EDTA Therapy for Vascular Disease

Chelation Natural Miracle For Protecting Your Heart

Chelation therapy has been conclusively shown to be up to 82 Percent Effective at dissolving the plaque that blocks arteries! In the ebook Chelation Natural Miracle For Protecting Your Heart you'll discover: The frustrating reason many doctors are ignoring Edta chelationor even openly rejecting it. The deadly heart surgeries even the American Heart Association admits are unnecessary. The hidden signs and symptoms of heart attacks and strokes? Are you in danger right now?. The average number of years stripped away by heart and vessel disease. Can you get them back?. The newest set of risk factors for heart disease (they'll likely surprise you!). Shady government practices that protect Big Pharma and keep Edta chelation out of the public eye. How the Roman Empire could have been savedif only they'd known about Edta chelation. Why Edta chelation is guaranteed to be safeeven in extremely high doses. (It puts aspirin to shame!). The shocking truth about plaque in young childrenand how to keep your little ones safer. Why dentists, artists and welders need Edta chelationand whether your workplace is dangerous too. The differences between IV and oral chelationand which kind of Edta is right for you. Other forms of chelationand how these little-known treatments can dramatically boost your health.

Chelation Natural Miracle For Protecting Your Heart Summary

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Author: Michael Cutler, M.D.
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Chelating agents

In clinical medicine, chelating agents are used in the treatment of metal poisoning, because they bind the metal more strongly than do the vulnerable components of the living organism. Chelating agents are heavy metal antagonists, since they compete with these reactive groups in the body for the metal and therefore they can prevent or treat toxic effects by permitting excretion. Clearly, a selective chelator is required so as not to bind the body's own essential metals, though it must distribute in the same body spaces. It must also have a greater affinity than the body's own ligands, it must produce non-toxic complexes pending excretion and it must be able to mobilize the metal after binding so as to permit excretion.

Solutions for Extraction of Genomic and Plasmid DNA

Digestion buffer 100 mMNaCl, 10 mMTris-HCl (pH 8.0), 25 mM ethylene-diaminetetraacetic acid (EDTA) (pH 8.0), 0.5 sodium dodecyl sulfate (SDS), 100 g mL proteinase K. 3. Tris-EDTA (TE) buffer 10 mM Tris-HCl, 1 mM EDTA. Adjust pH to 8.0. Autoclave. 5. Sucrose Triton X EDTA Tris (STET) solution 8 (w v) sucrose, 5 (w v) Triton X-100, 50 mM EDTA, 50 mM Tris-HCl (pH 8.0). Filter sterilize and store at 4 C. 6. Glucose Tris EDTA (GTE) solution 50 mM glucose, 25 mM Tris-HCl (pH 8.0), 10 mM EDTA (pH 8.0). Autoclave and store at 4 C.

Materials 21 Hybridization Filters

Yeast spheroplastmg solution 1M sorbitol, 20 mM EDTA, 10 mM Tris-HC1, pH 7.4, containing freshly added 0.1 mg mL zymolyase 20T (ICN, High Wycombe, UK) and 14 mM p-mercaptoethanol (see Notes 5 and 6). 3. 50 mM EDTA, pH 8.0. 4. Basic yeast spheroplastmg solution 1M sorbitol, 20 mM EDTA, 10 mM Tris-HCl, pH 7 4. 8. Filter sterilized or autoclaved yeast lysis solution (YLS) 1 lithium dodecyl sulfate, 100 mM EDTA, 10 mM Tris-HCl, pH 8.0 (see Note 7). 9. T0 ,E 10 mM Tris-HCl, pH 8, 0.1 mM EDTA.

A False Negative Results

(7) Some antibodies may be detected only in the presence of active complement. Anticoagulants such as ACD, CPD, or EDTA will chelate calcium, preventing activation of complement. Thus, the use of plasma rather than serum may lead to a false negative reaction. Old or improperly stored serum will also have impaired complement activity.

