EDTA treatments

Tumor cells. EMT6 cells were grown as a monolayer culture in DMEM medium containing 20% fetal calf serum (27). Cells were detached from the plate by trypsin-EDTA treatment and washed in PBS. A total of 5 x 103 cells were injected per mouse via the tail vein of Balb/c mice (6-8 weeks old) to induce experimental lung metastatic tumors. Immunoliposomes were injected iv 2 and 4 days after the tumor cell injection. The survival of mice was followed over the next 60 days. 

Water soluble proteins are present in shells in the amount of 0.1 to 27< of the total organic matter. EDTA treatment will release additional increments and in Mercenaria mercenaria shells, 15% of the total is EDTA extractable228. The material represents a highly sulfated glycoprotein with a molecular weight in the order of 160000. 

In the work-up of the diethyl ether reaction, the solvent is removed (in a hood) under a stream of nitrogen. Then the ether-free aqueous dispersion is mixed with 10 ml of a 5 mM EDTA solution to bind the calcium ions. Without the latter treatment, the calcium salt of the phosphatidic acid cannot be recovered (easily) from the reaction mixture. Thus, subsequent to the EDTA treatment, the sample is mixed with sufficient chloroform and methanol to make a mixture of chloroform-methanol-water (1 2 0.8, v/v). Then 0.5 volume chloroform and 0.5 volume water are added, and the mixture is vigorously shaken for 1 min and then allowed to separate into two phases. The upper, water-rich phase contains free nitrogen base—in this case, choline. It can be analyzed by the techniques described earlier. 

The above studies also give evidence of multiple localization of PolyP in the cells of cyanobacteria. This conclusion was confirmed by a 31P NMR spectroscopic study. In the cyanobacterium Synechocystis sp., two pools of soluble PolyP were identified in vivo by 31P NMR spectroscopy (Lawrence et al, 1998). One of these (PolyP-cation complexes) lost their association cations after EDTA treatment, while the other did not. 

Zeolite Y. We also substituted boron into dealuminated zeolite Y. We dealuminated zeolite Y by EDTA treatment using standard methods (4). The presence of hydroxyl nests in the product was confirmed using 29Si CPMAS NMR spectroscopy. The dealuminated material incorporated 33 times more boron than zeolite Y when treated with KOH/B2O3. These data are summarized in Table 3. The boron substituted faujasite exhibits a single sharp resonance in the NMR spectrum, consistent with structural substitution. Since the substitution level was low and would not be expected to cause large shifts in the diffraction pattern, no corrected XRD data were obtained on substituted zeolite Y. 

The biochemical reaction performed by the methyltransferase appears to be metal dependent, as determined by chemical inhibition studies, but the exact identity of this metal has yet to be defined [41]. EDTA treatment had no inhibitory effect on Stel4p, but incubation of the protein with 10 - 50 mM of the metal chelating agent 1,10-phenanthroline eliminated catalytic activity [37,41]. Additionally, even more hydrophobic metal sequesterants such as zincon [37], lysine nitriloacetic acid (Lys-NTA) [42], and cholesteryl-Lys-NTA [43] also inhibited the methyltransferase but at much lower concentrations. Together, these data suggest that the metal ion may be buried in a hydrophobic region of the enzyme. 

From the mere fact that CF, can be released from the membrane by EDTA treatment and the enzyme stays in solution without detergents, it is apparent that the catalytic sector has minimal, if any, direct interaction with the lipids of the chloroplast membrane. It is a globular protein that is held to the surface of the membrane via interaction with the membrane sector. Recently it was shown that the y subunit is in immediate contact with the membrane sector and the 8 and e subunits may induce proper binding for catalysis [17,18], The enzyme contains a few well-defined sites that were used for localization experiments by the method of fluorescent energy transfer [19,56-61], These studies revealed the position of those sites and helped to localize the various subunits of CF, in space relative to the chloroplast membranes (for a model of CF, see Refs. 61 and 62). These experiments are awaiting analysis of the amino acid sequence of the y subunit that is now under investigation in Herrmann s laboratory [148], Definite structural analysis could be obtained only after good crystals of the enzyme become available. 

Preparation of tumor cells. Cultured tumor cells are detached by EDTA treatment, resuspended and rinsed in HESS buffered with 10 mM HEPES at pH 7.3, and finally adjusted with the same buffer at a concentration of 10 /ml. 

Tumor cells are detached by EDTA treatment, rinsed with PBS and diluted in PBS at approximately 10 /ml. 100 /rl of the cell suspension are plated onto platelets and incubated for 1 h at 37 C. After washing three times with PBS, adherent tumor cells are removed by trypsin/EDTA treatment, and counted with an hemocytometer under phase microscopy. Alternatively, tumor cells can be radioac-... 

