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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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Related in: MedlinePlus

Protein profiles of whole FBS and HS.SDS-PAGE of decreasing amounts of whole FBS (lanes 1–5), and HS (lanes 6–10). FBS or HS was used at 1∶100 dilution, with the following amounts of protein loaded onto each lane: lanes 1–5: 4 µg, 3.5 µg, 3 µg, 2.5 µg, and 1.5 µg, respectively; and lanes 6–10: 3 µg, 1.8 µg, 1.2 µg, 0.6 µg, and 0.3 µg, respectively. The white dots shown in lanes 5 and 10 correspond to the bands excised for protein identification by MALDI-TOF mass fingerprint analysis.
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pone-0004417-g012: Protein profiles of whole FBS and HS.SDS-PAGE of decreasing amounts of whole FBS (lanes 1–5), and HS (lanes 6–10). FBS or HS was used at 1∶100 dilution, with the following amounts of protein loaded onto each lane: lanes 1–5: 4 µg, 3.5 µg, 3 µg, 2.5 µg, and 1.5 µg, respectively; and lanes 6–10: 3 µg, 1.8 µg, 1.2 µg, 0.6 µg, and 0.3 µg, respectively. The white dots shown in lanes 5 and 10 correspond to the bands excised for protein identification by MALDI-TOF mass fingerprint analysis.

Mentions: To confirm this notion, we analyzed next the protein profiles of whole sera that were used for the preparation of NLP and NB. Both whole FBS and HS were found to be enriched for the exact three major protein bands associated with NLP, and their predominant presence could be more easily assessed through a dose-dependent dilution of the kind shown in Figure 12. As detailed through protein sequencing shown in the next section, the three major proteins associated with serum turned out to be identical to the exact three proteins associated with both NLP and NB.


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)

Protein profiles of whole FBS and HS.SDS-PAGE of decreasing amounts of whole FBS (lanes 1–5), and HS (lanes 6–10). FBS or HS was used at 1∶100 dilution, with the following amounts of protein loaded onto each lane: lanes 1–5: 4 µg, 3.5 µg, 3 µg, 2.5 µg, and 1.5 µg, respectively; and lanes 6–10: 3 µg, 1.8 µg, 1.2 µg, 0.6 µg, and 0.3 µg, respectively. The white dots shown in lanes 5 and 10 correspond to the bands excised for protein identification by MALDI-TOF mass fingerprint analysis.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004417-g012: Protein profiles of whole FBS and HS.SDS-PAGE of decreasing amounts of whole FBS (lanes 1–5), and HS (lanes 6–10). FBS or HS was used at 1∶100 dilution, with the following amounts of protein loaded onto each lane: lanes 1–5: 4 µg, 3.5 µg, 3 µg, 2.5 µg, and 1.5 µg, respectively; and lanes 6–10: 3 µg, 1.8 µg, 1.2 µg, 0.6 µg, and 0.3 µg, respectively. The white dots shown in lanes 5 and 10 correspond to the bands excised for protein identification by MALDI-TOF mass fingerprint analysis.
Mentions: To confirm this notion, we analyzed next the protein profiles of whole sera that were used for the preparation of NLP and NB. Both whole FBS and HS were found to be enriched for the exact three major protein bands associated with NLP, and their predominant presence could be more easily assessed through a dose-dependent dilution of the kind shown in Figure 12. As detailed through protein sequencing shown in the next section, the three major proteins associated with serum turned out to be identical to the exact three proteins associated with both NLP and NB.

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