Preparation of Genomic DNA

Inactivate the restriction enzyme by the addition of proteinase K to 250 pg mL and EDTA, pH 8.0 to 0.05Mand incubating at 37 C for 30 min. Then inactivate the proteinase K by washing the blocks in 50 mL of 40 pg mL PMSF in TE twice for 30 mm at 50 C. Store the blocks in 0.5MEDTA while the quality of the digest is assessed.

Preparation of Mouse Embryonic Fibroblasts

Suspend each minced embryo in 5 mL of trypsin-EDTA and transfer it to a 15-mL tube. 10. When the flasks become highly confluent (2-4 days after initial seeding), expand the cultures. Treat the flasks individually as follows. Wash twice with PBS and add 2 mL trypsin-EDTA. After 5 min at 37 C, resuspend the cells in 40 mL MEF-DMEM. Seed in three 75-cm2 flasks. 11. When the cultures again are highly confluent, frozen stocks should be prepared. Wash twice with PBS and add 4 mL trypsin-EDTA. After 5 min at 37 C, resuspend cells in each 75-cm2 flask in 4 mL MEF-DMEM and transfer the trypsinized cells to 15-mL tubes.

Dealing with High Concentration of Heavy Metals Homeostasis Tolerance Detoxification

For most physiological and metabolic processes the maintenance and control of optimised metal concentration is necessary. For homeostasis as well as detoxification of supra-optimal metals concentration a number of cellular structures and physiological processes like accumulation in mycorrhiza, binding to the cell walls, exudation and a network of regulating metal transport, chelation, trafficking and sequestration are responsible in plants (Clemens 2001 Guerinot and Salt 2001 Clemens et al. 2002 Hall 2002).

Detergents Cleaners And Surfactants

Several different components may be present in a particular detergent formulation. These include alkalies, acids, surfactants, and chelating agents, in varying proportions. Such formulations are often referred to as built detergents or built cleaners and, depending on composition, may achieve multiple cleaning goals in a single application. Alternatively, formulations can be prepared in the winery using individual chemicals and ingredients. It is important to consult specific suppliers for information related to proper use of these chemicals because incorrect or improper mixing can create gases highly toxic to workers.

General Serologic Investigations

Collect clotted blood to provide serum and blood in EDTA, to provide RBCS for the direct antiglobulin test, grouping, and preparation of eluates. If it is not known whether the patient has AIHA associated with warm or cold antibodies, it is preferable to collect blood and separate it at 37 C. If powerful cold autoantibodies are present, several problems can arise if the blood is not kept at 37 C. (3) In vitro complement autosensitization may occur if EDTA blood is not used, leading to false increase in the strength of the direct antiglobulin test. EDTA will prevent any in vitro complement sensitization, even if the blood is cooled thus, any complement detected on the RBCS represents in vivo sensitization.

Determination of Amino Acids

Eluent A One liter of 0.1 M sodium phosphate buffer. Add 560 L of triethylamine and 200 L of a 1 mg mL solution of EDTA disodium salt. Adjust the pH to 5.70, and add 6 vol of acetonitrile (HPLC gradient grade) to 94 vol of the phosphate buffer with additives. 6. Eluent B acetonitrile water (60 40 v v ) to which is added 200 L of a 1 mg mL solution of EDTA disodium salt per liter.

Stabilization of Packaged DNA and Control of DNA Release Assembly and Structure of the SPP1 Connector

DNA is packaged at a high density inside the viral capsid of tailed bacteriophages. A direct correlation between the amount of encapsidated DNA and the sensitivity to chelating agents was demonstrated for a variety of phages including SPP1.3'30 Divalent cations thus play a central role to stabilise the DNA-filled capsid most probably by neutralizing the negative charges of closely packed DNA phosphate backbones. They might also relieve repulsion between DNA and the capsid lattice interior in case the latter is negatively charged as shown for phage HK97.26 The DNA packing generates an internal pressure that can lead to its spontaneous release from the phage capsid.8'31 34

Quantitation of Cochleate Associated DNA

Add 150 mM EDTA (pH 9.5) in 5- L aliquots (vortex after each addition), until sample becomes slightly alkaline (e.g., 10 L). Monitor pH by removing 0.5- L aliquots and depositing on pH paper (preferably pH 6.0-8.0 range with 0.1 U distinction). 4. Add 150 mM EDTA (pH 7.4) in 5- L aliquots, until cochleates are converted to liposomes (e.g., 10 L). Suspension turns from white particulate to opalescent. Check by light microscopy (x1000), oil immersion, phase contrast to confirm conversion of cochleate crystals to liposomes. Measure volume with a micropipetor.

