Nanobacteria--propagating calcifying nanoparticles.

Nanobacteria, also known as calcifying nanoparticles (CNP), are controversial infectious agents not matching the current criteria for 'living organism'. Despite the controversy of their classification, they propagate and cause cell death in vitro and are associated or found in many human diseases. Thus, more efforts should be focussed on research on pathogenicity of CNP.

Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method.

Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.

[Culture and identification of nanobacteria in bile].

OBJECTIVE: To study the distribution and identification of nanobacteria in bile and to evaluate the identifying methods of nanobacteria. METHODS: RPMI1640 culture or RPMI1640 culture with 10% heat-inactivated gamma-FBS was added into 75 samples of cystic bile from gallbladders resected in operation. Nanobacteria were identified by immunohistochemical staining, transmission electron microscopy (TEM), and calcific staining. RESULTS: Nanobacteria were found in 45 bile samples with a positive rate of 61.3%. The positive rate of nanobacteria was 75.7% among 37 bile samples with white precipitate adhering to the tube, and was 47.4% among the samples with flocculent precipitate or without precipitate (P < 0.05). The immunohistochemically confirmed presence of nanobacteria was re-confirmed by TEM in all the positive samples. The positive rate, sensitivity, specificity, false positive rate and false negative rate of calcific staining were 38.7%, 58.7%, 93.1%, 6.9% and 41.3% respectively. CONCLUSION: Immunohistochemistry with monoclonal antibody of nanobacteria associated with TEM is useful in identifying nanobacteria. Calcific staining is of great value to identification of nanobacteria. Precipitation of white floccules adhering to the tube is an important microbiological characteristic of nanobacteria.

Nanobacteria: controversial pathogens in nephrolithiasis and polycystic kidney disease.

Nanobacteria are unconventional agents 100-fold smaller than common bacteria that can replicate apatite-forming units. Nanobacteria are powerful mediators of biogenic apatite nucleation (crystal form of calcium phosphate) and crystal growth under conditions simulating blood and urine. Apatite is found in the central nidus of most kidney stones and in mineral plaques (Randall's plaques) in renal papilla. The direct injection of nanobacteria into rat kidneys resulted in stone formation in the nanobacteria-injected kidney during one month follow-up, but not in the control kidney injected with vehicle. After intravenous administration in rats and rabbits, nanobacteria are rapidly excreted from the blood into the urine, as a major elimination route, and damage renal collecting tubuli. Nanobacteria are cytotoxic to fibroblasts in vitro. Human kidney cyst fluids contain nanobacteria. Nanobacteria thus appear to be potential provocateurs and initiators of kidney stones, tubular damage, and kidney cyst formation. It is hypothesized that nanobacteria are the initial nidi on which kidney stone is built up, at a rate dependent on the supersaturation status of the urine. Those individuals having both nanobacteria and diminished defences against stone formation (i.e. genetic factors, diet and drinking habits) could be at high risk. Kidney cyst formation is hypothesized to involve nanobacteria-induced tubular damage and defective tissue regeneration yielding cyst formation, the extent of which is dependent on genetic vulnerability.

Endotoxin and nanobacteria in polycystic kidney disease.

BACKGROUND: Microbes have been suspected as provocateurs of polycystic kidney disease (PKD), but attempts to isolate viable organisms have failed. Bacterial endotoxin is the most often reported microbial product found in PKD fluids. We assessed potential microbial origins of endotoxin in cyst fluids from 13 PKD patients and urines of PKD and control individuals. METHODS: Fluids were probed for endotoxin and nanobacteria, a new bacterium, by the differential Limulus Amebocyte Lysate assay (dLAL), genus-specific antilipopolysaccharide (LPS) antibodies, monoclonal antibodies to nanobacteria, and hyperimmune serum to Bartonella henselae (HS-Bh). Selected specimens were also assessed by transmission electron microscopy (TEM) and nanobacterial culture methods. RESULTS: LPS or its antigenic metabolites were found in more than 75% of cyst fluids tested. Nanobacteria were cultured from 11 of 13 PKD kidneys, visualized in 8 of 8 kidneys by TEM, and immunodetected in all 13 PKD kidneys. By immunodetection, nanobacterial antigens were found in urine from 7 of 7 PKD males, 1 of 7 PKD females, 3 of 10 normal males, and 1 of 10 normal females. "Nanobacterium sanguineum" was dLAL positive and cross-reactive with antichlamydial LPS and HS-Bh. Some cyst fluids were also positive for LPS antigens from Escherichia coli, Bacteroides fragilis and/or Chlamydia, and HS-Bh, as were liver cyst fluids from one patient. Tetracycline and citrate inhibited nanobacterial growth in vitro. CONCLUSION: Nanobacteria or its antigens were present in PKD kidney, liver, and urine. The identification of candidate microbial pathogens is the first step in ascertaining their contribution, if any, to human disease.

