 4.0 (mM)². The amount of mineral in the precipitate was not altered, which suggests that tetracycline affects the primary nucleation of mineralization.     

A simple and defined method to study calcification by isolated matrix vesicles effect of ATP and vesicle phosphatase

A simplified and defined system was developed to study in vitro calcium phosphate deposition by isolated matrix vesicles from rabbit growth plate cartilage, and to examine the relationship between vesicle phosphatase and calcium deposition. Samples of suspended vesicles containing 25 μg of protein, were incubated for 2 h in a ⁴⁵Ca-labelled solution with 2.2 mM Ca²⁺, 1.6 mM PO₄^3? and 1 mM ATP at pH 7.6. Calcium deposition was related to the amount of PO₄ hydrolysed by matrix vesicle phosphatases from ATP and other phosphate esters. Ca²⁺ or Mg²⁺ was found to stimulate matrix vesicle. ATPase, but the hydrolysis of phosphoenolpyruvate, glucose 1-phosphate, β-glycerol phosphate and AMP was independent of either cation. All of the above substrates supported calcium deposition. 1 mM ATP was more effective than 5 mM in supporting calcium deposition, indicating inhibition of mineralization at higher ATP concentrations. Our results suggest that, in addition to concentrating calcium, veiscles provide phosphate from ATP for mineral formation and at the same time remove the inhibitory effect of ATP upon mineral deposition.     

Effect of verapamil and sulfhydryl reagents on calcium transport in bovine spermatozoa

Calcium uptake by intact bovine epididymal spermatozoa is not affected by low concentrations (up to 0.75 mM) of the calcium transport blocker verapamil. Under these conditions, calcium transport into sperm mitochondria is highly inhibited. At higher verapamil concentrations (1.0, 1.5 mM), calcium transport into intact sperm is also inhibited, and this inhibition cannot be relieved by disrupting the plasma membrane with filipin. Calcium uptake into intact sperm is highly inhibited by mersalyl and this inhibitory effect can be completely relieved when the plasma membrane is disrupted by filipin. This effect of mersalyl is not dependent on the presence of phosphate in the incubation medium. Phosphate itself, up to 2 mM, enhances calcium uptake into the cells; this effect decreases at higher concentrations and is depressed 57% at 10 mM phosphate. This inhibitory effect of high phosphate concentration can be blocked by mersalyl. It is suggested that the calcium carrier itself and not a phosphate carrier of the plasma membrane is inhibited by mersalyl. It is possible that there is a symporter for calcium and phosphate in the plasma membrane of bovine spermatozoa.     

Preparation and characterization of milk calcium salts by using casein phosphopeptide

Milk calcium salt solution was prepared by the following procedures using casein phosphopeptides (CPP). To CPP solution, 1 M citric acid, 1 M CaCl2 and 1 M K2HPO4 were added with stirring, while adjusting the pH to 6.7. The prepared solution was left for 12 hr at 25 degrees C and then used for subsequent experiments, or lyophilized. The concentrations of organic phosphate of CPP, calcium, inorganic phosphate, and citrate in the typical milk salt solution were 7, 30, 22, and 10 mM, respectively, which were close to those in bovine milk. The lyophilized sample was easily dissolved in water. No crystal structure of hydroxyapatite was shown in the lyophilized milk calcium salts by X-ray diffraction analysis, although the pattern of KCl crystal was observed. The X-ray diffraction pattern of commercial whey mineral, which was prepared by precipitation at alkaline pH from rennet whey, was similar to that of hydroxyapatite. It was confirmed by high-performance gel chromatographic analysis that the form of calcium phosphate in the milk calcium salts was similar to that of casein micelles.     

Mechanism of lysis by large granular lymphocyte granule cytolysin: generation of a stable cytolysin-RBC intermediate

The effect of ionic strength and pH on the hemolytic activity of large granular lymphocyte granule cytolysin was examined in detail. Cytolysin-mediated lysis of RBC was inhibited by either low ionic strength or low pH. Under these conditions a nonlytic cytolysin-RBC intermediate was formed as revealed by hemolysis when cytolysin pretreated cells were washed and resuspended at physiologic ionic strength and pH. Formation of the cytolysin-RBC intermediate at low ionic strength (250 mM sucrose), pH 7.3, required greater than 0.1 mM calcium. In contrast, formation of the intermediate at physiologic ionic strength (150 mM NaCl), pH 6.0, was calcium independent. Both types of intermediates were stable at 37 degrees C and required calcium to induce subsequent lysis. The degree of lysis of the intermediate generated at low ionic strength was similar to that measured under standard conditions with the use of either whole granule preparations or purified cytolysin. However, lysis of intermediates formed at pH 6.0 was much less efficient. Our data indicate that a stable cytolysin-RBC intermediate can be formed in which cytolysin is present in an unreactive state on the RBC surface; under conditions of physiologic ionic strength and calcium concentrations this intermediate rapidly lyses.     

Characterization of neuraminidase activity of cultured human fibroblasts.

Investigations have been carried out to establish the enzymatic properties and specificities of the neuraminidase of cultured human fibroblasts. Homogenates of these cells cleaved the actylated derivative of neuraminic acid from fetuin, N-acetylneuraminyllactose and 2-(3' methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Maximum activity occurred between pH 4.2 and 4.6 in sodium acetate buffer. The Km values were 3.6 . 10(-4) M, 3.0 . 10(-3) M and 1.1 . 10(-3) M, respectively, against fetuin, N-acetylneuraminyllactose and 2-(3'methoxyphenyl)-N-acetyl-alpha-neuraminic acid. Against the first two substrates, the rate of hydrolysis fell below the expected value as the cell homogenate was diluted with water or 10 mM NaCl. Dilution with 8 mg/ml bovine serum albumin prevented the deviation and yielded the expected linear decrease. After the first 2-h incubation, the rate of hydrolysis decreased from the initial linear rate. The enzyme(s) was partially or completely inactivated by sonication at 20 kHz, freeze-thaw treatment, incubation at 52 degrees C or storage for 48 h at -70 degrees C. Suspension of the fibroblasts in water for 10 min at room temperature, followed by homogenization with a tissue grinder, yielded preparations that were suitable for the assay of the neuraminidase activity.     

