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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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Negative-staining TEM and electron diffraction patterns of NLP and NB.(A–G) NLP were prepared as in Fig. 3 and submitted to TEM after incubation in DMEM at 37°C for either 2 days (A–C, G), 2 weeks (D), or 1 month (E and F). Round, dividing, or budding NLP are shown in close association with a crystalline phase, which shows either as a network of filamentous/membranous materials or as spindles/needles. The electron diffraction pattern characteristic of polycrystalline material obtained for both phases is shown in the inset. (B–D) show NLP with coccoid shapes and diameters smaller than 100 nm among larger particles. (D–F) Crystalline biofilms associated with NLP are seen with longer incubations. (F) is a magnified image of (E) depicting the transition between the round NLP and the crystalline matrix. (H and I) NB showing cell-dividing forms similar to NLP. (H) NB obtained from HS and (I) “nanons,” as in Fig. 3. Small crystalline projections can be distinguished on their surface. NB look virtually indistinguishable from NLP (compare with image G taken of NLP). Commercial (J) CaCO3, (K) Ca3(PO4)2, and (L) HAP produce a higher degree of crystallinity compared to NLP, NB, and “nanons” (insets). Scale bars: 100 nm (B–D, H–J, L); 200 nm (A, E–G, K).
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pone-0004417-g006: Negative-staining TEM and electron diffraction patterns of NLP and NB.(A–G) NLP were prepared as in Fig. 3 and submitted to TEM after incubation in DMEM at 37°C for either 2 days (A–C, G), 2 weeks (D), or 1 month (E and F). Round, dividing, or budding NLP are shown in close association with a crystalline phase, which shows either as a network of filamentous/membranous materials or as spindles/needles. The electron diffraction pattern characteristic of polycrystalline material obtained for both phases is shown in the inset. (B–D) show NLP with coccoid shapes and diameters smaller than 100 nm among larger particles. (D–F) Crystalline biofilms associated with NLP are seen with longer incubations. (F) is a magnified image of (E) depicting the transition between the round NLP and the crystalline matrix. (H and I) NB showing cell-dividing forms similar to NLP. (H) NB obtained from HS and (I) “nanons,” as in Fig. 3. Small crystalline projections can be distinguished on their surface. NB look virtually indistinguishable from NLP (compare with image G taken of NLP). Commercial (J) CaCO3, (K) Ca3(PO4)2, and (L) HAP produce a higher degree of crystallinity compared to NLP, NB, and “nanons” (insets). Scale bars: 100 nm (B–D, H–J, L); 200 nm (A, E–G, K).

Mentions: The crystalline nature of both NLP and NB was characterized further by X-ray diffraction pattern obtained on selected specimen areas magnified by TEM (Fig. 6A–I and insets). NLP and NB produced similar morphologies that will be discussed in more detail in a later section (compare Fig. 6A–G obtained for NLP with Fig. 6H and I for NB). NLP, formed by precipitation using different inputs of carbonate and phosphate, produced X-ray diffraction patterns characteristic of polycrystalline material as shown by the presence of several concentric rings seen irrespective of the conditions used for their preparation (Fig. 6A–G). The patterns obtained for human NB (Fig. 6H) and the NB strain “nanons” maintained in FBS (Fig. 6I) also revealed similar fuzzy rings indicative of polycrystalline material. It should be noted however that NLP, NB, and “nanons” produced electron diffraction patterns with a lower degree of crystallinity compared to commercial lots of calcium carbonate, tri-calcium phosphate, and HAP (Fig. 6J–L). These commercial preparations revealed preferred angles of diffraction by means of characteristic arrays of dot (Fig. 6J–L) which could not be distinguished in the patterns obtained for NLP or NB (Fig. 6A–I). With continued incubation of NLP at 37°C for several days, there was a gradual shift toward crystalline patterns, an observation similar to that made with the other spectroscopies. That is, the patterns obtained for NLP were seen to gradually show an increase in the number and the intensity of the concentric rings. However, such patterns were still lacking in the arrays of dots typical of synthetic preparations of HAP (not shown), indicating the relatively low levels of crystallinity seen associated with NLP and NB. This low crystallinity seen for NLP, NB, and “nanons” is consistent with the presence of an amorphous phase of calcium carbonate and calcium phosphate in these nanoparticles. Similar patterns have been obtained for biological HAP found in the bone [43]. Such amorphous-crystalline interfaces had earlier been attributed to the incorporation of carbonate ions into HAP, a process thought to alter the molecular organization and structure of the resultant HAP complex [44].