Materials see Note 1

10X Run-on buffer. 1.5MNaCl, 25 mMMgCl2, 50 mMMg acetate, 10 mM MnCl2, 20 mM DTT, 1 25 mM EDTA, 5 mM ATP (Pharmacia, Uppsala, Sweden), 5 mM GTP (Pharmacia), 5 mM CTP (Pharmacia), 20 mM creatine phosphate, 30 U mL creatine phosphokinase (Boehringer Mannheim, Mannheim, Germany), and 5 mg mL heparin. 19. 2X SSPE 360 mM NaCl, 20 mM Na phosphate buffer, pH 6.8, and 1 mM EDTA. 26. Hybridization buffer 500 mM Na phosphate buffer, pH 6.8,1 (w v) SDS, 1 mM EDTA, and 15 (v v) formamide (Fluka, Buchs, Switzerland) see Note 4).

Source of matrix effect

K3-EDTA Sarstedt Another study was designed to study the impact of different anticoagulants on LC-MS matrix effects using the same type of strategy. The markers were added to water and rat plasma containing different types and increasing amount of anticoagulants. No significant matrix effect was observed for all the test compounds with up to 29 of Na-heparin and Na2-EDTA in serum. However, an enhanced mass signal of CMPD 1 with increasing concentrations of Li-heparin in serum was observed, as shown in Figure 4.6. As shown in Table 4.4, the normalized mass responses of eight test compounds in serum are

Preparation of ISCOMs Conjugation to PreformedISCOMMatrix

Separate the ISCOM-matrix from SPDP by gel filtration, e.g., using prepacked desalting columns (PD-10 from Pharmacia and Upjohn, Kalamazoo, MI or Econo-Pac 10DG from Bio-Rad, Richmond, CA) equilibrated with N2-saturated 0.1 M phosphate buffer containing 0.1 M ethylenediaminetetraacetic acid (EDTA), pH 6.7. Collect fractions of 5-6 drops in an enzyme-linked immunosorbent assay (ELISA) plate and pool the ISCOM-matrix containing fractions (e.g., detected through 3H-cholesterol in the ISCOM-matrix). 3. Separate the ISCOM-matrix from SPDP DTT by gel filtration, e.g., using prepacked desalting columns (PD-10 from Pharmacia and Upjohn or Econo-Pac 10DG from Bio-Rad) equilibrated with N2-saturated 0.1 M phosphate buffer containing 0.1 M (EDTA), pH 6.7. Collect fractions of 5-6 drops in an ELISA plate and pool the ISCOM-matrix containing fractions (e.g., detected through 3H-cholesterol in the ISCOM-matrix). 2. Separate the ISCOM-matrix from MBS by gel filtration, e.g., using prepacked...

C Principles of the Procedure

(3) Both types of QBC tubes are internally coated with AO and potassium oxalate. The former, a supravital fluorochrome, stains the white cells and platelets as previously described. The potassium oxalate osmotically removes water from the erythrocytes, causing their density to increase and their volume to shrink. This tends to prevent commingling of certain equal-density erythrocytes and leukocytes at the interfacing boundary between these cell layers. QBC capillary-blood tubes additionally contain a coating of sodium heparin and dipotassium EDTA to inhibit the clotting of finger puncture blood.

Spillages and Decontamination

Any activity in the wrong place is a spill and must be dealt with immediately. The spilt material should be wiped up with a swab of tissues, which are placed in the appropriate waste receptacles. The area is then decontaminated. Try the following decontamination materials in the following order (a) distilled water, (b) water and detergent (decon 90), (c) a solution of EDTA, and (d) a+b+c+ion-exchange medium (fullers earth). When contamination occurs on clothing, the clothing should be removed as quickly as possible and washed by normal laundry procedures. A solution of EDTA is useful in difficult cases. Contaminated skin should be brushed for a long time with soap and warm water using a soft brush. If this is unsuccessful try titanium paste or EDTA soap. Skin decontamination should never be continued to the extent of damaging the skin.

