Lack of induction of somatic aneuploidy in the mouse by nitrilotriacetic acid (NTA)

Nitrilotriacetic acid (NTA) was tested for the induction of aneuploidy in mouse bone marrow cells. Doses of 138 or 275 mg/kg of body weight were intraperitoneally injected 24 h after implantation of a bromodeoxyuridine tablet. Cell-replication kinetics was assessed by comparing the relative percentages of first, second and third metaphases in control and treated samples. The hyperploidy incidence was estimated in second metaphases only, together with the SCE/cell level. Mice injected with 1.8 mg/kg vinblastine (VBL) were used as positive controls. A slight delay of cell cycle was induced by NTA, as shown by regression analysis applied to average generation time values. No increase over the control level was observed for hyperploidy or SCE induction in NTA-treated mice. VBL induced both cell-cycle alteration and a highly significant (P less than 0.001) increase of the hyperploid cell frequency. On the basis of these and previous (Costa et al., 1988) observations it seems that the non-disjunctional activity of NTA in the mouse is confined to meiotic processes.     

Facile synthesis of multivalent nitrilotriacetic acid (NTA) and NTA conjugates for analytical and drug delivery applications

High-affinity nitrilotriacetic acids (NTA) have great potential in the molecular manipulation of His-tagged proteins. We have developed a facile method to synthesize multivalent NTA and its conjugates. Starting with appropriately protected lysine, we synthesized the mono-NTA synthons functionalized with either an amino group or a carboxylic group. We then obtained tri-NTA through the condensation of the amino NTA and the carboxylic NTA. Using amino tri-NTA as the key intermediate, we synthesized a series of tri-NTA conjugates with a variety of functional units including biotin, dialkyl, fluorescein, and a hydroxybenzimidate moiety. The biotin-tri-NTA was employed to convert a Biacore streptavidin chip into a high-affinity tri-NTA chip. The equilibrium dissociation constants of tri-NTA/His-tagged protein complexes measured by surface plasmon resonance are in the 20 nM range. Histidine(6)-tagged yeast cytosine deaminase (His6-yCD) was incorporated onto the liposome surface by the lipid-tri-NTA conjugate without any activity loss. Fluorescein-tri-NTA formed a stable 1:1 complex with His6-yCD without significant fluorescence quenching. Specific tri-NTA derivatives for the radiolabeling and coupling of two His-tagged proteins to each other are described. Thus, we have added to the toolbox a number of high-affinity tri-NTA adaptors for the manipulation of His-tagged molecules.     

Immobilization of histidine-tagged proteins by magnetic nanoparticles encapsulated with nitrilotriacetic acid (NTA)-phospholipids micelle

We described the development of functionalized magnetic nanoparticles (MNPs) with PEG-modification, a phospholipids micelle coating, and their use in manipulating histidine-tagged proteins. Highly monodisperse MNPs were synthesized in an organic solvent and could be phase-transferred into an aqueous solution by encapsulating the nanoparticles with a phospholipids micelle. The phospholipids micelle coating rendered the nanoparticles highly water-soluble, and the functional groups of the phospholipids coating allowed for the bioconjugation of various moieties, such as fluorescent molecules and engineered proteins. Functionalized phospholipids, such as nitrilotriacetic acid (NTA)-phospholipids, caused the MNPs to bind and allowed for manipulation of histidine-tagged proteins. Due to their high surface/volume ratio, the MNPs showed better performance (about 100 times higher) in immobilizing engineered proteins than conventional micrometer-sized beads. This demonstrates that MNPs coated with phospholipids micelle can be a versatile platform for the effective manipulation of various kinds of engineered proteins, which is very important in the field of proteomics. It is expected that a combination of MNPs with optical fluorescent molecules can find applications in bimodal (magnetic and optical) molecular imaging nanoprobes.     

