Interaction of nanobacteria with cultured mammalian cells

Çiftçioğlu, Neva; Kajander, E. Olavi

doi:10.1016/s0928-4680(97)10001-3

Cited by 85 publications

(104 citation statements)

References 24 publications

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“…Nanobacteria were cultured in DMEM (GIBCO) under mammalian cell culture conditions (37°C; 5-10% CO 2 /90-95% air). Serum was used at a 10% final concentration as the supplement and source of nanobacteria, which were FBS ( lot 901045; Sera-Lab, Crawley Down, Sussex, U.K.) or human serum from a 29-years-old Finnish male, as described previously (5). The cultures were prepared using strict aseptic techniques in a cell culture facility.…”

Section: Methodsmentioning

confidence: 99%

“…3T6 cells (ATCC CCL 96) were cultured on coverslips as described before (5). SF nanobacterial cultures were scraped and 100-l portions were added to the cell cultures and incubated for 24 hr in the incubator.…”

Section: Methodsmentioning

confidence: 99%

“…According to the 16S rRNA gene sequence (EMBL X98418 and X98419), nanobacteria fall within the ␣-2 subgroup of Proteobacteria, which also includes Brucella and Bartonella species (10). The latter genera include human and animal pathogens that share similarities with nanobacteria, e.g., some antigens (our unpublished data), intra-and extracellular location in the host, and cytopathic effects (5).…”

mentioning

confidence: 95%

“…We have discovered nanobacteria in human and cow blood that are cytotoxic in vitro (5) and in vivo (6). They have been deposited in DSM (no.…”

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confidence: 99%

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Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation

Çiftçioğlu

1998

Proc. Natl. Acad. Sci. U.S.A.

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Calcium phosphate is deposited in many diseases, but formation mechanisms remain speculative. Nanobacteria are the smallest cell-walled bacteria, only recently discovered in human and cow blood and commercial cell culture serum. In this study, we identified with energydispersive x-ray microanalysis and chemical analysis that all growth phases of nanobacteria produce biogenic apatite on their cell envelope. Fourier transform IR spectroscopy revealed the mineral as carbonate apatite. The biomineralization in cell culture media resulted in biofilms and mineral aggregates closely resembling those found in tissue calcification and kidney stones. In nanobacteria-infected fibroblasts, electron microscopy revealed intra-and extracellular acicular crystal deposits, stainable with von Kossa staining and resembling calcospherules found in pathological calcification. Previous models for stone formation have led to an hypothesis that elevated pH due to urease and͞or alkaline phosphatase activity is a lithogenic factor. Our results indicate that carbonate apatite can be formed without these factors at pH 7.4, at physiological phosphate and calcium concentrations. Nanobacteria can produce apatite in media mimicking tissue f luids and glomerular filtrate and provide a unique model for in vitro studies on calcification.The formation of discrete and organized inorganic crystalline structures within macromolecular extracellular matrices is a widespread biological phenomenon generally referred to as biomineralization. Mammalian bone and dental enamel are examples of biomineralization involving apatite minerals. The molecular basis of mineralization remains largely unknown (1). Recently, bacteria have been implicated as factors in biogeochemical cycles for mineral formation in aqueous sediments (2, 3). The principal constituent of modern authigenic phosphate minerals in marine sediments is carbonate (hydroxy)fluorapatite Ca 10 (PO 4 ) 6-x (CO 3 ) x (F,OH) 2ϩx . Microorganisms are capable of depositing apatite outside thermodynamic equilibrium in sea water. They can segregate Ca from Mg and actively nucleate carbonate apatite by means of specific oligopeptides under conditions pH Ͻ 8.5 and [Mg]:[Ca] Ͼ 0.1 (4). Such conditions are also present in the human body.We have discovered nanobacteria in human and cow blood that are cytotoxic in vitro (5) and in vivo (6). They have been deposited in DSM (no. 5819-5821; Braunschweig, Germany). Nanobacteria possess unusual properties, making their detection difficult with standard microbiological methods. Although they typically had diameters of 0.2-0.5 m, they passed through 0.1-m filters probably because tiny forms (0.05-0.2 m) were also observed in transmission electron microscopy (TEM) (7). Nanobacteria were poorly disruptable, stainable, fixable, and exceptionally resistant to heat (8, 9). Their doubling time was about 3 days. High doses of ␥-irradiation or aminoglycoside antibiotics prevented their multiplication. According to the 16S rRNA gene sequence (EMBL X98418 and X98419), na...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 95%

“…We have discovered nanobacteria in human and cow blood that are cytotoxic in vitro (5) and in vivo (6). They have been deposited in DSM (no.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation

Çiftçioğlu

1998

Proc. Natl. Acad. Sci. U.S.A.

