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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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Sequence homology between fetuin-A and albumin.(A) Sequences were compared with the ClustalW program. The term “identity” refers to the presence of the same amino acids while “similarity” refers to the presence of different amino acids with similar chemical properties as assessed with the Gonnet matrix. Note limited homologies between fetuin-A and albumin from both bovine and human species. (B) Sequence alignment was done using the complete primary sequence of BSF (341 a.a.) as basis for comparison, while portions of the longer HSF (349 a.a.), BSA (583 a.a.), and HSA (585 a.a.) sequences were deleted at various sites for purposes of fitting the alignment. Identical amino acids are highlighted in yellow. The meter on top of the sequences refers to BSF only.
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pone-0004417-g017: Sequence homology between fetuin-A and albumin.(A) Sequences were compared with the ClustalW program. The term “identity” refers to the presence of the same amino acids while “similarity” refers to the presence of different amino acids with similar chemical properties as assessed with the Gonnet matrix. Note limited homologies between fetuin-A and albumin from both bovine and human species. (B) Sequence alignment was done using the complete primary sequence of BSF (341 a.a.) as basis for comparison, while portions of the longer HSF (349 a.a.), BSA (583 a.a.), and HSA (585 a.a.) sequences were deleted at various sites for purposes of fitting the alignment. Identical amino acids are highlighted in yellow. The meter on top of the sequences refers to BSF only.

Mentions: In line with this reasoning, it should be noted that fetuin-A (349 amino acids in the human form; 341 a.a., bovine) and albumin (585 a.a., human; 583 a.a., bovine) are known to share limited homology ranging from 11 to 13% identity and 17–19% similarity (Fig. 17A). The identity regions, while scattered throughout the length of the two sequences, appear to be a bit more clustered toward the N-terminus of both proteins (Fig. 17B). Accordingly, only fetuin-A is known to harbor a unique N-terminal cystatin-like domain which has been shown to be required for its high calcification inhibitory activity [25]. Mutational analysis and computer modeling of domain structures have suggested that a dense array of acidic residues on an extended β-sheet of the cystatin-like domain of fetuin-A mediates efficient inhibition of this type of mineralization [25]. The low homology between the two proteins may also explain earlier immunoblotting experiments that demonstrated weak immunological cross-reactivity between the two species that became apparent only following protein reduction and alkylation, which may have unfolded hidden homologies [61].


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)

Sequence homology between fetuin-A and albumin.(A) Sequences were compared with the ClustalW program. The term “identity” refers to the presence of the same amino acids while “similarity” refers to the presence of different amino acids with similar chemical properties as assessed with the Gonnet matrix. Note limited homologies between fetuin-A and albumin from both bovine and human species. (B) Sequence alignment was done using the complete primary sequence of BSF (341 a.a.) as basis for comparison, while portions of the longer HSF (349 a.a.), BSA (583 a.a.), and HSA (585 a.a.) sequences were deleted at various sites for purposes of fitting the alignment. Identical amino acids are highlighted in yellow. The meter on top of the sequences refers to BSF only.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004417-g017: Sequence homology between fetuin-A and albumin.(A) Sequences were compared with the ClustalW program. The term “identity” refers to the presence of the same amino acids while “similarity” refers to the presence of different amino acids with similar chemical properties as assessed with the Gonnet matrix. Note limited homologies between fetuin-A and albumin from both bovine and human species. (B) Sequence alignment was done using the complete primary sequence of BSF (341 a.a.) as basis for comparison, while portions of the longer HSF (349 a.a.), BSA (583 a.a.), and HSA (585 a.a.) sequences were deleted at various sites for purposes of fitting the alignment. Identical amino acids are highlighted in yellow. The meter on top of the sequences refers to BSF only.
Mentions: In line with this reasoning, it should be noted that fetuin-A (349 amino acids in the human form; 341 a.a., bovine) and albumin (585 a.a., human; 583 a.a., bovine) are known to share limited homology ranging from 11 to 13% identity and 17–19% similarity (Fig. 17A). The identity regions, while scattered throughout the length of the two sequences, appear to be a bit more clustered toward the N-terminus of both proteins (Fig. 17B). Accordingly, only fetuin-A is known to harbor a unique N-terminal cystatin-like domain which has been shown to be required for its high calcification inhibitory activity [25]. Mutational analysis and computer modeling of domain structures have suggested that a dense array of acidic residues on an extended β-sheet of the cystatin-like domain of fetuin-A mediates efficient inhibition of this type of mineralization [25]. The low homology between the two proteins may also explain earlier immunoblotting experiments that demonstrated weak immunological cross-reactivity between the two species that became apparent only following protein reduction and alkylation, which may have unfolded hidden homologies [61].

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