DNA damage and repair induced in vitro by nitrilotriacetic acid (NTA) in human lymphocytes

In cultured human lymphocytes we determined the ability of nitrilotriacetic acid (NTA) to inhibit DNA replication and to stimulate DNA repair synthesis (UDS), as well as to influence the UDS induced by UV irradiation. In phytohemagglutinin-stimulated lymphocytes a strong inhibition of DNA replication was induced by NTA concentrations above 10(-3) M, which was accompanied by a marked cell lethality, whereas at lower doses the incorporation of tritiated thymidine (3H-TdR) into DNA or treated cells was slightly increased in comparison to untreated cells. When, after NTA pretreatment, UDS was determined by scintillation spectrometry or autoradiography in unstimulated G0 lymphocytes, UV-irradiated or unirradiated, an increased incorporation of 3H-TdR was observed, positively correlated with the NTA doses. This effect was only partially due to the expansion of the intracellular TdR pool as a consequence of the stimulation of 3H-TdR uptake by NTA. Even after normalization of the scintillometric data by the radioactivities of the soluble nucleotide fraction, significant increase of DNA repair synthesis was detected after treatment with 7.5 x 10(-3)-10(-2) M NTA.     

Mutagenicity of lead chromate in Drosophila melanogaster in the presence of nitrilotriacetic acid (NTA)

By using the sex-linked recessive lethal mutation test in Drosophila melanogaster (standard Basc scheme) we analysed the mutagenic effects of treatments by feeding with nitrilotriacetic acid (NTA: 5 X 10(-2) M), with the insoluble Cr(VI) compound lead chromate, PbCrO4 (supernatant of 4.6 X 10(-4)-M suspension in which the actual concentration was 0.06 gamma/ml as Cr(VI)) and with both compounds preincubated at 3 relative ratios (NTA: 5 X 10(-2) M; PbCrO4: 4.6 X 10(-4), 4.6 X 10(-5) and 9.2 X 10(-6) M, respectively). The estimation of mutation frequencies was done at different developmental stages of the germ cells, namely spermatozoa, spermatids and spermatocytes. Ethyl methanesulphonate (EMS: 5 X 10(-3) M) was used as the reference positive control, with clearly mutagenic results. Treatments with NTA or with PbCrO4 alone did not induce any significant increase of the mutation frequency. PbCrO4 at the 3 concentrations tested was completely soluble in the 5 X 10(-2)-M NTA solution, and the mixture of NTA and PbCrO4 induced significant increases of the frequency of sex-linked lethal mutations, with a significant dose-effect relationship with respect to PbCrO4, apparently as a result of the interaction of the compounds and subsequent release of the genotoxic heavy-metal Cr(VI) ions. This result indicates an important synergistic action of NTA with PbCrO4 under the conditions described.     

Microbial degradation of chelating agents used in detergents with special reference to nitrilotriacetic acid (NTA)

The extensive use of phosphate-based detergents and agricultural fertilizers is one of the main causes of the world-wide eutrophication of rivers and lakes. To ameliorate such problems partial or total substitution of phosphates in laundry detergents by synthetic, non-phosphorus containing complexing agents is practiced in several countries. The physiological, biochemical and ecological aspects of the microbial degradation of the complexing agents most frequently used, such as polyphosphates, aminopolycarboxylates (especially of nitrilotriacetic acid), and phosphonates are reviewed.Abbreviations AODC Acridine orange direct counts - ATMP Aminotrimethylphosphonate - DTPA Diethylenetriaminepentaacetate - DTPMP Diethylenetriaminepentamethylphosphonate - EDTA Ethylenediaminetetraacetate - EDTMP Ethylenediaminetetramethylphosphonate - ED3A Ethylenediaminetriacetate - HEDP Hydroxyethylidenediphosphonate - HEDTA Hydroxyethylethylenediaminetriacetate - IDA Iminodiacetate - IFT Immunofluorescence test - MW Molecular weight - NTA Nitrilotriacetate - PA Polyacrylate - PHC Polyhydroxycarboxylate - PMS Phenazine methosulfate - SDS-PAGE Sodium dodecylsulfate polyacrylamide gel electrophoresis - SPP Tetrasodiumpyrophosphate - STP Pentasodiumtriphosphate     

The genotoxicity of nitrilotriacetic acid (NTA) in a somatic mutation and recombination test in Drosophila melanogaster

The genotoxicity of a chelating agent, the trisodium salt of nitrilotriacetic acid (NTA), was assessed in a somatic mutation and recombination test (SMART) in Drosophila melanogaster employing the wing hair markers mwh and flr3. The experiments were performed in parallel in two different laboratories (Padua, Italy and Schwerzenbach, Switzerland). The effectively absorbed doses of NTA, which was administered by feeding to larvae, were determined by a sensitive method employing [3H]leucine which allowed individual consumption levels to be measured. The particular pattern of clone induction produced by this compound suggests that NTA is active in inducing mitotic recombination and possibly aneuploidy in somatic cells of Drosophila. This is discussed in relation to the data present in the literature regarding the genotoxicity of NTA in a variety of experimental systems.     