Chromium Release Assay

Cell-lifting media for adherent targets (0.25 STV). For 1 L of 0.25 STV Potassium chloride (0.4 g), sodium chloride (8 g), glucose (1 g), sodium bicarbonate (0.58 g), trypsin (2.5 g), ethylenediamine tetraacetic acid (EDTA) (0.2 g) phenol red (0.004 g), hydrochloric acid, 6 N as required, sterile H2O up to 1 L. Add each chemical to the solution in the order listed, stir each component except for trypsin one at a time for at least 10 min. Stir trypsin for at least 30 min. Keep solution covered after adding phenol. Adjust pH to 7.05 0.05 using HCl 6 N. Sterile filter (0.22 im). Store at 4 C, expires in 6 mo. 5. Triton lytic mix. For 1 L of triton lytic mix Triton, 10 (in 0.01 M Tris-HCl, pH 8.0) (10 mL), 1 M Tris-HCl, pH 8.0 (50 mL), 0.25 M EDTA, pH 8.0 (250 mL), sterile H2O up to 1 L. Store at 2-30 C. Expires in 1 yr.

Molecular Engineering to Improve the Performance of Plants in Phytoremediation

Detoxification chelation, degradation, transformation Fig. 7.3. Several physiological mechanism for resistance against pollutants and targets for bioengineering and enhanced performance in phytoremediation. (1) uptake by the roots, (2) loading into the xylem, (3) transport by mass flow in the xylem to the shoot in the transpiration stream, (4) uptake into leaf tissue, (5) accumulation, storage and detoxification (chelation, degradation and transformation), and (6) sequestration compartmentation of pollutants 5. detoxification chelation, degradation, transformation Fig. 7.3. Several physiological mechanism for resistance against pollutants and targets for bioengineering and enhanced performance in phytoremediation. (1) uptake by the roots, (2) loading into the xylem, (3) transport by mass flow in the xylem to the shoot in the transpiration stream, (4) uptake into leaf tissue, (5) accumulation, storage and detoxification (chelation, degradation and transformation), and (6) sequestration...

Species of Dominant Bacteria

Bacterial reactions with heavy metals can occur extracellularly, pericellularly (surrounding the cell), and intracellularly. Metals may accumulate extracellularly through chelation by extracellular polysaccharides that are secreted by bacteria such as Bacillus and Zoogloea. Chelated metals do not cause toxicity.

Microneme secretion attachment and invasion of the host cell

A decrease in host invasion by tachyzoites is observed (Vieira and Moreno, 2000). Inhibition of microneme release by chelation of intracellular calcium with BAPTA-AM also inhibits parasite invasion of host cells (Carruthers et al., 1999b). In summary, these results indicate that a Ca2+ increase that occurs upon attachment of tachyzoites to the host-cell surface is possibly associated with conoid extrusion, microneme secretion, and invasion. The source of the Ca2+ increase needed for invasion is an intracellular store. Although previous reports using EGTA to chelate extracellular Ca2+ indicated that this was important for invasion (Pezzella et al., 1997), it was found that this effect was due to acidification of the culture medium by EGTA and not to chelation of Ca2+ (Lovett and Sibley, 2003).

Egress from the host cell

Exposure of tachyzoites to 5-mM dithiotretitol (DTT) was also shown to activate egress of previously non-motile intravacuolar parasites within 60 seconds. This was accompanied by an increase in the intra-parasitophorous vacuole (PV) fluorescence ratio of indo 1-loaded infected human fibrob-lasts. The parasite activation and Ca2+ increase were prevented by chelation of extracellular Ca2+ by EGTA

