Superoxide does react with peroxides : direct observation of the haber-weiss reaction in the gas phase

The fact that nature provides specific enzymes to selectively remove superoxide (O2.−) from aerobic organisms, namely, the superoxide dismutase enzymes,1 has led to the suggestion that this radical ion may cause the oxidative damage associated with degradative disease and aging.2 Intriguingly, however, superoxide itself is relatively unreactive toward most cellular components, which suggests that dismutase enzymes may ultimately protect the cell against more pernicious oxidants formed from superoxide. As such, there is increasing interest in the endogenous chemistry of superoxide and the pathways by which it might beget more reactive oxygen species. Protonation of superoxide to form the hydroperoxyl radical (HOO.) and dismutation of the same species to hydrogen peroxide (HOOH), with subsequent metal-catalyzed reduction to the hydroxyl radical (HO.), are well-characterized processes in which both the HOO. and HO. radicals are significantly more reactive than their common progenitor.2 Recent examples, however, have also linked superoxide to the putative production of singlet oxygen3 and ozone,4, 5 although the definitive characterization of these chemistries in the cellular milieu has proved challenging

Plasma-induced death of HepG2 cancer cells : intracellular effects of reactive species

Reports show that cold atmospheric-pressure plasmas can induce death of cancer cells in several minutes. However, very little is presently known about the mechanism of the plasma-induced death of cancer cells. In this paper, an atmospheric-pressure plasma plume is used to treat HepG2 cells. The experimental results show that the plasma can effectively control the intracellular concentrations of ROS, NO and lipid peroxide. It is shown that these concentrations are directly related to the mechanism of the HepG2 death, which involves several stages. First, the plasma generates NO species, which increases the NO concentration in the extracellular medium. Second, the intracellular NO concentration is increased due to the NO diffusion from the medium. Third, an increase in the intracellular NO concentration leads to the increase of the intracellular ROS concentration. Fourth, the increased oxidative stress results in more effective lipid peroxidation and consequently, cell injury. The combined action of NO, ROS and lipid peroxide species eventually results in the HepG2 cell death. The mechanism of death of human hepatocellular carcinoma cells (HepG2) induced by atmospheric-pressure room-temperature plasma, related to the plasma-controlled intracellular concentrations of reactive oxygen species (ROS), nitric oxide (NO) and lipid peroxide is revealed. Only 34.75 s are required to reduce the number of the viable HepG2 cells by 50%.

Straminipilan protists : a study of their taxonomic position, morphology and abiotic stress-mediated gene expression

