Metal Chelator Diethylenetriaminepentaacetic Acid Research Articles

Comparison of Local and Systemic DTPA Treatment Efficacy According to Actinide Physicochemical Properties Following Lung or Wound Contamination in the Rat.

Purpose: In cases of occupational accidents in nuclear facilities or subsequent to terrorist activities, the most likely routes of internal contamination with alpha-particle emitting actinides, such as plutonium (Pu) and americium (Am), are by inhalation or following wounding. Following contamination, actinide transfer to the circulation and subsequent deposition in skeleton and liver depends primarily on the physicochemical nature of the compound. The treatment remit following internal contamination is to decrease actinide retention and in consequence potential health risks, both at the contamination site and in systemic retention organs as well as to promote elimination. The only approved drug for decorporation of Pu and Am is the metal chelator diethylenetriaminepentaacetic acid (DTPA). However, a limited efficacy of DTPA has been reported following contamination with insoluble actinides, irrespective of the contamination route. The objectives of this work are to evaluate the efficacy of prompt local and/or systemic DTPA treatment regimens following lung or wound contamination by actinides with differing solubility. The conclusions are drawn from retrospective analysis of experimental studies carried out over 10 years. Materials and Methods: Rat lungs or wounds were contaminated either with poorly soluble Mixed OXide (U, Pu O2) or more soluble forms of Pu (nitrate or citrate). DTPA treatment was administered promptly after contamination, locally to lungs by insufflation of a powder or inhalation of aerosolized solution or by injection directly into the wound site. Intravenous injections of DTPA were given either once or repeated in combination with the local treatment. Doses ranged from 1 to 30 µmol/kg. Animals were euthanized from day 7–21 and alpha activity levels were measured in urine, lungs, wound, bone and liver for determination of decorporation efficacy. Results: Different experiments confirmed that whatever the route of contamination, most of the activity is retained at the entry site after insoluble MOX contamination as compared with contamination with more soluble forms which results in very low activities reaching the systemic compartment and subsequent retention in bone and liver. Several DTPA treatment regimens were evaluated that had no significant effect on either lung or wound levels compared with untreated animals. In contrast, in all cases systemic retention (skeleton and liver) was reduced and urinary excretion were enhanced irrespective of the contamination route or DTPA treatment regimen. Conclusion: The present study demonstrates that despite limitation of retention in systemic organs, different DTPA protocols were ineffective in removing insoluble actinides deposited in lungs or wound site. For moderately soluble actinides, local or intravenous DTPA treatment reduced activity levels both at contamination and at systemic sites.

Identifying the Sources and Sinks of CDOM/FDOM across the Mauritanian Shelf and Their Potential Role in the Decomposition of Superoxide (O2-)

Superoxide (O2-) is a short lived reactive oxygen species (ROS) formed in seawater by photochemical or biological sources, it is important in the redox cycling of trace elements and organic matter in the ocean. The photoproduction of O2- is now thought to involve reactions between O2 and reactive reducing (radical) intermediates formed from dissolved organic matter (DOM) via intramolecular reactions between excited singlet state donors and ground-state acceptors (Zhang et al., 2012). In seawater the main pathways identified for the decomposition of O2- into H2O2 and O2, involve reactions with Cu, Mn and DOM. In productive regions of the ocean, the reaction between DOM and O2- can be a significant sink for O2-. Thus DOM is a key component of both the formation and decomposition of O2- and formation of H2O2. In the present work we examined the relationships between O2- decay rates and parameters associated with chromophoric dissolved organic matter (CDOM) and fluorescent dissolved organic matter (FDOM) by using the thermal O2- source SOTS-1. Filtered samples (0.2 µm) were run both in the presence, and absence, of the metal chelator diethylenetriaminepentaacetic acid (DTPA) to determine the contribution from DOM. Samples were collected along a transect across the continental shelf of the Mauritanian continental shelf during a period of upwelling. In this region we found that reactions with DOM, are a significant sink for O2- in the Mauritanian Upwelling, constituting on average 58 ± 13 % of the O2- loss rates. Superoxide reactivity with organic matter showed no clear correlation with bulk CDOM or FDOM properties (as assessed by PARAFAC analysis) suggesting that future work should concentrate at the functional group level to clearly elucidate which molecular species are involved as bulk properties represent a wide spread of chemical moieties with different O2- reactivities. Analysis of FDOM parameters indicates that many of the markers used previously for terrestrial sources of DOM and FDOM are called into question as marine sources exist. In particular recent work (Rico et al., 2013) indicates that algal species may also produce syringic, vanillic and cinnamic acids, which had previously been ascribed solely to terrestrial vegetation.

