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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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NLP associated with fetuin-A or albumin are morphologically similar.NLP were produced as described in Fig. 19, with 3 mM each of CaCl2, Na2CO3, and NaH2PO4 incubated with either 10 µM of BSF (A–C), 50 µM of HSA (D–F), or a combination of 10 µM of BSF and 50 µM of HSA (G–I). Incubation was done for 3 days (A, B, D, E, G, and H) or 1 week (C, E, and I) at room temperature. The initially clear solutions increased in turbidity with time, and by 3 days showed noticeable precipitation, which were then pelleted by centrifugation and washed with HEPES buffer once, followed by another wash with water, prior to SEM analysis. (B, E, and H) represent enlarged views of (A, D, and G), respectively. By one week of incubation, the round particles had coalesced to form films. Round particles or films seen with the 3 types of treatments appear virtually identical. Scale bars: 100 nm (B,D,F); 500 nm (A,C,E).
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pone-0004417-g020: NLP associated with fetuin-A or albumin are morphologically similar.NLP were produced as described in Fig. 19, with 3 mM each of CaCl2, Na2CO3, and NaH2PO4 incubated with either 10 µM of BSF (A–C), 50 µM of HSA (D–F), or a combination of 10 µM of BSF and 50 µM of HSA (G–I). Incubation was done for 3 days (A, B, D, E, G, and H) or 1 week (C, E, and I) at room temperature. The initially clear solutions increased in turbidity with time, and by 3 days showed noticeable precipitation, which were then pelleted by centrifugation and washed with HEPES buffer once, followed by another wash with water, prior to SEM analysis. (B, E, and H) represent enlarged views of (A, D, and G), respectively. By one week of incubation, the round particles had coalesced to form films. Round particles or films seen with the 3 types of treatments appear virtually identical. Scale bars: 100 nm (B,D,F); 500 nm (A,C,E).

Mentions: When the same incubation mixtures were allowed to sit at room temperature or at 4°C, we noticed precipitation that increased with time. In the case of albumin, precipitation could be noticed within 2 hours of incubation, which increased over the next 24 hours. With fetuin-A, precipitation was slower, becoming more pronounced after overnight incubation and which continued to increase for the next three days. When examined by SEM, both fetuin-A and albumin complexes showed typical round, amorphous NLP structures (Fig. 20A, B, D, E, G, and H) that further coalesced into film-like crystalline structures with incubation (Fig. 20C, F, and I). Precipitates obtained from the three different types of treatment produced virtually identical morphologies. That both fetuin-A and albumin were associated with these precipitates could be ascertained by SDS-PAGE, which demonstrated a dose-dependent increase in the amount of proteins found in the precipitating NLP as a function of the input protein used (Fig. 21).


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)

NLP associated with fetuin-A or albumin are morphologically similar.NLP were produced as described in Fig. 19, with 3 mM each of CaCl2, Na2CO3, and NaH2PO4 incubated with either 10 µM of BSF (A–C), 50 µM of HSA (D–F), or a combination of 10 µM of BSF and 50 µM of HSA (G–I). Incubation was done for 3 days (A, B, D, E, G, and H) or 1 week (C, E, and I) at room temperature. The initially clear solutions increased in turbidity with time, and by 3 days showed noticeable precipitation, which were then pelleted by centrifugation and washed with HEPES buffer once, followed by another wash with water, prior to SEM analysis. (B, E, and H) represent enlarged views of (A, D, and G), respectively. By one week of incubation, the round particles had coalesced to form films. Round particles or films seen with the 3 types of treatments appear virtually identical. Scale bars: 100 nm (B,D,F); 500 nm (A,C,E).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004417-g020: NLP associated with fetuin-A or albumin are morphologically similar.NLP were produced as described in Fig. 19, with 3 mM each of CaCl2, Na2CO3, and NaH2PO4 incubated with either 10 µM of BSF (A–C), 50 µM of HSA (D–F), or a combination of 10 µM of BSF and 50 µM of HSA (G–I). Incubation was done for 3 days (A, B, D, E, G, and H) or 1 week (C, E, and I) at room temperature. The initially clear solutions increased in turbidity with time, and by 3 days showed noticeable precipitation, which were then pelleted by centrifugation and washed with HEPES buffer once, followed by another wash with water, prior to SEM analysis. (B, E, and H) represent enlarged views of (A, D, and G), respectively. By one week of incubation, the round particles had coalesced to form films. Round particles or films seen with the 3 types of treatments appear virtually identical. Scale bars: 100 nm (B,D,F); 500 nm (A,C,E).
Mentions: When the same incubation mixtures were allowed to sit at room temperature or at 4°C, we noticed precipitation that increased with time. In the case of albumin, precipitation could be noticed within 2 hours of incubation, which increased over the next 24 hours. With fetuin-A, precipitation was slower, becoming more pronounced after overnight incubation and which continued to increase for the next three days. When examined by SEM, both fetuin-A and albumin complexes showed typical round, amorphous NLP structures (Fig. 20A, B, D, E, G, and H) that further coalesced into film-like crystalline structures with incubation (Fig. 20C, F, and I). Precipitates obtained from the three different types of treatment produced virtually identical morphologies. That both fetuin-A and albumin were associated with these precipitates could be ascertained by SDS-PAGE, which demonstrated a dose-dependent increase in the amount of proteins found in the precipitating NLP as a function of the input protein used (Fig. 21).

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