Nanobacteria: an infectious cause for kidney stone formation.

BACKGROUND: Nanobacteria are cytotoxic, sterile-filterable, gram-negative, atypical bacteria detected in bovine and human blood. Nanobacteria produce carbonate apatite on their cell walls. Data on Randall's plaques suggest that apatite may initiate kidney stone formation. We assessed nanobacteria in 72 consecutively collected kidney stones from Finnish patients. METHODS: Nanobacteria and kidney stone units were compared using scanning electron microscopy (SEM). Demineralized kidney stones were screened for nanobacteria using a double-staining method and a specific culture method. Isolated nanobacteria were analyzed for mineral formation in vitro with Ca and 85Sr incorporation tests. RESULTS: SEM highlighted the resemblance in size and morphology of nanobacteria and the smallest apatite units in the kidney stones. Nanobacterial antigens could be detected after the demineralization of the stones in 1 N HCl. Nanobacteria were surprisingly resistant to this treatment, and cultures could be established from 93.1% of the stones. Only struvite stones had common bacteria, in addition to the nanobacteria. When the results of all of the assays were combined, 70 of the 72 stones (that is, 97.2%) were nanobacteria positive. Although apatite stones indicated highest nanobacteria antigen signals, the overall nanobacteria positivity did not depend on the stone type. The isolated nanobacteria produced apatite stones in vitro, measured by Ca and 85Sr incorporation. CONCLUSIONS: We propose that kidney stone formation is a nanobacterial disease analogous to Helicobacter pylori infection and peptic ulcer disease. Both diseases are initiated by bacterial infection and subsequently endogenous and dietary factors influence their progression.

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

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 energy-dispersive 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 and/or 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 fluids and glomerular filtrate and provide a unique model for in vitro studies on calcification.

Amphoterin, the 30-kDa protein in a family of HMG1-type polypeptides. Enhanced expression in transformed cells, leading edge localization, and interactions with plasminogen activation.

Amphoterin is a heparin-binding protein that is developmentally regulated in brain and functionally involved in neurite outgrowth. Unexpectedly, amphoterin has a high mobility group 1 (HMG1)-type sequence. In the present study we have expressed amphoterin cDNA in a baculovirus vector and produced antibodies against the recombinant protein and several synthetic peptides. It was found that the amphoterin cDNA encodes the 30-kDa form of the protein isolated from tissues, whereas the co-purifying 28- and 29-kDa proteins (p28 and p29) have closely related but distinct primary structures. Partial amino acid sequencing shows several local changes in the sequences of p28 and p29 compared with amphoterin, suggesting the occurrence of a multigene family that encodes at least three different HMG1-type sequences in the rat. Studies using the probes that discern amphoterin from the other HMG1-type proteins indicate a high level expression in various transformed cell lines. Immunostaining of cells with the amphoterin-specific antibodies indicates a cytoplasmic localization that becomes remarkably enriched at the leading edges in spreading and motile cells. An extracellular localization is suggested by immunostaining of nonpermeabilized cells and by a plasminogen-dependent degradation of amphoterin in the substratum-attached material of cells. Tissue-derived and recombinant amphoterins strongly enhance the rate of plasminogen activation and promote the generation of surface-bound plasmin both by tissue-type and urokinase-type plasminogen activators. The results suggest an extracellular function for amphoterin in the leading edge of various invasive cells.

Screening of effects of selenomethionine-enriched yeast supplementation on various immunological and chemical parameters of skin and blood in psoriatic patients.