Rifampin induces hydroxyl radical formation in Mycobacterium tuberculosis.

The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indicating that the ROS were induced after RpoB binding. To identify the ROS formed, lysates of RIF-treated bacilli were incubated with DMPO, a spin trap specific for ·OH and O2·(-), with or without pretreatment with SOD, catalase, THIO, or DTPA. Superoxide dismutase, catalase, and THIO decreased formation of the DMPO-OH adduct, and SOD plus DTPA completely suppressed it, suggesting that RIF activated metal-dependent O2·(-)-mediated mechanisms producing ·OH inside tubercle bacilli. The finding that the metal chelator DTPA reduced the bactericidal activity of RIF supported the possibility that ·OH was generated through these mechanisms and that it participated at least in part in M. tuberculosis killing by the drug.

Sequence-independent Control of Peptide Conformation in Liposomal Vaccines for Targeting Protein Misfolding Diseases

Synthetic peptide immunogens that mimic the conformation of a target epitope of pathological relevance offer the possibility to precisely control the immune response specificity. Here, we performed conformational analyses using a panel of peptides in order to investigate the key parameters controlling their conformation upon integration into liposomal bilayers. These revealed that the peptide lipidation pattern, the lipid anchor chain length, and the liposome surface charge all significantly alter peptide conformation. Peptide aggregation could also be modulated post-liposome assembly by the addition of distinct small molecule β-sheet breakers. Immunization of both mice and monkeys with a model liposomal vaccine containing β-sheet aggregated lipopeptide (Palm1-15) induced polyclonal IgG antibodies that specifically recognized β-sheet multimers over monomer or non-pathological native protein. The rational design of liposome-bound peptide immunogens with defined conformation opens up the possibility to generate vaccines against a range of protein misfolding diseases, such as Alzheimer disease.

Effects of DTPA on zinc flux in leukemia cell lines

Previous studies from our laboratory with Zn‐65 have shown that H4IIE (rat hepatoma) and GH3 (rat pituitary tumor) cell lines reduce zinc efflux in the presence of the extracellular metal chelator DTPA (diethylenetriaminepentaacetic acid). Primary rat hepatocytes and anterior pituitary cells give out more zinc under the same conditions. This may reflect the greater zinc requirements of the rapidly dividing transformed cells. Here, we examined the effects of DTPA on radioactive Zn‐65 flux in C58(NT) (rat thymic lymphoma) and HL‐60 (human promyelocytic leukemia) cell lines. When these cells were co‐incubated with DTPA (50 µM) and Zn‐65, uptake of label was reduced. But when cells were pre‐labeled with Zn‐65 and then incubated with DTPA, the chelator increased efflux of Zn‐65. Despite being transformed, these cell lines behaved similarly to primary cells. Since the leukemia cells grow in suspension unlike the monolayer cell lines previously examined, we determined whether non‐adherence contributed to the difference in response to zinc deprivation. Experiments were repeated with H4IIE cells kept in suspension by growth in roller bottles. However, even under these conditions, DTPA enhanced retention of cellular zinc in the hepatoma cells. Thus, leukemia cells differ from other transformed cell lines in their homeostatic response to zinc deprivation and this is not due to their inability to form a monolayer.

Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes.

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.

Bioactivation of Nitroglycerin by Purified Mitochondrial and Cytosolic Aldehyde Dehydrogenases

Metabolism of nitroglycerin (GTN) to 1,2-glycerol dinitrate (GDN) and nitrite by mitochondrial aldehyde dehydrogenase (ALDH2) is essentially involved in GTN bioactivation resulting in cyclic GMP-mediated vascular relaxation. The link between nitrite formation and activation of soluble guanylate cyclase (sGC) is still unclear. To test the hypothesis that the ALDH2 reaction is sufficient for GTN bioactivation, we measured GTN-induced formation of cGMP by purified sGC in the presence of purified ALDH2 and used a Clark-type electrode to probe for nitric oxide (NO) formation. In addition, we studied whether GTN bioactivation is a specific feature of ALDH2 or is also catalyzed by the cytosolic isoform (ALDH1). Purified ALDH1 and ALDH2 metabolized GTN to 1,2- and 1,3-GDN with predominant formation of the 1,2-isomer that was inhibited by chloral hydrate (ALDH1 and ALDH2) and daidzin (ALDH2). GTN had no effect on sGC activity in the presence of bovine serum albumin but caused pronounced cGMP accumulation in the presence of ALDH1 or ALDH2. The effects of the ALDH isoforms were dependent on the amount of added protein and, like 1,2-GDN formation, were sensitive to ALDH inhibitors. GTN caused biphasic sGC activation with apparent EC50 values of 42 ± 2.9 and 3.1 ± 0.4 μm in the presence of ALDH1 and ALDH2, respectively. Incubation of ALDH1 or ALDH2 with GTN resulted in sustained, chloral hydrate-sensitive formation of NO. These data may explain the coupling of ALDH2-catalyzed GTN metabolism to sGC activation in vascular smooth muscle.