Thraustochytrids have become of considerable industrial and scientific interest in the past decade due to their health benefits. They have been proven to be the principle source in marine and estuarine fish diets with high percentage of long chain (LC) or polyunsaturated fatty acids (PUFA). Therefore, the oil extracted from fish for human document.forms[0].elements[13].select();consumption is rich in PUFA with high omega-3 fatty acid content. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) of all of the omega-3 fatty acids, are considered beneficial essential oils for humans with a wide range of health benefits. These include brain and neural development in infants, general wellbeing of adults and drug delivery through precursor molecules. They have become one of the most extensively studied organisms for industrial oil preparations as PUFA extraction from fish becomes less profitable. Many forms of these Thraustochytrid oils are being trialled for human consumption all over the world. In Australia, there has been little research performed on these organisms in the past ten years. A few Australian studies have been conducted in the form of comparative studies related to PUFA production within the related genera, but not focussed on their identification or cellular and genomic characterisation. Therefore, the main aim of this study was to investigate the morphological and genetic characteristics of Australian Thraustochytrids in order to aid in their identification and characterisation, as well as to better understand the effect of environmental conditions in the regulation of PUFA production. It was also noted that there was a knowledge gap in the preservation and total genomic DNA extraction of these organisms for the purposes of scientific research. The cryopreservation of these organisms for studies around the world follows existing generic methods. However, it is well understood that many of these generic methods attract not only high costs for chemicals, but also uses considerable storage space and other resources, all of which can be improved with new or modified approaches. In this context, a simple and inexpensive bead preservation method is described, without compromising the storage shelf life. We also describe, for the first time, the effects of culture age on the successful cryopreservation of Thraustochytrids. It was evident in the literature that DNA and RNA extractions for molecular and genetic studies of Thraustochytrids follow the classical phenol-chloroform extraction methods. It was also observed that modern protocols failed to avoid the use of phenol-chloroform rather than improving preparation and cell disruption. In order to provide a high quantity and quality DNA extraction, a modified protocol has been introduced that employs the use of modern commercial extraction kits and standard laboratory equipment. Thraustochytrids have been shown to be highly conserved in their 18S rDNA gene sequences, which is used as the current standard for identification. It was demonstrated that the 18S rDNA gene sequence limits the recognition of closely related genera or within the genera from each member. Therefore, it was proposed that another profile, such as a randomly amplified polymorphic DNA (RAPD) based profiling system, be tested for use in the characterisation of Thraustochytrids. The RAPD profiles were shown to provide a unique DNA fingerprint for each isolate and small variations in their genome were able to be detected. This method involved the use of a minimum number of standard arbitrary primers and with an increase in the number of different primers used, a very high discrimination between organisms could be achieved. However, the method was not suitable for taxonomic purposes because the results did not correlate with other taxonomic features such as morphology. Another knowledge gap was found with respect to Australian Thraustochytrid growth characteristics, in that these had not been recorded and published. In order to rectify this, a record of colony and microscopic features of 12 selected isolates was performed. The results of preliminary studies indicated that further microbiological and biochemical studies are needed for full characterisation of these organisms. This information is of great importance to bio-prospecting of new Thraustochytrids from Australian ecosystems and would allow for their accurate identification, and so permit the prediction of their PUFA capability by comparison with related genera/species. It was well recognized that environmental stress plays a role in the PUFA production and is mainly due to the reactive oxygen species as abiotic stress (Chiou et al., 2001; Okuyama et al., 2008; Shabala et al., 2009; Shabala et al., 2001). In this aspect, this study makes the first attempt towards better understanding of this phenomenon by way of the use of real-time PCR for the detection of environmental effects on the regulation of PUFA production. Three main environmental conditions including temperature, pH and oxygen availability were monitored as stress inducers. In summary, this study provides novel approaches for the preservation and handling of Thraustochytrids, their molecular biological features, taxonomy, characterisation and responses to environmental factors with respect to their oil production enzymes. The information produced from this study will prove to be vital for both industrial and scientific investigations in the future.

Dynamic, invivo, real-time detection of retinal oxidative status in a model of elevated intraocular pressure using a novel, reversibly responsive, profluorescent nitroxide probe

Changes to the redox status of biological systems have been implicated in the pathogenesis of a wide variety of disorders including cancer, Ischemia-reperfusion (I/R) injury and neurodegeneration. In times of metabolic stress e.g. ischaemia/reperfusion, reactive oxygen species (ROS) production overwhelms the intrinsic antioxidant capacity of the cell, damaging vital cellular components. The ability to quantify ROS changes in vivo, is therefore essential to understanding their biological role. Here we evaluate the suitability of a novel reversible profluorescent probe containing a redox-sensitive nitroxide moiety (methyl ester tetraethylrhodamine nitroxide, ME-TRN), as an in vivo, real-time reporter of retinal oxidative status. The reversible nature of the probe's response offers the unique advantage of being able to monitor redox changes in both oxidizing and reducing directions in real time. After intravitreal administration of the ME-TRN probe, we induced ROS production in rat retina using an established model of complete, acute retinal ischaemia followed by reperfusion. After restoration of blood flow, retinas were imaged using a Micron III rodent fundus fluorescence imaging system, to quantify the redox-response of the probe. Fluorescent intensity declined during the first 60 min of reperfusion. The ROS-induced change in probe fluorescence was ameliorated with the retinal antioxidant, lutein. Fluorescence intensity in non-Ischemia eyes did not change significantly. This new probe and imaging technology provide a reversible and real-time response to oxidative changes and may allow the in vivo testing of antioxidant therapies of potential benefit to a range of diseases linked to oxidative stress

Atmospheric pressure gas plasma-induced colorectal cancer cell death is mediated by Nox2-ASK1 apoptosis pathways and oxidative stress is mitigated by Srx-Nrf2 anti-oxidant system