Selenium (Se) is known to affect the immune system, and decreased Se-levels in blood of patients with moderate or severe psoriasis have been reported. In this study, the effect of Se-supplementation (400 micrograms/day for 6 weeks as Se-yeast, containing about 70% selenomethionine, SeMet) on skin and blood Se-content, on skin glutathione peroxidase activity and on various chemical and immunological parameters of blood and skin was investigated in 7 psoriatic patients. Before the SeMet-supplementation, serum and blood Se-levels were at the normal range, but they increased 42-45% during the Se-dosage, while zinc levels remained unchanged. Se-dependent glutathione peroxidase activity in both normal and lesional psoriatic skin remained unchanged during the trial, although a small net Se-uptake was detected. At the same time, a slight but statistically significant increase in the number of CD4+ T-cells was observed in the reticular dermis of the psoriatic lesions whereas the numbers of CD8+, CD11c+, and CD1+ cells were not significantly altered. Also, a relatively high number of patients (3 out of 7) showed a strongly reduced number of gamma/delta T-lymphocytes or increased CD8+ T-cells (2 patients) in peripheral blood. However, SeMet-supplementation was not related to these abnormalities or to the number of other peripheral blood immunocytes or to serum immunoglobulin levels. In addition, no marked effect on the clinical condition of the patients was observed. This pilot study suggests that SeMet may be able to modulate the immunological mechanism of psoriatic lesions by increasing the number of CD4+ T-cells.

Metabolism, cellular actions, and cytotoxicity of selenomethionine in cultured cells.

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized to S-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 microM levels. Yet, low selenomethionine levels (10 microM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes in S-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.

Effects of selenomethionine on cell growth and on S-adenosylmethionine metabolism in cultured malignant cells.

The effects of selenomethionine (SeMet) on the growth of 17 cultured cell lines were studied. SeMet in the culture medium of three hepatoma cell lines promoted cell growth at subcytotoxic levels (1-20 microM), but the growth of malignant lymphoid and myeloid cells was not stimulated. L-SeMet was cytotoxic to all 17 cell lines when assayed after culture for 3-10 days. A 50% growth inhibition was observed by 30-160 microM-SeMet in a culture medium containing 100 microM-methionine. SeMet cytotoxicity to normal (fibroblasts) and malignant cells was rather similar, excluding specific antineoplastic cytotoxicity. Cytotoxicity was increased by decreasing concentrations of methionine. The DL form of SeMet was less cytotoxic than the L form. L-SeMet was metabolized to a selenium analogue of S-adenosylmethionine approximately as effectively as the natural sulphur analogue methionine in malignant R1.1 lymphoblasts. Concomitantly, S-adenosylmethionine pools were decreased. This occurred early and at cytotoxic SeMet levels. Methionine adenosyltransferase activity was not altered by SeMet treatment. ATP pools were not affected early, and decreases in the synthesis of DNA and protein took place late and were apparently related to cell death. RNA synthesis was slightly stimulated at low cytotoxic SeMet levels by 24 h, but was markedly inhibited after 48 h. The SeMet analogue of S-adenosylmethionine could be effectively utilized in a specific enzymic transmethylation. Neither S-adenosylhomocysteine nor its selenium analogue accumulated in the treated cells. These findings together suggest a direct or indirect involvement of S-adenosylmethionine metabolism in SeMet cytotoxicity, but exclude a gross blockage of transmethylations.

Synthesis and analysis of selenomethionine metabolites.

This paper describes the enzymatic synthesis of selenomethionine metabolites of the transmethylation and polyamine synthesis pathways: adenosylselenomethionine, adenosylselenohomocysteine, decarboxylated adenosylselenomethionine, and methylselenoadenosine. These compounds and the corresponding methionine metabolites were simultaneously separated by a single HPLC run. The sensitivity of the HPLC method is about 20 pmol per compound. The method may be used for direct analysis of the metabolite levels in tissues or cells treated with selenomethionine and it provides an assay method for the pulse-chase type of analysis of relative flows for both selenium- and sulfur-containing compounds in transmethylation and polyamine pathways.

Purification and partial characterization of human polyamine synthases.

Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.

Effect of drugs on histamine radio-enzyme assay.