Rapid homeostatic response of H4IIE cells to zinc deprivation is not due to changes in total amount of ZnT‐1 protein

Earlier studies with H4IIE (rat hepatoma) cells have shown that in the presence of an extracellular metal chelator DTPA (diethylenetriaminepentaacetic acid), efflux of zinc is shut down rapidly. Zinc efflux in cells is controlled by the SLC30 group of membrane transporters known as ZnTs. ZnT-1 is the only member of this family located in the plasma membrane. We have earlier reported that DTPA did not significantly change the concentration of ZnT-1 mRNA in transformed cells. To further understand the mechanism of DTPA-induced zinc retention, protein expression of ZnT-1 was assessed in H4IIE cells, at different time points, in the presence of either zinc or DTPA. Cells were incubated with or without 100μM ZnSO4 or 50μM DTPA for 2 h or 48 h and ZnT-1 protein concentrations were estimated using Western Blot analysis. Zinc increased (15%) whereas DTPA reduced (39%) the expression of ZnT-1 after 48 h treatment. However, ZnT-1 protein concentrations were not affected by either zinc or DTPA, after 2 h of treatment, the time at which zinc efflux was altered in our earlier studies. Thus we conclude that the rapid homeostatic response of cells to DTPA is not due to a change in the total amount of ZnT-1 protein, but could be due to a reduction in its plasma membrane localization.

Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: Implications for Parkinson's disease

Several studies on mitochondrial functions following brief exposure (5-15 min) to dopamine (DA) in vitro have produced extremely variable results. In contrast, this study demonstrates that a prolonged exposure (up to 2 h) of disrupted or lysed mitochondria to DA (0.1-0.4 mM) causes a remarkable and dose-dependent inhibition of complex I and complex IV activities. The inhibition of complex I and complex IV activities is not prevented by the antioxidant enzyme catalase (0.05 mg/ml) or the metal-chelator diethylenetriaminepentaacetic acid (0.1 mM) or the hydroxyl radical scavengers like mannitol (20 mM) and dimethyl sulphoxide (20 mM) indicating the non-involvement of *OH radicals and Fenton's chemistry in this process. However, reduced glutathione (5 mM), a quinone scavenger, almost completely abolishes the DA effect on mitochondrial complex I and complex IV activities, while tyrosinase (250 units/ml) which catalyses the conversion of DA to quinone products dramatically enhances the former effect. The results suggest the predominant involvement of quinone products instead of reactive oxygen radicals in long-term DA-mediated inactivation of complex I and complex IV. This is further indicated from the fact that significant amount of quinones and quinoprotein adducts (covalent adducts of reactive quinones with protein thiols) are formed during incubation of mitochondria with DA. Monoamine oxidase A (MAO-A) inhibitor clorgyline also provides variable but significant protection against DA induced inactivation of complex I and complex IV activities, presumably again through inhibition of quinoprotein formation. Mitochondrial ability to reduce tetrazolium dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) in presence of a respiratory substrate like succinate (10 mM) is also reduced by nearly 85% following 2 h incubation with 0.4 mM DA. This effect of DA on mitochondrial function is also dose-dependent and presumably mediated by quinone products of DA oxidation. The mitochondrial dysfunction induced by dopamine during extended periods of incubation as reported here have important implications in the context of dopaminergic neuronal death in Parkinson's disease (PD).

Improved radiolabeling of PEGylated protein: PEGylated annexin V for noninvasive imaging of tumor apoptosis.