Atmospheric pressure gas plasma (AGP) generates reactive oxygen species (ROS) that induce apoptosis in cultured cancer cells. The majority of cancer cells develop a ROS-scavenging anti-oxidant system regulated by Nrf2, which confers resistance to ROS-mediated cancer cell death. Generation of ROS is involved in the AGP-induced cancer cell death of several colorectal cancer cells (Caco2, HCT116 and SW480) by activation of ASK1-mediated apoptosis signaling pathway without affecting control cells (human colonic sub-epithelial myofibroblasts; CO18, human fetal lung fibroblast; MRC5 and fetal human colon; FHC). However, the identity of an oxidase participating in AGP-induced cancer cell death is unknown. Here, we report that AGP up-regulates the expression of Nox2 (NADPH oxidase) to produce ROS. RNA interference designed to target Nox2 effectively inhibits the AGP-induced ROS production and cancer cell death. In some cases both colorectal cancer HT29 and control cells showed resistance to AGP treatment. Compared to AGP-sensitive Caco2 cells, HT29 cells show a higher basal level of the anti-oxidant system transcriptional regulator Nrf2 and its target protein sulfiredoxin (Srx) which are involved in cellular redox homeostasis. Silencing of both Nrf2 and Srx sensitized HT29 cells, leads to ROS overproduction and decreased cell viability. This indicates that in HT29 cells, Nrf2/Srx axis is a protective factor against AGP-induced oxidative stress. The inhibition of Nrf2/Srx signaling should be considered as a central target in drug-resistant colorectal cancer treatments.

Particle emissions, volatility, and toxicity from an ethanol fumigated compression ignition engine

Particle emissions, volatility, and the concentration of reactive oxygen species (ROS) were investigated for a pre-Euro I compression ignition engine to study the potential health impacts of employing ethanol fumigation technology. Engine testing was performed in two separate experimental campaigns with most testing performed at intermediate speed with four different load settings and various ethanol substitutions. A scanning mobility particle sizer (SMPS) was used to determine particle size distributions, a volatilization tandem differential mobility analyzer (V-TDMA) was used to explore particle volatility, and a new profluorescent nitroxide probe, BPEAnit, was used to investigate the potential toxicity of particles. The greatest particulate mass reduction was achieved with ethanol fumigation at full load, which contributed to the formation of a nucleation mode. Ethanol fumigation increased the volatility of particles by coating the particles with organic material or by making extra organic material available as an external mixture. In addition, the particle-related ROS concentrations increased with ethanol fumigation and were associated with the formation of a nucleation mode. The smaller particles, the increased volatility, and the increase in potential particle toxicity with ethanol fumigation may provide a substantial barrier for the uptake of fumigation technology using ethanol as a supplementary fuel.

The synthesis of novel nitroxides and their application as small-molecule antioxidants and profluorescent probes for oxidative stress

The detection and potential treatment of oxidative stress in biological systems has been explored using isoindoline-based nitroxide radicals. A novel tetraethyl-fluorescein nitroxide was synthesised for its use as a profluorescent probe for redox processes in biological systems. This tetraethyl system, as well as a tetramethyl-fluorescein nitroxide, were shown to be sensitive and selective probes for superoxide in vitro. The redox environment of cellular systems was also explored using the tetramethylfluorescein species based on its reduction to the hydroxylamine. Flow cytometry was employed to assess the extent of nitroxide reduction, reflecting the overall cellular redox environment. Treatment of normal fibroblasts with rotenone and 2-deoxyglucose resulted in an oxidising cellular environment as shown by the lack of reduction of the fluorescein-nitroxide system. Assessment of the tetraethyl-fluorescein nitroxide system in the same way demonstrated its enhanced resistance to reduction and offers the potential to detect and image biologically relevant reactive oxygen species directly. Importantly, these profluorescent nitroxide compounds were shown to be more effective than the more widely used and commercially available probes for reactive oxygen species such as 2’,7’-dichlorodihydrofluorescein diacetate. Fluorescence imaging of the tetramethyl-fluorescein nitroxide and a number of other rhodamine-nitroxide derivatives was undertaken, revealing the differential cellular localisation of these systems and thus their potential for the detection of redox changes in specific cellular compartments. As well as developing novel methods for the detection of oxidative stress, a number of novel isoindoline nitroxides were synthesised for their potential application as small-molecule antioxidants. These compounds incorporated known pharmacophores into the isoindoline-nitroxide structure in an attempt to increase their efficacy in biological systems. A primary and a secondary amine nitroxide were synthesised which incorporated the phenethylamine backbone of the sympathomimetic amine class of drugs. Initial assessment of the novel primary amine derivative indicated a protective effect comparable to that of 5-carboxy-1,1,3,3- tetramethylisoindolin-2-yloxyl. Methoxy-substituted nitroxides were also synthesised as potential antioxidants for their structural similarity to some amphetamine type stimulants. A copper-catalysed methodology provided access to both the mono- and di-substituted methoxy-nitroxides. Deprotection of the ethers in these compounds using boron tribromide successfully produced a phenolnitroxide, however the catechol moiety in the disubstituted derivative appeared to undergo reaction with the nitroxide to produce quinone-like degradation products. A novel fluoran-nitroxide was also synthesised from the methoxy-substituted nitroxide, providing a pH-sensitive spin probe. An amino-acid precursor containing a nitroxide moiety was also synthesised for its application as a dual-action antioxidant. N-Acetyl protection of the nitroxide radical was necessary prior to the Erlenmeyer reaction with N-acetyl glycine. Hydrolysis and reduction of the azlactone intermediate produced a novel amino acid precursor with significant potential as an effective antioxidant.