The effect of over 200 drugs and other compounds on histamine radio-enzyme assay was studied. Some muscle relaxants (e.g. alcuronium), some sympathomimetics (e.g. dopamine, isoxsuprine, tyramine and possibly phenylethylamine), antimalarial drugs, procaine, procainamide, Berenil and serotonin were potent compounds to interfere with this assay. In some special cases still potentially inhibitory drugs seemed to be some muscle relaxants (e.g. vecuronium, pancuronium and tubocurarine), antidepressants, antihistamines (e.g. cimetidine, ranitidine and diphenhydramine), chinidin, disopyramide, tolazoline and salazosulfapyridine.

Effect of handling of plasma samples on histamine radio-enzyme assay.

1. Freeze-thawing of plasma samples increased the histamine level at physiological histamine concentrations as analyzed by radio-enzyme assay, but not by high performance liquid chromatography. 2. Heating of plasma samples decreased the histamine levels. 3. Enzymatic or non-enzymatic formation of histamine during handling of plasma samples was not detected. 4. Some albumin preparations contain histamine.

Modulation of diphthamide synthesis by 5'-deoxy-5'-methylthioadenosine in murine lymphoma cells.

The histidine derivative diphthamide occurs uniquely in eukaryotic elongation factor 2 (EF-2), and is the specific target for the diphtheria toxin mono(ADP-ribosyl)transferase. The first step in diphthamide biosynthesis may involve the transfer of aminocarboxypropyl moiety from S-adenosylmethionine to the imidazole ring of histidine in EF-2, to yield 2-(3-carboxy-3-aminopropyl)histidine and 5'-deoxy-5'-methylthioadenosine (MeSAdo). As the possible nucleoside product of the initial reaction in the diphthamide biosynthetic pathway, MeSAdo could be an inhibitor of diphthamide formation. In the present experiments, we have analyzed the effects of MeSAdo on diphthamide synthesis in a MeSAdo phosphorylase-deficient mutant murine lymphoma cell line (R1.1, clone H3). As measured by susceptibility to diphtheria toxin-induced ADP-ribosylation, MeSAdo inhibited the formation of diphthamide in EF-2. The inhibition was not due to a nonspecific effect on protein synthesis. Indeed, exogenous MeSAdo substantially protected the lymphoma cells from the lethal effects of diphtheria toxin. These results suggest that MeSAdo can specifically modulate the biosynthesis of diphthamide in EF-2 in murine malignant lymphoma cells.

Resistance to multiple adenine nucleoside and methionine analogues in mutant murine lymphoma cells with enlarged S-adenosylmethionine pools.

Adenosine and many adenosine analogues exert toxicity to mammalian cells at the nucleoside level. The mechanism of action of these agents is controversial. Previous experiments suggested that adenosine toxicity could be mediated by the accumulation of S-adenosylhomocysteine (AdoHcy), a potent inhibitor of S-adenosylmethionine (AdoMet) dependent methylation reactions. To analyze this question genetically, adenosine resistant, adenosine kinase deficient mutant clones of a murine T-lymphoma cell line (R1.1) have been selected and analyzed. Compared to parental lymphoma cells, the adenosine resistant mutants had severalfold elevated levels of AdoMet and an increased AdoMet:AdoHcy ratio. The activity of methionine adenosyltransferase was also raised in the mutants. The mutant cells were cross-resistant to agents postulated to cause accretion of AdoHcy, formation of AdoHcy analogues, impairment of AdoMet synthesis, or direct interference with AdoMet dependent reactions. These included 3-deazaadenosine, carbocyclic adenosine, carbocyclic 3-deazaadenosine, formycin A, 8-azaadenosine, 5'-deoxy-5'-methylthiotubercidin, 5'-deoxy-5'-methylthioadenosine, 5'-deoxy-5'-S-isobutylthioadenosine, adenine, cycloleucine, L-ethionine, seleno-DL-ethionine, and (+/-)-2-aminobicyclo[2.1.1]hexane-2-carboxylic acid. These results suggest that diverse purine nucleoside and methionine analogues may block the growth of adenosine kinase deficient cells by interference with AdoMet synthesis and degradation. An increase in AdoMet pools can render mammalian cells cross-resistant to multiple drugs affecting this essential metabolic pathway.