We have developed a one-step procedure to introduce both polyethylene glycol (PEG) and the metal chelator diethylenetriaminepentaacetic acid (DTPA) to proteins through a heterofunctional PEG precursor. The PEG precursor contains DTPA at one end and an amine-reactive isothiocyanate (SCN-) functional group at the other end. It was obtained as lyophilized powder and could be stored at 4 degrees C for several months. Protein conjugation was achieved by simply mixing the proteins and the PEG precursor SCN-PEG-DTPA in an aqueous solution. As exemplified by the PEGylation and radiolabeling of annexin V, the resulting conjugate 111In-DTPA-PEG-annexin V showed selective binding to apoptotic cells in vitro and increased blood half-life in vivo. The PEGylated, radiolabeled annexin V may be useful in the noninvasive imaging of apoptosis.

Ghost protein damage by peroxynitrite and its protection by melatonin.

We have studied the effect of peroxynitrite (ONOO-) on the membrane cytoskeleton of red blood cells and its protection by melatonin. Analysis of the protein fraction of the preparation by SDS-PAGE revealed a dose-dependent (0-600 microM ONOO-) disappearance at pH 7. 4 of the main proteins: spectrin, band 3, and actin, with the concomitant formation of high-molecular weight aggregates resistant to reduction by ss-mercaptoethanol (2%) at room temperature for 20 min. These aggregates were not solubilized by 8 M urea. Incubation of the membrane cytoskeleton with ONOO- was characterized by a marked depletion of free sulfhydryl groups (50% at 250 microM ONOO-). However, a lack of effect of ss-mercaptoethanol suggests that, under our conditions, aggregate formation is not mediated only by sulfhydryl oxidation. The lack of a protective effect of the metal chelator diethylenetriaminepentaacetic acid confirmed that (ONOO-)-induced oxidative damage does not occur only by a transition metal-dependent mechanism. However, we demonstrated a strong protection against cytoskeletal alterations by desferrioxamine, which has been described as a direct scavenger of the protonated form of peroxynitrite. Desferrioxamine (0.5 mM) also inhibited the loss of tryptophan fluorescence observed when the ghosts were treated with ONOO-. Glutathione, cysteine, and Trolox (1 mM), but not mannitol (100 mM), were able to protect the proteins against the effect of ONOO- in a dose-dependent manner. Melatonin (0-1 mM) was especially efficient in reducing the loss of spectrin proteins when treated with ONOO- (90% at 500 microM melatonin). Our findings show that the cytoskeleton, and in particular spectrin, is a sensitive target for ONOO-. Specific antioxidants can protect against such alterations, which could seriously impair cell dynamics and generate morphological changes.

Immunomodulatory properties of the metal chelators desferrioxamine and diethylenetriamine penta-acetic acid in vitro.

In this study, we investigated the effects of the intracellular metal chelator desferrioxamine (DFO) and the extracellular metal chelator diethylenetriamine penta-acetic acid (DTPA), which were previously shown to have strong anticytomegalovirus potencies, on their ability to elicit immunomodulatory effects in vitro[fcn,3]. The results showed that nontoxic and in vivo attainable concentrations of both DFO and DTPA inhibited mitogen- and allogen-induced proliferation of peripheral blood lymphocytes. The immunomodulatory effects of DFO/DTPA seem to be due to the impaired expression of interleukin-2 receptor and the reduced secretion of interleukin-2. However, metal chelators were more effective than cyclosporine or tacrolimus (FK506) in our in vitro experiments. Moreover, cytotoxicity mediated by lymphokine-activated killer cells and natural killer cells and the expression of HLA and adhesion molecules on cytokine-stimulated endothelial cells were differentially impaired by DFO/DTPA. These results warrant further study of the immunological effects of metal chelators in vivo.

Modulatory Role of Nitric Oxide on Superoxide-Dependent Luminol Chemiluminescence

