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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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Effect of trypsin on NLP formation.NLP were prepared by adding 1 mM of CaCl2, Na2CO3, and NaH2PO4 to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).
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pone-0004417-g010: Effect of trypsin on NLP formation.NLP were prepared by adding 1 mM of CaCl2, Na2CO3, and NaH2PO4 to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).

Mentions: The inhibitory effects of serum on NLP formation could be overcome in the presence of trypsin. Figure 10A and C show the serum-dependent inhibition on NLP precipitation produced by 1 mM each of calcium, carbonate, and phosphate inoculated into DMEM, followed by overnight incubation at 37°C. As expected from earlier experiments, increasing amounts of FBS (Fig. 10A) or HS (Fig. 10C) were seen to inhibit NLP formation. Treatment with 0.5% trypsin under these conditions not only removed the serum-mediated inhibition but appeared to facilitate NLP formation as a function of the amount of serum added, with more serum inducing more precipitation (Fig. 10B and D). That is, rather than being inhibitory, both FBS and HS added in the presence of trypsin now induced a dose-dependent accumulation of NLP. As an additional control, the initial serum-induced inhibition on NLP formation could be seen to be sustained for at least 3 days in the absence of trypsin (Fig. 10E and G), an inhibition that was again more noticeable with FBS compared to HS. By comparison, the trypsin effect was stable, resulting in steady accumulation of NLP with incubation time (Fig. 10F and H).


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)

Effect of trypsin on NLP formation.NLP were prepared by adding 1 mM of CaCl2, Na2CO3, and NaH2PO4 to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636888&req=5

pone-0004417-g010: Effect of trypsin on NLP formation.NLP were prepared by adding 1 mM of CaCl2, Na2CO3, and NaH2PO4 to DMEM containing the indicated amounts of serum, in the presence or absence of 0.5% trypsin, followed by incubation at 37°C for 1 day (A–D) or 3 days (E–H). Trypsin was shown not only to release the inhibition caused by FBS or HS, but also to produce a dose-dependent increase in the amount of precipitation that increased with the serum dosage. (I) Trypsin treatment on NLP as demonstrated by SDS-PAGE. NLP formed as in Fig. 9 from 3 mM each of the 3 precipitating reagents as well as 1% FBS in DMEM, either in the absence (lane 1) or presence (lane 2) of 0.5% trypsin. After overnight incubation of the 1 ml incubation mixture at 37°C, 100 µl was removed, pelleted by centrifugation, and washed twice in DMEM, and resuspended in 16 µl of 50 mM EDTA in double-distilled water for SDS-PAGE. Lane 3 contains 5 µg of trypsin, as control, which shows as a 25 kDa band. Note a prominent 72 kDa band and two fainter 54 kDa and 30 kDa bands associated with FBS NLP (lane 1), all of which disappears with trypsin treatment (lane 2).
Mentions: The inhibitory effects of serum on NLP formation could be overcome in the presence of trypsin. Figure 10A and C show the serum-dependent inhibition on NLP precipitation produced by 1 mM each of calcium, carbonate, and phosphate inoculated into DMEM, followed by overnight incubation at 37°C. As expected from earlier experiments, increasing amounts of FBS (Fig. 10A) or HS (Fig. 10C) were seen to inhibit NLP formation. Treatment with 0.5% trypsin under these conditions not only removed the serum-mediated inhibition but appeared to facilitate NLP formation as a function of the amount of serum added, with more serum inducing more precipitation (Fig. 10B and D). That is, rather than being inhibitory, both FBS and HS added in the presence of trypsin now induced a dose-dependent accumulation of NLP. As an additional control, the initial serum-induced inhibition on NLP formation could be seen to be sustained for at least 3 days in the absence of trypsin (Fig. 10E and G), an inhibition that was again more noticeable with FBS compared to HS. By comparison, the trypsin effect was stable, resulting in steady accumulation of NLP with incubation time (Fig. 10F and H).

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