Aggregation assay. Tumor cells are detached from the culture dish by EDTA treatment (if trypsin has to be used, then a time for recovery in serum containing medium should be allowed before the aggregation assay) and diluted at 10 /ml in DMEM-2%FCS. 

Fluorescent labeling. Tumor cells to be labeled with fluorescent dyes are first put in suspension by EDTA treatment (cell concentration ranging from 2 to 5 x 10 /ml), and then incubated with the chosen dye for 15-60 min. 

Cells are harvested from the dish by EDTA treatment, resuspended and rinsed in PBS-CMF, and finally resuspended at the desired concentration in serum free medium buffered at pH 7.2 with Hepes and filtered through 0.22 fim MiUipore membranes. Cell viability is checked by trypan blue dye exclusion test, and aggregates, if present, can be removed by filtration through cell strainers (Costar). 

EDTA treatment of intermittent claudication—a doubleblind, placebo-controlled study. J Intern Med 1992 231(3) 261-7. 

The concentration of (EDTA) ", and thus the ability to complex metal ions, will depend upon the pH. A decrease in pH results in an increase in the deprotonation of EDTA and hence an increase in the concentration of the ED I A ion. The effect of this is that only metal ions with a very high affinity for EDTA will be able to form stable complexes. The stability constants for the EDTA and [diethylenetriaminepentaacetic acid] - (DTPA ) complexes with some important metal ions that are of particular interest for chelation therapy are listed in Table 7.3. It is important to note that the stability of the EDTA and DTPA complexes with toxic metals, such as lead, mercury, cadmium, or plutonium are quite similar to those with essential metals such as zinc, cobalt or copper however, the Ca complex is many orders of magnitude lower. This has important implications for chelation therapy. First, the mobilization and excretion of zinc and other essential metals are likely to be increased, along with that of the toxic metal during EDTA treatment and secondly, the chelation of the ionic calcium in the blood, that can cause tetany and even death, can be avoided by administering the chelator as the calcium salt. 

Insulin shock may also occur due to the EDTA treatment lowering the serum glucose. Regular food intake during the chelation treatment will help to prevent this. 

Fig. 5. Rate of cholesterol synthesis in the mucosa along the villus-crypt axis in the rat intestine. Enriched mucosal cell fractions were obtained from the mucosa by EDTA treatment and these viable cells were subsequently incubated in the presence of [ HJwater. Panels A and B represent the rate of cholesterol synthesis expressed as the nmoles of acetyl-CoA units incorporated into cholesterol/h/mg of cell protein in the jejunum and ileum, respectively. Panels C and D represent the total activity found in the isolated cell fractions talcing into account the cell protein recovered in each fraction. The columns and bars represent means + 1 S.E.M.

Ibim SE, Trotman J, Musey PI, et al. 1992. Depletion of essential elements by calcium disodium EDTA treatment in the dog. Toxicology 73 229-237. 

The IR spectra for chelate treated samples still consist of the two bands that were observed for the original samples but now vary in intensity. The absorbance ratios of the IR bands at 2115 and 2050 cm increase as shown in table 2. These facts suggest that the higher coordinated species is removed form the zeolite by EDTA treatment. To resolve the problem of whether or not effect is due to the chelating ability of EDTA or water solubility, the Fe(CN)-Y(l) and Fe(CN)-Y(2) zeolites were stirred with a NaCl solution. In each case the absorbance ratio of IR bands at 2115 and 2050 cm" increases several times whereas the C and N content decreases (table 2). From these results it appears that a decrease in the intensity of the IR band at 2050 cm" can account for the solubility of higher coordinated species in... 

Of the agents listed, lead is most likely to cause a decrease in heme biosynthesis. Exposure to inorganic arsenic may also cause anemia. The urinary concentrations of lead before and after EDTA treatment may confirm the diagnosis. The answer is (C). 

E. Treatment courses should be separated by a minimum of 2 days, and an interval of 2 or more weeks may be indicated to assess the extent of posttreatment rebound in blood lead levels. An additional course of calcium EDTA treatment may be considered based on posttreatment blood lead concentrations and the persistence or recurrence of symptoms. 

F. Consider changing to orai succimer (see p 501) or orai unithioi (p 506) after 3 to 5 days of caicium EDTA treatment, provided that encephaiopathy or coiic has resoived, the biood iead ievei has faiien to iess than than 100 mcg/dL, and the patient is abie to absorb an orai formuiation. 

Leive, L. (1965) Release of lipopolysaccharide by EDTA treatment of E. coli. Biochem. Biophys. Res. Commun. 21, 290-296. 

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