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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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Powder X-ray diffraction analysis of NLP and NB.(A–D) Various amorphous and crystalline patterns are associated with NLP. NLP were prepared from the inoculation of 3 mM each of CaCl2, Na2CO3, and NaH2PO4 into DMEM. NLP were examined by XRD (A,B) immediately after formation, which revealed either an amorphous pattern (A) or the presence of the mono-calcium form CaHPO4(H2O)2, or they were examined after the following periods of incubation in DMEM at 37°C: (C) 1 day; and (D) 4 days. (E) NB obtained from DMEM containing 10% HS. (F) “Nanons” after one week in DMEM without serum. Peaks were assigned the specified chemical structure after comparing peaks with the database. With the exception of (C), which revealed the Ca9 form of HAP, the peaks seen in (D–F) all corresponded to Ca10 HAP. Peaks at 32.0 degrees and 31.8 degrees correspond to the Ca9 and Ca10 forms of HAP, respectively. Notice also the small peaks at 66.2 degrees seen only with the Ca10 form (D–F). Commercial powders of (G) CaCO3, (H) Ca3(PO4)2, and (I) HAP were included as controls.
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pone-0004417-g003: Powder X-ray diffraction analysis of NLP and NB.(A–D) Various amorphous and crystalline patterns are associated with NLP. NLP were prepared from the inoculation of 3 mM each of CaCl2, Na2CO3, and NaH2PO4 into DMEM. NLP were examined by XRD (A,B) immediately after formation, which revealed either an amorphous pattern (A) or the presence of the mono-calcium form CaHPO4(H2O)2, or they were examined after the following periods of incubation in DMEM at 37°C: (C) 1 day; and (D) 4 days. (E) NB obtained from DMEM containing 10% HS. (F) “Nanons” after one week in DMEM without serum. Peaks were assigned the specified chemical structure after comparing peaks with the database. With the exception of (C), which revealed the Ca9 form of HAP, the peaks seen in (D–F) all corresponded to Ca10 HAP. Peaks at 32.0 degrees and 31.8 degrees correspond to the Ca9 and Ca10 forms of HAP, respectively. Notice also the small peaks at 66.2 degrees seen only with the Ca10 form (D–F). Commercial powders of (G) CaCO3, (H) Ca3(PO4)2, and (I) HAP were included as controls.

Mentions: Conversion to HAP could be ascertained by X-ray diffraction (XRD) analysis (Fig. 3), which also revealed a time and temperature dependent process. Immediately after formation, some of the NLP complexes revealed either amorphous patterns with no discernible crystalline peaks (Fig. 3A) or mono-calcium phosphate peaks (Fig. 3B). Following incubation at room temperature or 37°C, peaks eventually shifted to correspond to larger crystalline forms, reaching eventually Ca9 calcium phosphate compounds or Ca10 compounds corresponding to HAP within 1–2 days (Fig. 3C and D; compare signals with the CaCO3, calcium phosphate, HAP controls shown in Fig. 3G–I). Accumulation of HAP showed marked variability as a function of time, with some samples displaying HAP peaks after only 30 minutes of incubation, but typically more extensive incubations varying between several hours to 2 days were needed for this conversion. Even at room temperature or at 4°C, conversion to HAP could still be seen, albeit with much slower kinetics (not shown). In fact, samples that had initially shown only amorphous patterns and that had been washed and suspended in water alone at 4°C also showed conversion to HAP when examined two weeks later (not shown). Based on serial observations made during regular intervals, there appeared again to be a rapid conversion to HAP within a short span of time; however, the onset of this sharp HAP conversion could not be defined accurately given the wide margin of error seen with each experiment.


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)

Powder X-ray diffraction analysis of NLP and NB.(A–D) Various amorphous and crystalline patterns are associated with NLP. NLP were prepared from the inoculation of 3 mM each of CaCl2, Na2CO3, and NaH2PO4 into DMEM. NLP were examined by XRD (A,B) immediately after formation, which revealed either an amorphous pattern (A) or the presence of the mono-calcium form CaHPO4(H2O)2, or they were examined after the following periods of incubation in DMEM at 37°C: (C) 1 day; and (D) 4 days. (E) NB obtained from DMEM containing 10% HS. (F) “Nanons” after one week in DMEM without serum. Peaks were assigned the specified chemical structure after comparing peaks with the database. With the exception of (C), which revealed the Ca9 form of HAP, the peaks seen in (D–F) all corresponded to Ca10 HAP. Peaks at 32.0 degrees and 31.8 degrees correspond to the Ca9 and Ca10 forms of HAP, respectively. Notice also the small peaks at 66.2 degrees seen only with the Ca10 form (D–F). Commercial powders of (G) CaCO3, (H) Ca3(PO4)2, and (I) HAP were included as controls.
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Related In: Results  -  Collection

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pone-0004417-g003: Powder X-ray diffraction analysis of NLP and NB.(A–D) Various amorphous and crystalline patterns are associated with NLP. NLP were prepared from the inoculation of 3 mM each of CaCl2, Na2CO3, and NaH2PO4 into DMEM. NLP were examined by XRD (A,B) immediately after formation, which revealed either an amorphous pattern (A) or the presence of the mono-calcium form CaHPO4(H2O)2, or they were examined after the following periods of incubation in DMEM at 37°C: (C) 1 day; and (D) 4 days. (E) NB obtained from DMEM containing 10% HS. (F) “Nanons” after one week in DMEM without serum. Peaks were assigned the specified chemical structure after comparing peaks with the database. With the exception of (C), which revealed the Ca9 form of HAP, the peaks seen in (D–F) all corresponded to Ca10 HAP. Peaks at 32.0 degrees and 31.8 degrees correspond to the Ca9 and Ca10 forms of HAP, respectively. Notice also the small peaks at 66.2 degrees seen only with the Ca10 form (D–F). Commercial powders of (G) CaCO3, (H) Ca3(PO4)2, and (I) HAP were included as controls.
Mentions: Conversion to HAP could be ascertained by X-ray diffraction (XRD) analysis (Fig. 3), which also revealed a time and temperature dependent process. Immediately after formation, some of the NLP complexes revealed either amorphous patterns with no discernible crystalline peaks (Fig. 3A) or mono-calcium phosphate peaks (Fig. 3B). Following incubation at room temperature or 37°C, peaks eventually shifted to correspond to larger crystalline forms, reaching eventually Ca9 calcium phosphate compounds or Ca10 compounds corresponding to HAP within 1–2 days (Fig. 3C and D; compare signals with the CaCO3, calcium phosphate, HAP controls shown in Fig. 3G–I). Accumulation of HAP showed marked variability as a function of time, with some samples displaying HAP peaks after only 30 minutes of incubation, but typically more extensive incubations varying between several hours to 2 days were needed for this conversion. Even at room temperature or at 4°C, conversion to HAP could still be seen, albeit with much slower kinetics (not shown). In fact, samples that had initially shown only amorphous patterns and that had been washed and suspended in water alone at 4°C also showed conversion to HAP when examined two weeks later (not shown). Based on serial observations made during regular intervals, there appeared again to be a rapid conversion to HAP within a short span of time; however, the onset of this sharp HAP conversion could not be defined accurately given the wide margin of error seen with each experiment.

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