Effects of combined secretagogues and extracellular calcium on neonatal insulin release

The insulin response of 3-day old neonatal rat islets was evaluated following a 1 h incubation with glucose alone and in the presence of 30 nM sulfated cholecystokinin octapeptide (CCK) and/or 20 microM carbachol (CCh). Insulin secretion was found to be incrementally increased from the lowest glucose concentration and enhanced several fold in the presence of CCK and/or CCh. In combination, CCK and CCh increased glucose-stimulated insulin secretion by an amount equivalent to the sum of their individual increases. The presence of either CCK alone or CCK plus CCh increased phosphoinositide hydrolysis by the same relative amount that they increased insulin secretion when compared to 8.3 mM glucose. Glucose-stimulated insulin secretion was totally inhibited when calcium was omitted from the incubation buffer; this effect was partially negated by CCK alone and more so by CCK combined with CCh. Insulin secretion in response to 8.3 mM glucose alone was unchanged when calcium in the incubation buffer was increased from 1 to 5 mM; however, the insulin response to 16.7 mM glucose alone and 8.3 mM glucose in the presence of CCK and/or CCh was increased under this condition. Thus, we have shown that, even at 3 days postpartum, insulin secretion from isolated islets is a complex response capable of being molded by several secretagogues at once and ultimately determined by interplay of different signaling systems activated.     

Inhibition of aspartate aminotransferase by glycation in vitro under various conditions

Incubation of 50 mM D-glucose with aspartate aminotransferase (AST, EC 2.6.1.1) preparations (purified pig heart enzyme or a rat liver 20,000 x g supernatant) at 25 degrees C had no effect on enzyme activity. 50 mM D-fructose or D-ribose gradually inhibited pig heart AST under the same conditions to zero activity after 14 days. 50 mM DL-glyceraldehyde decreased enzyme activity to zero after 6 days of incubation. The inhibition of pig heart AST by 50 mM D-fructose or D-ribose was marked even at a temperature of 4 degrees C but it was less pronounced than at 25 degrees C. There was no effect of 0.5 mM 2-oxoglutarate on AST activity during incubation, while the presence of 25 mM L-aspartate decreased it rapidly. 0.5 mM 2-oxoglutarate partly prevented inhibition of AST by D-ribose or D-fructose, while an analogous experiment with 25 mM aspartate resulted in a rapid decline similar to that in the absence of sugars.     

Biomineralization Studies on Cellulose Membrane Exposed to Biological Fluids of Anodonta cygnea

The present work proposes to analyse the results obtained under in vitro conditions where cellulose artificial membranes were incubated with biological fluids from the freshwater bivalve Anodonta cygnea. The membranes were mounted between two half ‘Ussing chambers’ with different composition solutions in order to simulate epithelial surfaces separating organic fluid compartments. The membrane surfaces were submitted to two synthetic calcium and phosphate solutions on opposite sides, at pH 6.0, 7.0 or 9.0 during a period of 6 hours. Additional assays were accomplished mixing these solutions with haemolymph or extrapallial fluid from A. cygnea, only on the calcium side. A selective ion movement, mainly dependent on the membrane pore size and/or cationic affinity, occurred with higher permeability for calcium ions to the opposite phosphate chamber supported by calcium diffusion forces across the cellulose membrane. In general, this promoted a more intense mineral precipitation on the phosphate membrane surface. A strong deposition of calcium phosphate mineral was observed at pH 9.0 as a primary layer with a homogeneous microstructure, being totally absent at pH 6.0. The membrane showed an additional crystal phase at pH 7.0 exhibiting a very particular hexagonal or cuttlebone shape, mainly on the phosphate surface. When organic fluids of A. cygnea were included, these crystal forms presented a high tendency to aggregate under rosaceous shapes, also predominantly in the phosphate side. The cellulose membrane was permeable to small organic molecules that diffused from the calcium towards the phosphate side. In the calcium side, very few similar crystals were observed. The presence of organic matrix from A. cygnea fluids induced a preliminary apatite–brushite crystal polymorphism. So, the present results suggest that cellulose membranes can be used as surrogates of biological epithelia with preferential ionic diffusion from the calcium to the phosphate side where the main mineral precipitation events occurred. Additionally, the organic fluids from freshwater bivalves should be also thoroughly researched in the applied biomedical field, as mineral nucleators and crystal modulators on biosynthetic systems.     

Stabilization of the colchicine-binding activity of tubulin by organic acids

A number of carboxylic acids and organic phosphates were found to be highly effective in stabilizing the colchicine-binding activity of calf brain tubulin. The most active were glutamate, glutarate, delta-aminovalerate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-(bis)phosphate, creatine phosphate and 6-phosphogluconate Maximum effects occurred at high concentrations. Combinations of agents were also examined, and the most effective mixture for stabilizing tubulin found thus far was the combination of 1.0 M glutamate, 100 mM glucose 1-phosphate, 1 mM GTP and 0.5 mg/ml of albumin. No loss of activity occurred over 48 h at 37 degrees C with tubulin was present at a concentration of 100 microgram/ml.