Interactions of nitrilotriacetic acid (NTA) with Cr(VI) compounds in the induction of gene mutations in cultured mammalian cells

We used the V79 Chinese hamster cell line to detect the induction by NTA of 6-thioguanine resistance, due to mutation at the HGPRT locus, with direct and indirect mutagens as positive controls. NTA was tested within the 10(-4)-1.5 X 10(-2) M concentration range: although it was cytotoxic above the 10(-2) M dose, it did not increase the frequency of mutations at any of the tested concentrations, independently of metabolic activation (rat-liver S9 fraction). NTA is known to dissolve heavy metals and therefore to increase their genotoxicity. We found that an insoluble Cr(VI) compound, lead chromate (PbCrO4), was not cytotoxic nor mutagenic on V79 cells, probably because it is taken up by the cells very slowly, whereas the presence of NTA (2.5 X 10(-3) M in water) elicited a direct cytotoxicity and mutagenicity, which was dose-dependent from 5 X 10(-5) M to 10(-4) M PbCrO4. This effect was due to solubilization of the chromate anion by NTA, as determined by comparing spectrophotometric determinations of Cr(VI) in PbCrO4 treatment solutions with a mutagenicity titration curve obtained with a completely soluble Cr(VI) salt (potassium dichromate, K2Cr2O7).     

The metabolism of cresols by species of Pseudomonas

1. A comparison of rates of oxidation of various compounds by whole cells indicated that protocatechuate was a reaction intermediate when a non-fluorescent species of Pseudomonas oxidized p-cresol. In contrast, a fluorescent Pseudomonas oxidized 3-methylcatechol and 4-methylcatechol when grown with p-cresol, but did not oxidize protocatechuate. 2. Heat-treated extracts of the fluorescent Pseudomonas oxidized catechol, 3-methylcatechol and 4-methylcatechol to ring-fission products, the spectroscopic properties of which were recorded. Rates of enzymic degradation of these products were also measured. 3. Acetic acid and formic acid were obtained by the action of a Sephadex-treated extract on 3-methylcatechol and 4-methylcatechol respectively. In each case 0.8mol. of the carboxylic acid was formed from 1.0mol. of substrate. 4. Dialysed extracts converted 3-methylcatechol into acetaldehyde and pyruvate, with 4-hydroxy-2-oxovalerate as a reaction intermediate. 4-Methylcatechol was converted first into 4-hydroxy-2-oxohexanoate and then into propionaldehyde and pyruvate. 5. The ring-fission product of catechol was formed from phenol by a fluorescent Pseudomonas, that of 3-methylcatechol was formed from o-cresol and m-cresol, and the ring-fission product of 4-methylcatechol was given from p-cresol. Propionate was readily oxidized by these cells after growth with p-cresol, but this compound was not attacked when phenol, o-cresol or m-cresol served as source of carbon. 6. Cell extracts appeared to attack only one enantiomer of synthetic 4-hydroxy-2-oxohexanoate.     

Fructose metabolism in four Pseudomonas species

1. ATP-Dependent phosphorylation of fructose could not be detected in extracts of fructose-grown cells of Pseudomonas extorquens strain 16, Pseudomonas 3A2, Pseudomonas acidovorans and Pseudomonas fluorescens. Instead, phosphorylation of fructose to fructose-1-phosphate was found to occur when cell-free extracts were incubated with fructose and phosphoenolpyruvate. Such an activity could not be detected in cell-free extracts of succinate-grown cells. 2. High levels of 1-phosphofructokinase were found in extracts of the above organisms when growth on fructose. 3. Mutants of Pseudomonas extorquens strain 16 lacking 1-phosphofructokinase were unable to grow on fructose. Revertants to growth on fructose had regained the capacity to synthesize this enzyme, indicating its necessary involvement in fructose metabolism. 4. A survey has been carried out of enzymes involved in carbohydrate metabolism in the species listed above.     

