Limits...
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.

Show MeSH

Related in: MedlinePlus

Protein profiles of NLP and NB as determined by their passage through serum-free or serum-containing medium.(A) Protein profile of HS NB maintained in DMEM containing 5% HS (lane 1) followed by transfer to serum-free DMEM and incubation for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), after which proteins were analyzed by SDS-PAGE. A gradual disappearance of proteins bands is seen with increased incubation time in serum-free medium. (B) Protein profile of NB strain DSM 5821 after 1 day in DMEM containing 2% FBS (lane 1), followed by transfer to serum-free DMEM for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), again showing a gradual disappearance of bands. (C) Protein profiles of NB obtained from 10% HS as in Fig. 3 (lane 1) and HS NLP formed as in Fig. 3 except that 5% HS was present in the precipitating mixture (lane 2). HS NLP were then transferred to DMEM containing 5% HS and incubated for the following periods of time: 1 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). A gradual increase of bands of low molecular weight could be seen along with a fading of the high molecular weight bands, especially the 70–72 kDa band. (D) Protein profiles of NB strain DSM 5820 maintained in 10% γ-irradiated FBS (lane 1) and FBS NLP obtained as in Fig. 3, except that 5% FBS was added to the precipitating mixture (lane 2). FBS NLP were inoculated into DMEM containing 5% FBS and incubated for the following periods of time: 2 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). By day 14, the protein profile of these FBS NLP, with an increase in the number of bands and a loss of the 70–72 kDa band, closely resembled that of DSM 5820 (lane 1).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2636888&req=5

pone-0004417-g013: Protein profiles of NLP and NB as determined by their passage through serum-free or serum-containing medium.(A) Protein profile of HS NB maintained in DMEM containing 5% HS (lane 1) followed by transfer to serum-free DMEM and incubation for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), after which proteins were analyzed by SDS-PAGE. A gradual disappearance of proteins bands is seen with increased incubation time in serum-free medium. (B) Protein profile of NB strain DSM 5821 after 1 day in DMEM containing 2% FBS (lane 1), followed by transfer to serum-free DMEM for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), again showing a gradual disappearance of bands. (C) Protein profiles of NB obtained from 10% HS as in Fig. 3 (lane 1) and HS NLP formed as in Fig. 3 except that 5% HS was present in the precipitating mixture (lane 2). HS NLP were then transferred to DMEM containing 5% HS and incubated for the following periods of time: 1 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). A gradual increase of bands of low molecular weight could be seen along with a fading of the high molecular weight bands, especially the 70–72 kDa band. (D) Protein profiles of NB strain DSM 5820 maintained in 10% γ-irradiated FBS (lane 1) and FBS NLP obtained as in Fig. 3, except that 5% FBS was added to the precipitating mixture (lane 2). FBS NLP were inoculated into DMEM containing 5% FBS and incubated for the following periods of time: 2 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). By day 14, the protein profile of these FBS NLP, with an increase in the number of bands and a loss of the 70–72 kDa band, closely resembled that of DSM 5820 (lane 1).

Mentions: Since serum has been routinely used both as a source progenitor as well as a nutrient basis for NB, we next sought to study further the influence of the serum milieu on the protein profiles of both NLP and NB. We reasoned that the protein coating of NLP/NB should mirror their passage history with regards to the presence or absence of serum. Figure 13A shows the protein gel profile of human NB maintained in 5% HS (lane 1) that had been inoculated into serum-free DMEM (lanes 2–4). The gradual disappearance of protein bands was obvious over a course of several days, with the three more predominant bands remaining by the end of day 6 (lane 4). The same phenomenon could be seen with the DSM 5821 strain, cultured in 2% FBS, that was subsequently inoculated into serum-free FBS (Fig. 13B). Again, over a period of several days, only a few major bands remained (contrast lane 4 obtained on day 6 of incubation with lane 1, obtained for DSM 5821 particles maintained in 2% FBS). Conversely, similar passage histories could be established with human NLP obtained with exogenous calcium-carbonate-phosphate ions (Fig. 13C). Here, human NLP showing only three major bands (Fig. 13C, lane 2) were first contrasted with the smear of bands seen with NB obtained by inoculation of 10% HS into DMEM using published protocols as before (Fig. 13C, lane 1). Figure 13C shows that these same NLP could be made to acquire many other protein bands when inoculated into DMEM containing 10% HS (lanes 3–7). By day 10, there was a large increase of lower molecular weight bands accompanied by a decrease of the 70 kDa (Fig. 13C, lane 6). After 14 days of incubation in the presence of serum, the protein profile seen associated with the precipitating particles (Fig. 13C, lane 7) closely resembled the protein profile of NB (lane 1). Similar results were obtained with NLP formed in the absence of serum and that had been inoculated subsequently into DMEM containing 5% FBS (Fig. 13D, lanes 2–7). There was a gradual smearing of the protein bands, with a loss of the 70 kDa band and an increase of low molecular weight bands (see Fig. 13D, lanes 6 and 7, corresponding respectively to days 10 and 14 of incubation in the presence of 5% FBS). Again, this profile was virtually indistinguishable from that obtained for DSM 5820 maintained in 10% FBS (Fig. 13D, lane 1).