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444

343

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“…Both bases are incorporated into tRNA of the respective cells. Thus, to minimize contamination by serum-containing thiolated nucleotides produced by nanobacteria (15), only dialyzed serum (cutoff 10,000) was used. In fact, standard qualitative HPLC analysis that was performed on RNase-treated samples showed the presence of two closely located peaks, one of them indicative for s4U (SI Fig.…”

Section: Thiolated Rna Can Specifically Be Isolated By An Organomercumentioning

confidence: 99%

Microarray analysis of newly synthesized RNA in cells and animals

Kenzelmann

Maertens

Hergenhahn

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Current methods to analyze gene expression measure steady-state levels of mRNA. To specifically analyze mRNA transcription, we have developed a technique that can be applied in vivo in intact cells and animals. Our method makes use of the cellular pyrimidine salvage pathway and is based on affinity-chromatographic isolation of thiolated mRNA. When combined with data on mRNA steady-state levels, this method is able to assess the relative contributions of mRNA synthesis and degradation/stabilization. It overcomes limitations associated with currently available methods such as mechanistic intervention that disrupts cellular physiology, or the inability to apply the techniques in vivo. Our method was first tested in serum response of cultured fibroblast cells and then applied to the study of renal ischemia reperfusion injury, demonstrating its applicability for whole organs in vivo. Combined with data on mRNA steady-state levels, this method provided a detailed analysis of regulatory mechanisms of mRNA expression and the relative contributions of RNA synthesis and turnover within distinct pathways, and identification of genes expressed at low abundance at the transcriptional level.newly transcribed RNA ͉ posttranscriptional regulation ͉ RNA degradation/stabilization I n response to environmental changes, cells alter their gene expression program. Eukaryotic gene expression is a complex process that is fine-tuned at different levels, such as transcription, turnover, and translation of mRNAs. Microarray-based measurements of steady-state mRNA levels do not distinguish between different regulatory processes. Both transcriptional and posttranscriptional regulation, however, can profoundly influence steady-state mRNA levels (1-4). To evaluate de novo transcription and the relative contributions of mRNA transcription and turnover, nuclear transcription run-on and its modifications have long been the methods of choice (4-6). These methods, however, are either inherently cell-invasive and/or are not compatible with the assay formats currently used to analyze global gene expression. To avoid mechanistic intervention that disrupts cellular physiology for example by cell permeabilization, we modified a method in which we take advantage of the cellular pyrimidine salvage pathway. Pyrimidine ribonucleotides may be de novo synthesized from simple molecules by energetically expensive multistep pathways. Alternatively, ribonucleotide pools are sustained by recycling of the basic components derived from their catabolism during normal RNA turnover, the socalled salvage pathway, a mechanism that is energetically less expensive. By this more favorable pathway, it is possible to label and selectively enrich newly transcribed (nascent) RNA molecules (NT-RNA; refs. 7-9) in vivo by incorporation of exogenous 4-thiouridine (s4U) and subsequent RNA purification by affinity chromatography. The RNA can then be further processed by standard protocols for hybridization to oligonucleotide microarrays. We refer to our method with the acronym NIAC-...

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A preliminary study on calcifying nanoparticles in dental plaque: Isolation, characterization, and potential mineralization mechanism

Wang,

Yang,

Bai

et al. 2024

Clinical & Exp Dental Res

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ObjectivesCalcifying nanoparticles (CNPs), referred to as nanobacteria (NB), are recognized to be associated with ectopic calcification. This study aims to isolate and culture CNPs from the dental plaque of patients with periodontal disease and investigate their possible role in unravelling the aetiology of periodontal disease.Material and MethodsSupragingival and subgingival plaques were sampled from 30 periodontitis patients for CNPs isolation and culture. Alkaline phosphatase (ALP) content changes were tracked over time. Positive samples underwent thorough morphological identification via hematoxylin and eosin (HE) staining, Alizarin red S (ARS), and transmission electron microscopy (TEM). The chemical composition of CNPs analysis involved calcium (Ca) and phosphorus (P) content determination, Fourier transform infrared spectroscopy (FTIR), and X‐ray diffraction (XRD).ResultsThe subgingival plaque dental group exhibited a higher CNPs isolation rate at 36.67% (11/30) compared to the supragingival dental plaque group at 66.67% (20/30). ALP activity varied among the positive, negative and control groups. Morphological observation characterized the CNPs as round, oval, and ellipsoid particles with Ca deposits. Chemical analysis revealed the Ca/P ratio was 0.6753. Hydroxyl, methyl, carbonate, phosphate, hydrogen phosphate, and dihydrogen phosphate were detected by FTIR; the main chemical components detected by XRD were hydroxyapatite and tricalcium phosphate.ConclusionCNPs were found in periodontitis‐related dental plaque and exhibited the potential to develop calcified structures resembling dental calculus. However, the potential involvement of ALP in CNPs formation requires deeper exploration, as does the precise nature of its role and the interrelation with periodontitis demand a further comprehensive investigation.

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Interaction of nanobacteria with cultured mammalian cells

Cited by 85 publications

References 24 publications

Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation

Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation

Microarray analysis of newly synthesized RNA in cells and animals

A preliminary study on calcifying nanoparticles in dental plaque: Isolation, characterization, and potential mineralization mechanism

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