The metabolism of 2-furoic acid by Pseudomonas F2

1. Pseudomonas F2 isolated by enrichment culture on 2-furoic acid and grown with it as carbon source oxidized the compound with a Q(o) (2) of 170mul./mg. dry wt./hr. and the overall consumption of 2.5mumoles of oxygen/mumole of substrate. 2. In the presence of 1mm-sodium arsenite, oxygen uptake was restricted to 0.54mumole/mumole of 2-furoate oxidized, with the formation of 0.86mumole of 2-oxoglutarate/mumole of 2-furoate. 3. Cell suspensions, disrupted in a French pressure cell and centrifuged at 27000g, yielded supernatants capable of catalysing the slow oxidation of 2-furoate (0.17mumole/mg. of protein/hr.). 4. Fractionation of 27000g supernatants at 200000g yielded a soluble enzyme fraction capable of catalysing the oxidation of 2-furoate only in the presence of added 200000g pellet or of Methylene Blue. 5. The 2-furoate-stimulated uptake of oxygen or the anaerobic reduction of Methylene Blue by dialysed 27000g supernatant required the addition of ATP and CoA, and the rate of oxygen uptake was further enhanced by the addition of magnesium chloride and NAD(+). 6. The role of ATP and CoA in the formation of 2-furoyl-CoA was demonstrated by the accumulation of 2-furoylhydroxamic acid in the presence of hydroxylamine. 7. Dialysed 200000g supernatant, treated with Dowex 1, required the addition of ATP, CoA and Methylene Blue before it could oxidize 2-furoate to 2-oxoglutarate, which was trapped in unitary stoicheiometric yield as its phenylhydrazone. Magnesium chloride and NAD(+) were not stimulatory in this system. The oxidation of 2-furoate to 2-oxoglutarate was not inhibited by substrate analogues, metal ion-chelating agents, thiol-active compounds or inhibitors of cytochrome-mediated electron transport. 8. No evidence was obtained for the intervention of 2,5-dioxovalerate as an intermediate in 2-oxoglutarate formation.     

The novel chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) promotes stable binding of His-tagged proteins to liposomal membranes: potent anti-tumor responses induced by simultaneously targeting antigen, cytokine and costimulatory signals to T cells

Recent studies indicate that the chelator lipid nitrilotriacetic acid ditetradecylamine (NTA-DTDA) can be used to engraft T cell costimulatory molecules onto tumor cell membranes, potentially circumventing the need for genetic manipulation of the cells for development of cell- or membrane-based tumor vaccines. Here, we show that a related lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA, which has three NTA moieties in its headgroup instead of one) is several-fold more effective than NTA-DTDA at promoting stable His-tagged protein engraftment. IAsys biosensor studies show that binding of His-tagged B7.1 (B7.1-6H) to NTA(3)-DTDA-containing membranes, exhibit a faster on-rate and a slower off-rate, compared to membranes containing NTA-DTDA. Also, NTA(3)-DTDA-containing liposomes and plasma membrane vesicles (PMV) engrafted with B7.1-6H and CD40-6H exhibit greater binding to T cells, in vitro and in vivo. Engrafted NTA(3)-DTDA-containing PMV encapsulated cytokines such as IL-2, IL-12, GM-CSF and IFN-gamma, allowing targeted delivery of both antigen and cytokine to T cells, and stimulation of antigen-specific T cell proliferation and cytotoxicity. Importantly, use of B7.1-CD40-engrafted PMV containing IL-2 and IL-12 as a vaccine in DBA/2J mice induced protection against challenge with syngeneic tumor cells (P815 mammary mastocytoma), and regression of established tumors. The results show that stable protein engraftment onto liposomal membranes using NTA(3)-DTDA can be used to simultaneously target associated antigen, costimulatory molecules and cytokines to T cells in vivo, inducing strong anti-tumor responses and immunotherapeutic effect.     

Degradation of 4-chlorophenylacetic acid by a Pseudomonas species.

Pseudomonas sp. strain CBS3 was able to utilize 4-chlorophenylacetic acid as the sole source of carbon and energy. When this strain was grown with 4-chlorophenylacetic acid, homoprotocatechuic acid was found to be an intermediate which was further metabolized by the meta-cleavage pathway. Furthermore, three isomers of chlorohydroxyphenylacetic acid, two of them identified as 3-chloro-4-hydroxyphenylacetic acid and 4-chloro-3-hydroxyphenylacetic acid, were isolated from the culture medium. 4-Hydroxyphenylacetic acid was catabolized in a different manner by the glutathione-dependent homogentisate pathway. Degradation enzymes of both of these pathways were inducible.