PMC:2779105 / 35709-44942 JSON TXT

Annnotations TAB JSON ListView MergeView

2_test

{"project":"2_test","denotations":[{"id":"19956594-19198665-88162775","span":{"begin":3821,"end":3822},"obj":"19198665"},{"id":"19956594-19198665-88162776","span":{"begin":7340,"end":7341},"obj":"19198665"},{"id":"19956594-19412552-88162777","span":{"begin":7345,"end":7346},"obj":"19412552"},{"id":"19956594-11374033-88162778","span":{"begin":8154,"end":8156},"obj":"11374033"},{"id":"T94973","span":{"begin":3821,"end":3822},"obj":"19198665"},{"id":"T93656","span":{"begin":7340,"end":7341},"obj":"19198665"},{"id":"T12130","span":{"begin":7345,"end":7346},"obj":"19412552"},{"id":"T43950","span":{"begin":8154,"end":8156},"obj":"11374033"}],"text":"Role of Proteins in the Formation of NB-Like Calcium Particles: Effect of Heat Treatment and Distinction between Metastable and Supersaturated Calcium Solutions\nOther treatments were sought to verify the role of proteinaceous serum factors in creating (or inhibiting) NB-like particles in metastable solutions like DMEM. Heat treatment of proteins is commonly used as a way of denaturing proteins or inducing conformational changes. Figure 3 shows the results obtained with boiling FBS or HS followed by inoculation into DMEM. Heat treatment of membrane-filtered serum was done at 95°C for various periods of time (10, 30, and 120 min). While the boiled serum became more opalescent and occasionally showed white precipitations, the entire serum solution was used for inoculation without further processing. As can be seen, there is an increase in seeding by at least several fold as compared with non-boiled control serum (Fig. 3A). This increase in turbidity seen with serum that had been boiled for 10 or 30 minutes occurred gradually and was not the result of an immediate precipitation due to heat treatment of serum alone (note the low turbidity seen on “Day 1”). However, with longer boiling times (120 min), some immediate precipitations were seen on “Day 1,” which in this particular experiment were more noticeable with FBS as compared to the HS sample used (Fig. 2A). With even longer boiling times (overnight), mineral precipitation was obvious in the serum even prior to seeding (data not shown). Nonetheless, from Figure 3A, it can be seen that there was a slow and gradual increase in turbidity associated with the higher amounts of boiled serum used over the course of several weeks of incubation (compare “1 Month” and “Day 1” readings). However, as evidenced with protease-treated serum, the same bell-shaped, dose-dependent precipitation seen earlier with control, untreated serum was no longer apparent with serum that had been boiled. That is, it appears that the inhibition seen with higher amounts of serum (exceeding 1–3% in the case of FBS and HS) was somehow released through the boiling treatment.\n10.1371/journal.pone.0008058.g003 Figure 3 Formation of NB-like particles from boiled serum in metastable versus supersaturated medium.\n(A) Metastable medium: FBS and 25% HS were boiled at 95°C for the time indicated on the left panel (0, 10, 30, and 120 min). “0 min” refers to control, untreated serum. The boiled sera were then inoculated into DMEM to the concentrations shown on the top heading. (B) Supersaturated medium: both FBS and HS were treated as described in (A), except that 1 mM each of CaCl2 and NaH2PO4 (labeled as “Calcium+Phosphate Added”) was added following the inoculation of the boiled serum into DMEM. Note the marked differences in turbidity changes between the various panels shown in (A) versus (B). See the text for explanation and interpretation. To further verify whether there are differences seen between metastable versus supersaturated calcium and phosphate solutions with respect to the effects of boiled serum, we repeated the same seeding experiment with DMEM shown in Figure 3A, supplemented however with 1 mM calcium and phosphate. Mineral precipitation, along with an increase in turbidity, was immediate under these conditions (Fig. 3B, “Day 1”). Untreated, control serum (both FBS and HS) at higher concentrations (1% for FBS and 3% for HS) was capable of exerting immediate, partial inhibition of this same mineral precipitation (Fig. 3B, see the row “0 min Boiling Time” within the “Day 1” panel), an inhibition that could be sustained for several days (Fig. 3B, compare with “Day 4” panel). Thus, this precipitation showed a prominent bell-shaped, dual relationship as a function of the serum amount added, in line with the data published earlier [2].