Some Specialists Can Deal with High Levels of Heavy Metals Hyperaccumulators

Metal-rich soils (Salt et al. 1998 Meagher 2000 Clemens 2001 Guerinot and Salt 2001 Hall 2002 Clemens et al. 2002 Pollard et al. 2002 Cobbett 2003 McGrath and Zhao 2003 Freeman et al. 2004). This was observed for essential nutrients (Cu, Fe, Zn, and Se) or non-essential metals (Cd, Hg, Pb, Al and As) which can cause toxicity already at low concentrations. The metal concentration in hyperaccumulators can be some orders of magnitudes higher than in not accumulating plants (100-1000-fold) 1 for Zn (up to 4 ) and Mn 0.1 for Co (up to 1.2 ), Cu, Ni (up to 3.8 ), As (up to 0.75 ) and Se (up to 0.4 ) and 100 ppm for Cd (up to 0.2 ). Hyperaccumulators have an unusually high metal uptake and highly efficient chelation and compartmen-tation (Pilon-Smits and Pilon 2002 Pollard et al. 2002).

Preparation of EF Cells for ESCell Culturing

Place the embryos in a dish containing 15 mL of 0.25 Trypsin EDTA and mechanically dissociate by mincing with fine iris scissors. Draw up and down six times with a 10-mL pipet and place the minced embryo suspension in a 37 C incubator for 10 min. Pipet up and down six times with a 5-mL pipet and return the dish to the incubator for a further 10 min. 7. To freeze these cells at passage 2, split the cells in the original T-150 flask into 3 T-150 flasks. Incubate for 24 h, refeed and incubate for another 24 h. Dissociate the cells in these flasks with 0.05 trypsin EDTA and pool. Pellet the cells and bring up the pellet in 10-mL EFM. 1. Trypsinize the cells with 3 mL 0.05 Trypsin-EDTA (w o HEPES) for about 1 min.

Preparation of Transgene DNA

Be injected must be digested with the appropriate restriction endonuclease(s) to free the transgene from prokaryotic vector sequences (8). The incorporation of prokaryotic sequences inhibits expression of transgenes in the murine genome. The transgene is usually separated from the vector sequences by agarose-gel electrophoresis. The appropriate fragment is excised, purified, and quantitated by measuring the optical density (OD) of the sample at 260 nm. The purity of the fragment is estimated by measuring the OD at 280 nm and comparing the ratio of OD 260 OD 280. Once the isolated DNA is verified as the correct fragment and quantitated, the DNA is diluted to 2 ng L in TE buffer (10 mM Tris pH 7.5, 0.25 mM ethylenediaminetetraacetic acid EDTA ). The DNA is then microinjected into the male pronucleus of one-cell mouse embryos. This buffer and concentration has been shown to be optimal for incorporation of the transgene into the murine genome (8).

Cytolytic Assay

0.25 g mL amphotericin-B and 10 fetal bovine serum (see Note 8). The cells are detached with a trypsin-EDTA solution and expanded three times per week. The protocol described below takes about 3 d to be performed and can be used for both hTNF and mTNF. Other, more sensitive bioassays have been described using different cell lines (10-13).

Transgene Release

Run a REN digestion check (10-15 jiL digested DNA) and stop the reaction by adding 2 jiL of 0.5 M EDTA. At this stage the DNA may be mixed with loading buffer and directly loaded onto a preparative agarose gel, or, alternatively, extracted by phenol-chloroform-isoamylalcohol extracted first.

DNA Purification

Adjust DNA solution to solute concentrations of Elutip buffer I to 100 jL DNA in TE buffer add 9.0 jL 1 M Tris-HCl, pH 7.4, 0.8 jL 0.5 M EDTA, 20 jL 5 M NaCl, 370 jL dH2O. 14. Dissolve pellet in 50 jL filtered (Uniflo, 0.45 jm) special TE buffer for microinjection 10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA.

