Calcium and apatite granulations are demonstrated here to form in both human and fetal bovine serum in response to the simple addition of either calcium or phosphate, or a combination of both. These granulations are shown to represent precipitating complexes of protein and hydroxyapatite (HAP) that display marked pleomorphism, appearing as round, laminated particles, spindles, and films. These same complexes can be found in normal untreated serum, albeit at much lower amounts, and appear to result from the progressive binding of serum proteins with apatite until reaching saturation, upon which the mineralo-protein complexes precipitate. Chemically and morphologically, these complexes are virtually identical to the so-called nanobacteria (NB) implicated in numerous diseases and considered unusual for their small size, pleomorphism, and the presence of HAP. Like NB, serum granulations can seed particles upon transfer to serum-free medium, and their main protein constituents include albumin, complement components 3 and 4A, fetuin-A, and apolipoproteins A1 and B100, as well as other calcium and apatite binding proteins found in the serum. However, these serum mineralo-protein complexes are formed from the direct chemical binding of inorganic and organic phases, bypassing the need for any biological processes, including the long cultivation in cell culture conditions deemed necessary for the demonstration of NB. Thus, these serum granulations may result from physiologically inherent processes that become amplified with calcium phosphate loading or when subjected to culturing in medium. They may be viewed as simple mineralo-protein complexes formed from the deployment of calcification-inhibitory pathways used by the body to cope with excess calcium phosphate so as to prevent unwarranted calcification. Rather than representing novel pathophysiological mechanisms or exotic lifeforms, these results indicate that the entities described earlier as NB most likely originate from calcium and apatite binding factors in the serum, presumably calcification inhibitors, that upon saturation, form seeds for HAP deposition and growth. These calcium granulations are similar to those found in organisms throughout nature and may represent the products of more general calcium regulation pathways involved in the control of calcium storage, retrieval, tissue deposition, and disposal.
Recent evidence suggests a role for nanobacteria in a growing number of human diseases, including renal stone formation, cardiovascular diseases, and cancer. This large body of research studies promotes the view that nanobacteria are not only alive but that they are associated with disease pathogenesis. However, it is still unclear whether they represent novel life forms, overlooked nanometer-size bacteria, or some other primitive self-replicating microorganisms. Here, we report that CaCO 3 precipitates prepared in vitro are remarkably similar to purported nanobacteria in terms of their uniformly sized, membrane-delineated vesicular shapes, with cellular division-like formations and aggregations in the form of colonies. The gradual appearance of nanobacteria-like particles in incubated human serum as well as the changes seen with their size and shape can be influenced and explained by introducing varying levels of CO 2 and NaHCO 3 as well as other conditions known to influence the precipitation of CaCO 3 . Western blotting reveals that the monoclonal antibodies, claimed to be specific for nanobacteria, react in fact with serum albumin. Furthermore, nanobacteria-like particles obtained from human blood are able to withstand high doses of γ-irradiation up to 30 kGy, and no bacterial DNA is found by performing broad-range PCR amplifications. Collectively, our results provide a more plausible abiotic explanation for the unusual properties of purported nanobacteria.
Serum-derived granulations and purported nanobacteria (NB) are pleomorphic apatite structures shown to resemble calcium granules widely distributed in nature. They appear to be assembled through a dual inhibitory-seeding mechanism involving proteinaceous factors, as determined by protease (trypsin and chymotrypsin) and heat inactivation studies. When inoculated into cell culture medium, the purified proteins fetuin-A and albumin fail to induce mineralization, but they will readily combine with exogenously added calcium and phosphate, even in submillimolar amounts, to form complexes that will undergo morphological transitions from nanoparticles to spindles, films, and aggregates. As a mineralization inhibitor, fetuin-A is much more potent than albumin, and it will only seed particles at higher mineral-to-protein concentrations. Both proteins display a bell-shaped, dose-dependent relationship, indicative of the same dual inhibitory-seeding mechanism seen with whole serum. As ascertained by both seeding experiments and gel electrophoresis, fetuin-A is not only more dominant but it appears to compete avidly for nanoparticle binding at the expense of albumin. The nanoparticles formed in the presence of fetuin-A are smaller than their albumin counterparts, and they have a greater tendency to display a multi-layered ring morphology. In comparison, the particles seeded by albumin appear mostly incomplete, with single walls. Chemically, spectroscopically, and morphologically, the protein-mineral particles resemble closely serum granules and NB. These particles are thus seen to undergo an amorphous to crystalline transformation, the kinetics and completeness of which depend on the protein-to-mineral ratios, with low ratios favoring faster conversion to crystals. Our results point to a dual inhibitory-seeding, de-repression model for the assembly of particles in supersaturated solutions like serum. The presence of proteins and other inhibitory factors tend to block apatite nuclei formation or to stabilize the nascent nuclei as amorphous or semi-crystalline spherical nanoparticles, until the same inhibitory influences are overwhelmed or de-repressed, whereby the apatite nuclei grow in size to coalesce into crystalline spindles and films—a mechanism that may explain not only the formation of calcium granules in nature but also normal or ectopic calcification in the body.