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 NLP and NB as determined by their passage through serum-free or serum-containing medium.(A) Protein profile of HS NB maintained in DMEM containing 5% HS (lane 1) followed by transfer to serum-free DMEM and incubation for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), after which proteins were analyzed by SDS-PAGE. A gradual disappearance of proteins bands is seen with increased incubation time in serum-free medium. (B) Protein profile of NB strain DSM 5821 after 1 day in DMEM containing 2% FBS (lane 1), followed by transfer to serum-free DMEM for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), again showing a gradual disappearance of bands. (C) Protein profiles of NB obtained from 10% HS as in Fig. 3 (lane 1) and HS NLP formed as in Fig. 3 except that 5% HS was present in the precipitating mixture (lane 2). HS NLP were then transferred to DMEM containing 5% HS and incubated for the following periods of time: 1 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). A gradual increase of bands of low molecular weight could be seen along with a fading of the high molecular weight bands, especially the 70–72 kDa band. (D) Protein profiles of NB strain DSM 5820 maintained in 10% γ-irradiated FBS (lane 1) and FBS NLP obtained as in Fig. 3, except that 5% FBS was added to the precipitating mixture (lane 2). FBS NLP were inoculated into DMEM containing 5% FBS and incubated for the following periods of time: 2 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). By day 14, the protein profile of these FBS NLP, with an increase in the number of bands and a loss of the 70–72 kDa band, closely resembled that of DSM 5820 (lane 1).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0004417-g013: Protein profiles of NLP and NB as determined by their passage through serum-free or serum-containing medium.(A) Protein profile of HS NB maintained in DMEM containing 5% HS (lane 1) followed by transfer to serum-free DMEM and incubation for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), after which proteins were analyzed by SDS-PAGE. A gradual disappearance of proteins bands is seen with increased incubation time in serum-free medium. (B) Protein profile of NB strain DSM 5821 after 1 day in DMEM containing 2% FBS (lane 1), followed by transfer to serum-free DMEM for 1 day (lane 2), 4 days (lane 3), and 6 days (lane 4), again showing a gradual disappearance of bands. (C) Protein profiles of NB obtained from 10% HS as in Fig. 3 (lane 1) and HS NLP formed as in Fig. 3 except that 5% HS was present in the precipitating mixture (lane 2). HS NLP were then transferred to DMEM containing 5% HS and incubated for the following periods of time: 1 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). A gradual increase of bands of low molecular weight could be seen along with a fading of the high molecular weight bands, especially the 70–72 kDa band. (D) Protein profiles of NB strain DSM 5820 maintained in 10% γ-irradiated FBS (lane 1) and FBS NLP obtained as in Fig. 3, except that 5% FBS was added to the precipitating mixture (lane 2). FBS NLP were inoculated into DMEM containing 5% FBS and incubated for the following periods of time: 2 hr (lane 3), 1 day (lane 4), 4 days (lane 5), 10 days (lane 6), and 14 days (lane 7). By day 14, the protein profile of these FBS NLP, with an increase in the number of bands and a loss of the 70–72 kDa band, closely resembled that of DSM 5820 (lane 1).
Mentions: Since serum has been routinely used both as a source progenitor as well as a nutrient basis for NB, we next sought to study further the influence of the serum milieu on the protein profiles of both NLP and NB. We reasoned that the protein coating of NLP/NB should mirror their passage history with regards to the presence or absence of serum. Figure 13A shows the protein gel profile of human NB maintained in 5% HS (lane 1) that had been inoculated into serum-free DMEM (lanes 2–4). The gradual disappearance of protein bands was obvious over a course of several days, with the three more predominant bands remaining by the end of day 6 (lane 4). The same phenomenon could be seen with the DSM 5821 strain, cultured in 2% FBS, that was subsequently inoculated into serum-free FBS (Fig. 13B). Again, over a period of several days, only a few major bands remained (contrast lane 4 obtained on day 6 of incubation with lane 1, obtained for DSM 5821 particles maintained in 2% FBS). Conversely, similar passage histories could be established with human NLP obtained with exogenous calcium-carbonate-phosphate ions (Fig. 13C). Here, human NLP showing only three major bands (Fig. 13C, lane 2) were first contrasted with the smear of bands seen with NB obtained by inoculation of 10% HS into DMEM using published protocols as before (Fig. 13C, lane 1). Figure 13C shows that these same NLP could be made to acquire many other protein bands when inoculated into DMEM containing 10% HS (lanes 3–7). By day 10, there was a large increase of lower molecular weight bands accompanied by a decrease of the 70 kDa (Fig. 13C, lane 6). After 14 days of incubation in the presence of serum, the protein profile seen associated with the precipitating particles (Fig. 13C, lane 7) closely resembled the protein profile of NB (lane 1). Similar results were obtained with NLP formed in the absence of serum and that had been inoculated subsequently into DMEM containing 5% FBS (Fig. 13D, lanes 2–7). There was a gradual smearing of the protein bands, with a loss of the 70 kDa band and an increase of low molecular weight bands (see Fig. 13D, lanes 6 and 7, corresponding respectively to days 10 and 14 of incubation in the presence of 5% FBS). Again, this profile was virtually indistinguishable from that obtained for DSM 5820 maintained in 10% FBS (Fig. 13D, lane 1).

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