Desferrioxamine

Calcium disodium edetate (sodium calciumedetate, ethylene-diaminetetra-acetate, EDTA) is chiefly used in lead poisoning, though it is also of value in enhancing the clearance of cadmium (and also chromium, cobalt, copper, magnesium, nickel, selenium, uranium, vanadium, zinc) and (in double dosage) of the radioactive products from nuclear accidents (e.g. plutonium). The toxic metal displaces calcium from the chelator. It has also been used in atherosclerosis but without evidence to support this indication. EDTA has a half-life of about 40 min, distributes in the extracellular fluid and is excreted in the urine (50 in the first hour), so that it is contraindicated in anuria. The side-effects of EDTA include

Answers

The answer is a. (Levinson, p 4.) The periplasm is the space between the outer membrane and plasma membrane of bacteria. The periplasmic space in Escherichia coli has been shown to contain a number of proteins, sugars, amino acids, and inorganic ions. Ethylenediamine-tetraacetic acid (EDTA) is a chelating agent that disrupts the cell walls of Gram-negative bacteria.

Cytotoxicity

The presence during lipoprotein preparation of general free radical scavengers (vitamin E, butylated hydroxytoluene) or the divalent cation chelator EDTA prevented the formation of cytotoxic oxLDL. However, the toxic action of oxLDL could not be prevented by the addition of any of these agents during incubation of the oxLDL with cells (Morel et al., 1983). These observations indicate that an oxidized lipid is responsible for cytotoxicity rather than free radicals generated in culture by the action of oxLDL. Hughes et al. (1994) extracted the toxic components from lipid extracts of oxLDL and identified them as the oxysterols 7-ketocholesterol and 7-hydroxycholester-ol. When the two toxins were added to native LDL at concentrations equivalent to those present in oxLDL, the resultant LDL was not rendered cytotoxic. This suggests that the oxysterols are released more rapidly from oxLDL than from native LDL because of the severe disruption of the lipoprotein's lipid environment during...

Water Quality

Absent a water-softener system, many detergent formulations include special adjuncts that can help mitigate water hardness. Known as chelating agents, these chemicals physically remove metals from solution, thus softening the water. One group of chemicals, the polyphosphates, are widely used due to their ability to chelate calcium and magnesium and prevent precipitation. Specific examples of polyphosphates are sodium hexametaphosphate (e.g., Calgon ) and sodium tetraphosphate (Quadro-fos). With the exception of trisodium phosphate, these chemicals are noncorrosive. Another compound used as a chelator is ethylenediamine tetraacetic acid (EDTA). Although more expensive than polyphosphates, EDTA has the advantage of being relatively heat stable. Because chelating agents bind water hardness minerals (metals), these will improve the subsequent effectiveness of cleaners.

Anticoagulants

These anticoagulants prevent clotting by binding calcium and thus preventing the complete chemical reaction that produces fibrin. Ethylene-diamine-tetra-acetate (EDTA) is the most often used others include sodium citrate, sodium fluoride, and oxalates.

Cathy Abbott

1 STE 50 mM Tris-HCl, pH 8, 100 mM NaCl, 100 mM EDTA, and 1 (w v) SDS. (25'24 1) Phenol should be equilibrated in TE (10 mM Tris-HCl, pH 7.5, 1 mM EDTA). An equal vol of TE is mixed with phenol melted at 65 C. The phases are allowed to separate, and then the buffer aqueous phase is removed. Repeat the procedure until the pH of the phenol is 7.5.

Porphyria

The enzymic steps from the initial components, glycine and succinyl-CoA, to the eventual production of haem give rise to ring structures via progressive deamination, decarboxylation, oxidation and finally iron chelation. These steps clearly provide multiple sites for enzymic defects, and indeed an abnormality at each of

Mouse Models

The B16BL6 melanoma subline, selected from a spontaneous melanoma B16F10 line, was used in our studies. Tumor cells were expanded in vitro in Dulbecco's MEM containing 10 FCS, 2 mM l-glutamine, 1 mM sodium pyruvate, MEM-nonessential amino acid, MEM vitamins, 50 U mL penicillin, and 50 g mL streptomycin. On d 0 (the day of inoculation), cells were detached from the culture flask by a short EDTA treatment, washed once in complete medium, and twice in PBS. A total of 6 x 105 cells were suspended in