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)

Negative-staining TEM and electron diffraction patterns of NLP and NB.(A–G) NLP were prepared as in Fig. 3 and submitted to TEM after incubation in DMEM at 37°C for either 2 days (A–C, G), 2 weeks (D), or 1 month (E and F). Round, dividing, or budding NLP are shown in close association with a crystalline phase, which shows either as a network of filamentous/membranous materials or as spindles/needles. The electron diffraction pattern characteristic of polycrystalline material obtained for both phases is shown in the inset. (B–D) show NLP with coccoid shapes and diameters smaller than 100 nm among larger particles. (D–F) Crystalline biofilms associated with NLP are seen with longer incubations. (F) is a magnified image of (E) depicting the transition between the round NLP and the crystalline matrix. (H and I) NB showing cell-dividing forms similar to NLP. (H) NB obtained from HS and (I) “nanons,” as in Fig. 3. Small crystalline projections can be distinguished on their surface. NB look virtually indistinguishable from NLP (compare with image G taken of NLP). Commercial (J) CaCO3, (K) Ca3(PO4)2, and (L) HAP produce a higher degree of crystallinity compared to NLP, NB, and “nanons” (insets). Scale bars: 100 nm (B–D, H–J, L); 200 nm (A, E–G, K).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2636888&req=5

pone-0004417-g006: Negative-staining TEM and electron diffraction patterns of NLP and NB.(A–G) NLP were prepared as in Fig. 3 and submitted to TEM after incubation in DMEM at 37°C for either 2 days (A–C, G), 2 weeks (D), or 1 month (E and F). Round, dividing, or budding NLP are shown in close association with a crystalline phase, which shows either as a network of filamentous/membranous materials or as spindles/needles. The electron diffraction pattern characteristic of polycrystalline material obtained for both phases is shown in the inset. (B–D) show NLP with coccoid shapes and diameters smaller than 100 nm among larger particles. (D–F) Crystalline biofilms associated with NLP are seen with longer incubations. (F) is a magnified image of (E) depicting the transition between the round NLP and the crystalline matrix. (H and I) NB showing cell-dividing forms similar to NLP. (H) NB obtained from HS and (I) “nanons,” as in Fig. 3. Small crystalline projections can be distinguished on their surface. NB look virtually indistinguishable from NLP (compare with image G taken of NLP). Commercial (J) CaCO3, (K) Ca3(PO4)2, and (L) HAP produce a higher degree of crystallinity compared to NLP, NB, and “nanons” (insets). Scale bars: 100 nm (B–D, H–J, L); 200 nm (A, E–G, K).
Mentions: The crystalline nature of both NLP and NB was characterized further by X-ray diffraction pattern obtained on selected specimen areas magnified by TEM (Fig. 6A–I and insets). NLP and NB produced similar morphologies that will be discussed in more detail in a later section (compare Fig. 6A–G obtained for NLP with Fig. 6H and I for NB). NLP, formed by precipitation using different inputs of carbonate and phosphate, produced X-ray diffraction patterns characteristic of polycrystalline material as shown by the presence of several concentric rings seen irrespective of the conditions used for their preparation (Fig. 6A–G). The patterns obtained for human NB (Fig. 6H) and the NB strain “nanons” maintained in FBS (Fig. 6I) also revealed similar fuzzy rings indicative of polycrystalline material. It should be noted however that NLP, NB, and “nanons” produced electron diffraction patterns with a lower degree of crystallinity compared to commercial lots of calcium carbonate, tri-calcium phosphate, and HAP (Fig. 6J–L). These commercial preparations revealed preferred angles of diffraction by means of characteristic arrays of dot (Fig. 6J–L) which could not be distinguished in the patterns obtained for NLP or NB (Fig. 6A–I). With continued incubation of NLP at 37°C for several days, there was a gradual shift toward crystalline patterns, an observation similar to that made with the other spectroscopies. That is, the patterns obtained for NLP were seen to gradually show an increase in the number and the intensity of the concentric rings. However, such patterns were still lacking in the arrays of dots typical of synthetic preparations of HAP (not shown), indicating the relatively low levels of crystallinity seen associated with NLP and NB. This low crystallinity seen for NLP, NB, and “nanons” is consistent with the presence of an amorphous phase of calcium carbonate and calcium phosphate in these nanoparticles. Similar patterns have been obtained for biological HAP found in the bone [43]. Such amorphous-crystalline interfaces had earlier been attributed to the incorporation of carbonate ions into HAP, a process thought to alter the molecular organization and structure of the resultant HAP complex [44].

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