Reactive oxygen species are involved in luminol chemiexcitation induced in biological systems, but the contribution of nitrogen-derived oxidants in the process still remains unclear. Herein, we report that luminol chemiluminescence (LCL) induced by a superoxide[formula]- and hydrogen peroxide (H2O2)-generating system (2–25 mU/ml xanthine oxidase plus acetaldehyde and oxygen) was markedly inhibited by nitric oxide ([formula]NO) added either as bolus (0–10 μm) or a continuous flow (0–10 μm/min). However, the inhibition of LCL was followed by an overshoot in light emission after most[formula]NO was consumed or the infusion stopped and was due to reactions of remaining peroxynitrite, the product of the reaction between[formula]and[formula]NO, with luminol. Nitric oxide also inhibited peroxynitrite- and glucose oxidase-induced LCL, but no overshoot was observed. On the other hand, a continuous flux of pure peroxynitrite, at 2 to 10 μm/min, induced LCL with quantum yields close to those obtained by identical micromolar fluxes of[formula], while peroxynitrite formed from the decomposition of the sydnonimine SIN-1 yielded 76% of the chemiluminescence obtained with authentic peroxynitrite. Peroxynitrite- induced LCL was 80 and 55% inhibitable by SOD and catalase, respectively, showing that there were[formula]and H2O2-dependent routes of chemiexcitation. The hydroxyl radical scavengers dimethyl sulfoxide, mannitol, and ethanol and the metal chelator diethylenetriaminepentaacetic acid did not inhibit peroxynitrite-induced LCL while desferrioxamine was an efficient inhibitor of light emission by reaction with an activated state of peroxynitrous acid which is responsible of performing the initial one-electron oxidation of luminol. Our results are consistent with a dual role of[formula]NO in[formula]-induced LCL: (I) formation of peroxynitrite which in turn promotes the light-emitting route and (II) reaction with luminol radical intermediates directing the system toward a dark pathway. These considerations are of critical importance when analyzing cell- and tissue-derived LCL in[formula]NO-,[formula]-, and peroxynitrite-producing systems.

Desferrioxamine inhibition of the hydroxyl radical-like reactivity of peroxynitrite: Role of the hydroxamic groups

Nitric oxide reacts with superoxide to form peroxynitrite, a strong oxidizing species. Peroxynitrite can either directly oxidize molecules such as thiols or protonate to peroxynitrous acid, which can yield an oxidant with a reactivity similar to that of hydroxyl radical in a transition metal-independent mechanism. This oxidative chemistry of peroxynitrite, however, is inhibited by the metal chelator desferrioxamine. Indeed, desferrioxamine, was a potent inhibitor of dimethylsulfoxide, hydrogen peroxide, 5,5-dimethyl-l-pyrroline-N-oxide, and luminol oxidation, whereas the metal chelator diethylenetriaminepentaacetic acid, and ferrioxamine, the iron complex of desferrioxamine, were not. Two other hydroxamates, acetohydroxamate and salicylhydroxamate, were also effective inhibitors. Stopped-flow experiments showed that there is no direct reaction between peroxynitrite anion or cis-peroxynitrous acid with desferrrioxamine. Electron paramagnetic resonance (EPR) studies showed the formation of the desferrioxamine nitroxide radical in incubations containing desferrioxamine, but not ferrioxamine, indicating that the hydroxamic group acts as a one-electron donor to peroxynitrite-derived oxidants. Taken together, our results led us to propose that desferrioxamine can inhibit the oxidative chemistry of peroxynitrite by reaction of the hydroxamic acid moieties with trans-peroxynitrous acid.

Diaziquone-glutathione conjugates: characterization and mechanisms of formation.

The antitumor agent diaziquone (AZQ) reacts with reduced glutathione (GSH) in aqueous solutions and under aerobic conditions to give rise to the glutathionyl and hydroxyl free radicals, as well as the AZQ semiquinone. Under anaerobic conditions, the only radical observed was the glutathionyl radical. These radicals are quickly abrogated when superoxide dismutase and catalase are coincubated. Separately, superoxide dismutase favors the formation of thiyl radicals while catalase favors the formation of hydroxyl radicals and AZQ semiquinone. The metal chelator diethylenetriaminepentaacetic acid favors the production of hydroxyl radicals and AZQ semiquinone. The reaction of AZQ with GSH at pH 7.2 and 5.5 results in a variety of conjugates. These conjugates include addition of glutathione to both aziridines, displacement of the aziridines by GSH, and a combination of both. The majority of the conjugates are formed by nucleophilic attack of GSH to the AZQ aziridines or by 1,4-Michael addition to the AZQ quinone or a combination of both. There may be a small free radical component in conjugate formation, but the majority of the free radicals observed are from redox reactions that involve the oxidation of glutathione and the reduction and autoxidation of AZQ to produce oxygen radicals and hydrogen peroxide, a process that is enhanced by trace metal ions.

Formation of reactive oxygen species during one-electron reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase.