The spermostatic and microbicidal actions of quinones and malemides : towards a dual-purpose contraceptive agent

There is an urgent need to develop safe, effective, dual-purpose contraceptive agents that combine the prevention of pregnancy with protection against sexually transmitted diseases. Here we report the identification of a group of compounds that on contact with human spermatozoa induce a state of “spermostasis,” characterized by the extremely rapid inhibition of sperm movement without compromising cell viability. These spermostatic agents were more active and significantly less toxic than the reagent in current clinical use, nonoxynol 9, giving therapeutic indices (ratio of spermostatic to cytotoxic activity) that were orders of magnitude greater than this traditional spermicide. Although certain compounds could trigger reactive oxygen species generation by spermatozoa, this activity was not correlated with spermostasis. Rather, the latter was associated with alkylation of two major sperm tail proteins that were identified as A Kinase-Anchoring Proteins (AKAP3 and AKAP4) by mass spectrometry. As a consequence of disrupted AKAP function, the abilities of cAMP to drive protein kinase A-dependent activities in the sperm tail, such as the activation of SRC and the consequent stimulation of tyrosine phosphorylation, were suppressed. Furthermore, analysis of microbicidal activity using Chlamydia muridarum revealed powerful inhibitory effects at the same low micromolar doses that suppressed sperm movement. In this case, the microbicidal action was associated with alkylation of Major Outer Membrane Protein (MOMP), a major chlamydial membrane protein. Taken together, these results have identified for the first time a novel set of cellular targets and chemical principles capable of providing simultaneous defense against both fertility and the spread of sexually transmitted disease.

Interactive processes link the multiple symptoms of fatigue in sport competition

Muscle physiologists often describe fatigue simply as a decline of muscle force and infer this causes an athlete to slow down. In contrast, exercise scientists describe fatigue during sport competition more holistically as an exercise-induced impairment of performance. The aim of this review is to reconcile the different views by evaluating the many performance symptoms/measures and mechanisms of fatigue. We describe how fatigue is assessed with muscle, exercise or competition performance measures. Muscle performance (single muscle test measures) declines due to peripheral fatigue (reduced muscle cell force) and/or central fatigue (reduced motor drive from the CNS). Peak muscle force seldom falls by >30% during sport but is often exacerbated during electrical stimulation and laboratory exercise tasks. Exercise performance (whole-body exercise test measures) reveals impaired physical/technical abilities and subjective fatigue sensations. Exercise intensity is initially sustained by recruitment of new motor units and help from synergistic muscles before it declines. Technique/motor skill execution deviates as exercise proceeds to maintain outcomes before they deteriorate, e.g. reduced accuracy or velocity. The sensation of fatigue incorporates an elevated rating of perceived exertion (RPE) during submaximal tasks, due to a combination of peripheral and higher CNS inputs. Competition performance (sport symptoms) is affected more by decision-making and psychological aspects, since there are opponents and a greater importance on the result. Laboratory based decision making is generally faster or unimpaired. Motivation, self-efficacy and anxiety can change during exercise to modify RPE and, hence, alter physical performance. Symptoms of fatigue during racing, team-game or racquet sports are largely anecdotal, but sometimes assessed with time-motion analysis. Fatigue during brief all-out racing is described biomechanically as a decline of peak velocity, along with altered kinematic components. Longer sport events involve pacing strategies, central and peripheral fatigue contributions and elevated RPE. During match play, the work rate can decline late in a match (or tournament) and/or transiently after intense exercise bursts. Repeated sprint ability, agility and leg strength become slightly impaired. Technique outcomes, such as velocity and accuracy for throwing, passing, hitting and kicking, can deteriorate. Physical and subjective changes are both less severe in real rather than simulated sport activities. Little objective evidence exists to support exercise-induced mental lapses during sport. A model depicting mind-body interactions during sport competition shows that the RPE centre-motor cortex-working muscle sequence drives overall performance levels and, hence, fatigue symptoms. The sporting outputs from this sequence can be modulated by interactions with muscle afferent and circulatory feedback, psychological and decision-making inputs. Importantly, compensatory processes exist at many levels to protect against performance decrements. Small changes of putative fatigue factors can also be protective. We show that individual fatigue factors including diminished carbohydrate availability, elevated serotonin, hypoxia, acidosis, hyperkalaemia, hyperthermia, dehydration and reactive oxygen species, each contribute to several fatigue symptoms. Thus, multiple symptoms of fatigue can occur simultaneously and the underlying mechanisms overlap and interact. Based on this understanding, we reinforce the proposal that fatigue is best described globally as an exercise-induced decline of performance as this is inclusive of all viewpoints.

