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Putative nanobacteria represent physiological remnants and culture by-products of normal calcium homeostasis.

Young JD, Martel J, Young L, Wu CY, Young A, Young D - PLoS ONE (2009)

Bottom Line: Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB.Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification.The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nanomaterials, Chang Gung University, Gueishan, Taiwan, Republic of China. dingeyoung@hotmail.com

ABSTRACT
Putative living entities called nanobacteria (NB) are unusual for their small sizes (50-500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.

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Inhibition of NLP formation by fetuin-A and albumin.(A) NLP were prepared from 0.5 mM to 6 mM each of CaCl2, Na2CO3, and NaH2PO4 added to water. A650 readings using a spectrophotometer show dose-dependent increase in optical density with NLP formation that displays a linear relationship seen between 2 and 4 mM of precipitating reagents used. (B) The inhibitory effects of fetuin-A and albumin on NLP formed in water were studied using 3 mM each of the three precipitating reagents. BSF or HSA were added to the indicated concentrations prior to the addition of the precipitating reagents. Compared to HSA, BSF showed markedly higher inhibitory potency on NLP formation.
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pone-0004417-g019: Inhibition of NLP formation by fetuin-A and albumin.(A) NLP were prepared from 0.5 mM to 6 mM each of CaCl2, Na2CO3, and NaH2PO4 added to water. A650 readings using a spectrophotometer show dose-dependent increase in optical density with NLP formation that displays a linear relationship seen between 2 and 4 mM of precipitating reagents used. (B) The inhibitory effects of fetuin-A and albumin on NLP formed in water were studied using 3 mM each of the three precipitating reagents. BSF or HSA were added to the indicated concentrations prior to the addition of the precipitating reagents. Compared to HSA, BSF showed markedly higher inhibitory potency on NLP formation.

Mentions: Since both fetuin-A and albumin are known inhibitors of calcium biomineralization, we next sought to verify whether these same proteins can inhibit the formation of NLP. We first developed a simple turbidity assay based on the propensity of calcium, carbonate and phosphate to combine and form insoluble complexes that can be read at 650 nm by means of color spectrophotometry (Materials and Methods). Initial studies were met with variable results and wide margins of error which were corrected after the incubation times were properly controlled and the readings consistently done. As shown in Figure 19A, the concentrations of the various precipitating reactants (calcium, carbonate, and phosphate) could be conveniently adjusted and chosen to provide a dose-dependent reaction yielding a linear component stretching between 2 and 4 mM of calcium-carbonate-phosphate that could be used in turn to monitor the role of calcium-binding proteins. Based on these considerations, a final concentration of 3 mM was chosen for calcium, carbonate, and phosphate in the experiment shown in Figure 19B, while the amount of BSF or HSA was varied. The inhibition achieved with BSF was much more pronounced and efficient than that seen with HSA. The protein concentration needed to obtain 50% inhibition was estimated at 0.3 µM for BSF, compared to 6 µM for HSA. We found that Heiss et al. had earlier performed a comparable assay to study the formation of “calciprotein particles” that were obtained by mixing CaCl2 and Na2HPO4 in the presence of 50 mM Tris-HCl and 140 mM NaCl at pH 7.4 [58]. In their study, the concentration of bovine fetuin-A needed to obtain 50% inhibition was estimated at 7.4 µM, which is significantly higher than what we obtained here. This difference may be attributed in part to the presence of carbonate ions in our reaction assay which, through their own binding to calcium, may have enhanced the inhibition seen with the added proteins. Since carbonate groups appear to constitute an important part of the NB scaffold, they were included in our assay so as to reproduce more precisely the NB phenomenology.


Putative nanobacteria represent physiological remnants and culture by-products of normal calcium homeostasis.

Young JD, Martel J, Young L, Wu CY, Young A, Young D - PLoS ONE (2009)

Inhibition of NLP formation by fetuin-A and albumin.(A) NLP were prepared from 0.5 mM to 6 mM each of CaCl2, Na2CO3, and NaH2PO4 added to water. A650 readings using a spectrophotometer show dose-dependent increase in optical density with NLP formation that displays a linear relationship seen between 2 and 4 mM of precipitating reagents used. (B) The inhibitory effects of fetuin-A and albumin on NLP formed in water were studied using 3 mM each of the three precipitating reagents. BSF or HSA were added to the indicated concentrations prior to the addition of the precipitating reagents. Compared to HSA, BSF showed markedly higher inhibitory potency on NLP formation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2636888&req=5