One-electron reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase resulted primarily in the formation of o-semiquinone and, probably, also o-hydroquinone. Under aerobic conditions these reduced form(s) autoxidize, accompanied by the formation of reactive oxygen species, as revealed by continuous NADPH oxidation and oxygen consumption. The presence of manganese-pyrophosphate complex contributed to autoxidation of the o-semiquinone during the reduction of noradrenochrome catalyzed by NADPH-cytochrome P-450 reductase, since the addition of the metal chelator diethylenetriamine pentaacetic acid (DETAPAC) resulted in a 34% inhibition of NADPH oxidation. Oxygen in the ground state was found to be predominantly involved in the autoxidation of o-semiquinone during the reduction of noradrenochrome catalyzed by NADPH-cytocbrome P-450 reductase, since the addition of superoxide dismutase (SOD) to the incubation mixture only inhibited NADPH oxidation 13% and 6% in the absence and presence of DETAPAC, respectively. The addition of catalase to the incubation mixture resulted in a slight increase in NADPH oxidation, both in the absence and in the presence of diethylenetriamine pentaacetic acid. However, no effect of catalase and SOD together on NADPH oxidation was observed, either in the absence or presence of DETAPAC.

The role of free radicals in the decomposition of the phytoalexin desoxyhemigossypol

The cotton phytoalexin desoxyhemigossypol (dHG) decomposed rapidly in solution to give hemigossypol (HG). The rate of decomposition was retarded by the reducing agents ascorbic acid, reduced glutathione and cysteine, by the metal chelator diethylenetriaminepentaacetic acid, and by the enzyme catalase. However, the chelator, ethylenediaminetetraacetic acid did not reduce the rate of decomposition and the enzyme superoxide dismutase increased the rate of decomposition. Solutions of the phytoalexin desoxyhemigossypol-6-methyl ether were significantly more stable than were those of dHG. Oxygen-18 from water but not from oxygen gas was incorporated into HG during this decomposition. A hydroperoxynaphthalenone which loses hydrogen peroxide is proposed as an intermediate to explain this observation. The formation of hydrogen peroxide may be involved in the toxicity of this phytoalexin to plant pathogens such as Verticillium dahliae.

Rotational correlation times of peptides determined by perturbed angular correlations of ?-rays

The technique of Perturbed Angular Correlations of γ-rays has been used to study the rotational correlation times in aqueous solution of the peptides: oxytocin, glycyltryptophan, cholecystokinin and the glycopeptide ristocetin. These peptides were labelled with excited 111mCd through the covalent coupling of the metal chelator diethylenetriaminepentaacetic acid (DTPA) to the primary amines-of the peptides. The experimental correlation times are in good accordance with calculations based on the molecular weight. This indicates that the 111mCd-DTPA is rigidly bound to the molecules. In the case of ristocetin, the correlation time was measured at 2°C, 25°C and 38°C. These experiments show the expected linear dependence on the viscosity divided by temperature. The feasibility of determining rotational correlation times for peptides without lysines and with correlation times in the ns region is thus demonstrated. Also, the correlation time of 111mCd-DTPA coupled to the lysines of bovine serum albumin was determined. The measured correlation time is about 5 times less than the calculated correlation time. This effect is assigned to local motion. In spite of this, experiments show that 111mCd-DTPA-bovine-serum-albumin is significantly immobilised by aggregation with immunoglobulins. The nuclear quadrupole interactions, necessary for determining the correlations times, were determined for 111mCd-DTPA-ristocetin and 111mCd-DTPA-bovine-serum-albumin by adding sucrose to a concentration of 63% and cooling to 2°C. This showed a small but significant difference between the two molecules. We interpret this as due to different conformations, possibly different coordination numbers.

Efficient conjugation of DTPA to an IgM monoclonal antibody in ascites fluid

We have developed a simple, rapid, and reproducible method for conjugating the bifunctional metal chelator diethylenetriaminepentaacetic acid (DTPA) to an IgM monoclonal antibody (MoAb) without first isolating the MoAb from the ascites fluid. Treatment of the protein mixture in the ascites fluid with cyclic DTPA anhydride (cDTPAA) followed by HPLC purification on a size exclusion column allowed isolation of the DTPA-IgM conjugate which could then be labeled with 111 In ⩾80% yield. Over the range of total protein concentrations used (11–44 mg/mL), the number of DTPA molecules per molecule of IgM was approximately one-half the molar ratio of cDTPAA to total protein. We have used this method to prepare an 111In labeled anti-Thy 1.2 IgM, a MoAb with specificity for a murine cell-surface antigen found on normal and malignant T cells and neuroectodertmal tissues. Analysis of the DTPA-IgM conjugate prior to and after 111In labeling using indirect immunofluorescence flow cytometry and a target-cell binding assay showed that the antigen specificity of this anti-Thy 1.2 MoAb is not substantially altered by the presence of up to 8 DTPA molecules per IgM molecule.