\nIn the presence of boiled serum, however, the same DMEM challenged with exogenous calcium and phosphate (1 mM) displayed a pattern of response that differed markedly from that seen with DMEM alone (e.g. without the presence of additional, exogenously added calcium and phosphate). As can be seen in Figure 3B, the addition of 1 mM calcium and phosphate to DMEM produced immediate precipitation associated with turbidity increase (wells 1, “Day 1”). Turbidity did not increase significantly thereafter (compare with “Day 4”). As noted before, the presence of control FBS or HS in DMEM resulted in partial inhibition of this same turbidity produced by 1 mM calcium and phosphate (seen from control rows identified by a boiling time of “0” min, Fig. 3B). Moreover, both FBS and HS that had been boiled for 10 and 30 minutes produced the same partial inhibition of turbidity immediately after seeding (“Day 1”). In the case of FBS, this same partial inhibition could still be seen on Day 4 (Fig. 3B, “Boiled FBS” column). For HS, on the other hand, this inhibition was released after 3 days, being replaced by a straight, dose-dependent change in turbidity that increased with the amount of HS present (Fig. 3B, “Boiled HS, Day 4”). Upon using either FBS or HS that had been boiled for 120 minutes, however, this same inhibition was immediately released and a dose-dependent increase in turbidity could be seen both on “Day 1” and “Day 4” (Fig. 3B). In fact, the addition of boiled serum produced an additive precipitation effect on top of the precipitation seen with the exogenous addition of 1 mM of calcium and phosphate alone that was in itself dose-dependent (Fig. 3B, more evident with all the boiled HS samples tested, but also seen with FBS that had been boiled for 120 min). Upon longer incubation (up to 1 month), a progressive increase of turbidity was observed with all serum treatments, resulting in the reversal or overcoming of the seeding-inhibition seen at the earlier time points (data not shown).\nWith respect to supersaturated calcium and phosphate solutions, these results would seem to indicate that the calcification-inhibitory influences seen associated with serum are only partially sensitive to heat exposure and that extensive boiling and denaturation are required to effect their inactivation. In this same context, our results indicate that, for FBS, the same calcification-inhibitory factors show a higher resistance to heat inactivation as compared with HS. Our findings also reveal that the “seeding” and “inhibitory” tendencies associated with serum can be clearly distinguished by the experimental set-up used here. Thus, in the case of metastable medium (DMEM), the “seeding” aspects of the boiled serum can be more readily revealed. On the other hand, with supersaturated calcium and phosphate solutions displaying high natural propensity to calcify, it is the “inhibitory” aspects of the serum that are more readily evident and that in turn are either partially or completely removed through boiling.\nThese results further indicate that the use of metastable and supersaturated calcium and phosphate solutions may allow for the elucidation of certain mechanistic aspects of nanoparticle physiology that would not have been possible by making use of only metastable conditions. Earlier, we used exogenous precipitating ions to enhance the biomineralization process many fold—a strategy that was adopted to amplify precipitation and to obtain more quantifiable and reproducible data [2], [3]. Here, we have noticed that in addition to enhancing the calcification process, the addition of small amounts of calcium and phosphate could be used as a tool to study the role of serum and proteins in the context of the dual inhibition-seeding model proposed here for nanoparticle assembly. It should be further noted that DMEM contains 1.8 mM calcium and 0.9 mM phosphate. The addition of up to 1 mM of calcium and phosphate, as we have done in the experiment depicted in Figure 3B, gives final concentrations of 2.8 mM calcium and 1.9 mM phosphate, resulting in an ion product of 5.32×10−6 M2, still below the cut-off value of 6×10−6 M2, above which any solution at physiological pH, ionic strength, and body temperature—serum in body conditions for example—has been found to precipitate spontaneously [56]. Yet, the ion product obtained with such low amounts of calcium and phosphate is sufficient to reveal important differences between this ionic environment and metastable solutions like DMEM. In fact, we have noticed that as little as 0.7 mM calcium and phosphate, and at times, as little as 0.3 mM calcium phosphate, inoculated into DMEM will result in gradual mineral precipitation, giving results that are similar to those seen here with 1 mM calcium and phosphate except for a slower kinetics (data not shown). We have noticed further that the level of precipitating ions required to induce mineral deposition was clearly dependent on the age of the medium, its pH, and other environmental factors, such as the frequency with which the plates were examined and how long they were left out at room temperature (unpublished observations). These results indicate that one can easily simulate supersaturated calcium and phosphate conditions with minimal additions of exogenous calcium and phosphate, enabling one to minimize the ionic perturbation of the medium being studied."}

NEUROSES

PMC:2779105 / 35709-44942 JSONTXT

Annnotations TAB JSON ListView MergeView

2_test

NEUROSES

PMC:2779105 / 35709-44942 JSON TXT