pone-0004417-g019: Inhibition of NLP formation by fetuin-A and albumin.(A) NLP were prepared from 0.5 mM to 6 mM each of CaCl2, Na2CO3, and NaH2PO4 added to water. A650 readings using a spectrophotometer show dose-dependent increase in optical density with NLP formation that displays a linear relationship seen between 2 and 4 mM of precipitating reagents used. (B) The inhibitory effects of fetuin-A and albumin on NLP formed in water were studied using 3 mM each of the three precipitating reagents. BSF or HSA were added to the indicated concentrations prior to the addition of the precipitating reagents. Compared to HSA, BSF showed markedly higher inhibitory potency on NLP formation.
Mentions: Since both fetuin-A and albumin are known inhibitors of calcium biomineralization, we next sought to verify whether these same proteins can inhibit the formation of NLP. We first developed a simple turbidity assay based on the propensity of calcium, carbonate and phosphate to combine and form insoluble complexes that can be read at 650 nm by means of color spectrophotometry (Materials and Methods). Initial studies were met with variable results and wide margins of error which were corrected after the incubation times were properly controlled and the readings consistently done. As shown in Figure 19A, the concentrations of the various precipitating reactants (calcium, carbonate, and phosphate) could be conveniently adjusted and chosen to provide a dose-dependent reaction yielding a linear component stretching between 2 and 4 mM of calcium-carbonate-phosphate that could be used in turn to monitor the role of calcium-binding proteins. Based on these considerations, a final concentration of 3 mM was chosen for calcium, carbonate, and phosphate in the experiment shown in Figure 19B, while the amount of BSF or HSA was varied. The inhibition achieved with BSF was much more pronounced and efficient than that seen with HSA. The protein concentration needed to obtain 50% inhibition was estimated at 0.3 µM for BSF, compared to 6 µM for HSA. We found that Heiss et al. had earlier performed a comparable assay to study the formation of “calciprotein particles” that were obtained by mixing CaCl2 and Na2HPO4 in the presence of 50 mM Tris-HCl and 140 mM NaCl at pH 7.4 [58]. In their study, the concentration of bovine fetuin-A needed to obtain 50% inhibition was estimated at 7.4 µM, which is significantly higher than what we obtained here. This difference may be attributed in part to the presence of carbonate ions in our reaction assay which, through their own binding to calcium, may have enhanced the inhibition seen with the added proteins. Since carbonate groups appear to constitute an important part of the NB scaffold, they were included in our assay so as to reproduce more precisely the NB phenomenology.

Bottom Line: Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB.Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification.The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Nanomaterials, Chang Gung University, Gueishan, Taiwan, Republic of China. dingeyoung@hotmail.com

ABSTRACT
Putative living entities called nanobacteria (NB) are unusual for their small sizes (50-500 nm), pleomorphic nature, and accumulation of hydroxyapatite (HAP), and have been implicated in numerous diseases involving extraskeletal calcification. By adding precipitating ions to cell culture medium containing serum, mineral nanoparticles are generated that are morphologically and chemically identical to the so-called NB. These nanoparticles are shown here to be formed of amorphous mineral complexes containing calcium as well as other ions like carbonate, which then rapidly acquire phosphate, forming HAP. The main constituent proteins of serum-derived NB are albumin, fetuin-A, and apolipoprotein A1, but their involvement appears circumstantial since so-called NB from different body fluids harbor other proteins. Accordingly, by passage through various culture media, the protein composition of these particles can be modulated. Immunoblotting experiments reveal that antibodies deemed specific for NB react in fact with either albumin, fetuin-A, or both, indicating that previous studies using these reagents may have detected these serum proteins from the same as well as different species, with human tissue nanoparticles presumably absorbing bovine serum antigens from the culture medium. Both fetal bovine serum and human serum, used earlier by other investigators as sources of NB, paradoxically inhibit the formation of these entities, and this inhibition is trypsin-sensitive, indicating a role for proteins in this inhibitory process. Fetuin-A, and to a lesser degree albumin, inhibit nanoparticle formation, an inhibition that is overcome with time, ending with formation of the so-called NB. Together, these data demonstrate that NB are most likely formed by calcium or apatite crystallization inhibitors that are somehow overwhelmed by excess calcium or calcium phosphate found in culture medium or in body fluids, thereby becoming seeds for calcification. The structures described earlier as NB may thus represent remnants and by-products of physiological mechanisms used for calcium homeostasis, a concept which explains the vast body of NB literature as well as explains the true origin of NB as lifeless protein-mineralo entities with questionable role in pathogenesis.

Show MeSH
Related in: MedlinePlus