The ninth component of complement (C9) and the pore-forming protein (PFP or perforin) from cytotoxic T lymphocytes polymerize to tubular lesions having an internal diameter of 100 A and 160 A, respectively, when bound to lipid bilayers. Polymerized C9, assembled by slow spontaneous or rapid Zn2+-induced polymerization, and polyperforin, which is assembled only in the presence of Ca2+, constitute large aqueous pores that are stable, nonselective for solutes, and insensitive to changes of membrane potential. Monospecific polyclonal antibodies to purified C9 and PFP show cross-reactivity, suggesting structural homology between the two molecules. The structural and functional homologies between these two killer molecules imply an active role for pore formation during cell lysis.
The major outer membrane protein from Neisseria gonorrhoeae was incorporated into artificial planar bilayer membranes by a detergent-dilution procedure. The integrated protein forms voltage-dependent aqueous pores with a minimal pore diameter estimated to be 11 A. A pore of this size suggests a role for this protein in macromolecular sieving at the level of the outer membrane. This protein self-associates preferentially in triplets of three equal unit conductance steps of 130 pS (in 0.1 M NaCl) each. The two-state model may be applied to explain the voltage-dependent conductance. The average lifetime of the open state of single channels is strongly dependent on the applied voltage, the channels shifting to the closed state at higher voltages. The pore is anion selective, differing from porins of other Gramnegative bacteria studied so far but resembling the voltage-dependent anion-selective channel of the outer membrane of mitochondria.The outer membranes of a number of Gram-negative bacteria contain highly organized passive diffusion pathways for macromolecules between 600 and 3,000 daltons (Da) (1). Porin, the protein responsible for these molecular sieving properties, has been characterized for several different types of bacteria (1-14). Porin from Escherichia coli is available now in crystalline forms amenable to ultrastructural studies (15). Porins from E. coli and Salmonella typhimurium have been incorporated into model lipid membranes (1,3,(11)(12)(13)(14). Membranes containing porin develop aqueous channels with pore sizes large enough to account for the observed permeability of the bacteria to certain nonelectrolytes (1). Crosslinking (4, 5), ultrastructural (6, 7), physicochemical (8,9), and electrophysiological (10-14) studies have indicated that porin molecules preferentially aggregate as stable trimers.Similar to that of other Gram-negative bacteria, the outer membrane of Neisseria gonorrhoeae contains a major protein (or protein I) that by itself accounts for more than 60% by weight of the protein in the outer membrane (16,17). The molecular mass of this protein is strain dependent and lies between 32 and 39 kDa (17). Previous studies have shown that the outer membranes of N. gonorrhoeae are more permeable to macromolecules than are those of E. coli and other wild-type Gram-negative bacteria (18,19). The relationship of protein I to the permeability of the gonococcal outer membrane is not clear.In this report, we have incorporated the purified gonococcal protein I into high-resistance planar lipid bilayer membranes by a simple procedure that may be applicable to other integral membrane proteins. The high resolution of electrical measurements achieved on planar bilayers allows the dissection of molecular properties associated with permeability changes conferred by channel-forming proteins. We present evidence here that the major outer membrane protein from N. gonorrhoeae is a pore-forming protein that may be responsible for the permeability properties of the outer membrane of this bacterium....
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