Treatment

A wide range of pharmacological agents and alternative therapies have been utilized to treat the symptoms of mild to moderate limb ischemia. Unfortunately, controlled clinical trials coupled with careful evaluation of long-term improvement have demonstrated a consistent lack of benefit for the vast majority of agents tested. This includes vasodilator drugs, pentoxifylline, antiplatelet and antithrombotic drugs, chelation therapy, and a variety of herbal medications such as gingko. The agents that have been shown to be of some value in controlled clinical trials include cilostazol, a phos-phodiesterase inhibitor that cannot be used in patients with cardiac dysfunction, the Tibetan herbal supplement Padma Basic, and high doses of L-arginine, the amino acid precursor of the endogenous vasodilator nitric oxide. The benefit in walking distance with these agents, although statistically significant in clinical trials, is often minimal in terms of functional improvement for the patient. For...

Antioxidant activity

Belguendouz et al. (1997) carried out an extensive examination of the inhibition by resveratrol of porcine LDL oxidation in the presence of the free radical generator 2,2'-azobis (2-amidinopropane dihydrochloride) (AAPH) or copper ions. The slope of the propagation phase and the prolongation of the lag phase were much greater with the latter than with the former. Formation of TBARS was completely inhibited by up to 200 min in the copper-mediated system by 1 mol l resveratrol, more effective than trolox or the flavonoids tested. The relevant mechanisms appeared to be a combination of copper chelation and free radical scavenging surprisingly, resveratrol was unable to chelate iron. In a subsequent report it proved to be more effective than flavonoids as a chelator of copper and less effective as a free-radical scavenger (Fremont et al. 1999), but later authors found it to be very potent in scavenging superoxide and peroxyl radicals, and in blocking lipid peroxidation (Martinez and...

Microneme secretion

Microneme secretion is a regulated process (also known as stimulus-coupled secretion). The external stimulus that triggers MICs secretion is unknown, but fluorescence imaging studies have revealed that parasites in association with host cells show elevated levels of cytoplasmic calcium (Vieira and Moreno, 2000). Chelation of extracellular calcium with EGTA or BAPTA or addition of excess calcium has little effect on microneme secretion, parasite motility, or cell invasion (Lovett and Sibley, 2003). Chelation of host-cell intracellular calcium prior to invasion with the cell-permeable chelator BAPTA-AM also has no effect on attachment and invasion (Lovett and Sibley, 2003). In addition, host-cell calcium levels remain relatively constant during invasion (Lovett and Sibley, 2003). These observations indicate that parasite invasion occurs independently of host calcium. By contrast, secretion of micronemes is inhibited by chelation of parasite intracellular calcium (by pre-loading of...

Manipulation of [Ca2

With AM esters of BAPTA and a calcium indicator simultaneously, since the same conditions can be used (Kao, 1994). This BAPTA buffering method has been widely used to understand the role of calcium in microneme secretion by T. gondii (Carruthers and Sibley, 1999), conoid extrusion (Mondragon and Frixione, 1996), gliding motility (Wetzel et al., 2004) and invasion (Vieira and Moreno, 2000). It is important to load the cells with BAPTA analogs unable to chelate Ca2+, as a control that the effect of BAPTA is because of Ca2+ chelation and not because of toxicity. Analogs such as 'half-BAPTA' (Vieira and Moreno, 2000) or D-BAPTA (Saoudi et al., 2004) can be used, although half-BAPTA is not currently available. It is important to know that BAPTA can display side effects. It has a potent microtubule depolymerizing effect, and decreases the ATP pool of the cells (Saoudi et al., 2004). It is also important to provide controls showing that the concentrations of BAPTA-AM used are able to chelate...

Northern Blot

RNA denaturation buffer 1X MOPS buffer, 6.5 formaldehyde, and 50 formamide. 10X MOPS buffer consists of 0.4 M 3-N-morpholino-propanesulfonic acid, pH 7.0 0.1 M sodium acetate and 0.01 M EDTA-Na2 (see Note 4). 4. RNA gel-loading buffer 1 mM EDTA, pH 8.0, 0.25 bromophenol blue, 0.25 xylene cyanol, 50 glycerol. 6. RNA transfer solution (10X SSPE) 1.5 M NaCl, 96 mM NaH2PO4, 10 mM EDTA-Na2.