A physico-chemical characterisation of particulate emissions from a compression ignition engine : the influence of biodiesel feedstock

This study undertook a physico-chemical characterisation of particle emissions from a single compression ignition engine operated at one test mode with 3 biodiesel fuels made from 3 different feedstocks (i.e. soy, tallow and canola) at 4 different blend percentages (20%, 40%, 60% and 80%) to gain insights into their particle-related health effects. Particle physical properties were inferred by measuring particle number size distributions both with and without heating within a thermodenuder (TD) and also by measuring particulate matter (PM) emission factors with an aerodynamic diameter less than 10 μm (PM10). The chemical properties of particulates were investigated by measuring particle and vapour phase Polycyclic Aromatic Hydrocarbons (PAHs) and also Reactive Oxygen Species (ROS) concentrations. The particle number size distributions showed strong dependency on feedstock and blend percentage with some fuel types showing increased particle number emissions, whilst others showed particle number reductions. In addition, the median particle diameter decreased as the blend percentage was increased. Particle and vapour phase PAHs were generally reduced with biodiesel, with the results being relatively independent of the blend percentage. The ROS concentrations increased monotonically with biodiesel blend percentage, but did not exhibit strong feedstock variability. Furthermore, the ROS concentrations correlated quite well with the organic volume percentage of particles – a quantity which increased with increasing blend percentage. At higher blend percentages, the particle surface area was significantly reduced, but the particles were internally mixed with a greater organic volume percentage (containing ROS) which has implications for using surface area as a regulatory metric for diesel particulate matter (DPM) emissions.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6 h, 12 h and 24 h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24 h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48 h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

Physicochemical characterization of particulate emissions from a compression ignition engine employing two injection technologies and three fuels

Alternative fuels and injection technologies are a necessary component of particulate emission reduction strategies for compression ignition engines. Consequently, this study undertakes a physicochemical characterization of diesel particulate matter (DPM) for engines equipped with alternative injection technologies (direct injection and common rail) and alternative fuels (ultra low sulfur diesel, a 20% biodiesel blend, and a synthetic diesel). Particle physical properties were addressed by measuring particle number size distributions, and particle chemical properties were addressed by measuring polycyclic aromatic hydrocarbons (PAHs) and reactive oxygen species (ROS). Particle volatility was determined by passing the polydisperse size distribution through a thermodenuder set to 300 °C. The results from this study, conducted over a four point test cycle, showed that both fuel type and injection technology have an impact on particle emissions, but injection technology was the more important factor. Significant particle number emission (54%–84%) reductions were achieved at half load operation (1% increase–43% decrease at full load) with the common rail injection system; however, the particles had a significantly higher PAH fraction (by a factor of 2 to 4) and ROS concentrations (by a factor of 6 to 16) both expressed on a test-cycle averaged basis. The results of this study have significant implications for the health effects of DPM emissions from both direct injection and common rail engines utilizing various alternative fuels.

Concentration and oxidative potential of on-road particle emissions and their relationship with traffic composition : Relevance to exposure assessment

Particles emitted by vehicles are known to cause detrimental health effects, with their size and oxidative potential among the main factors responsible. Therefore, understanding the relationship between traffic composition and both the physical characteristics and oxidative potential of particles is critical. To contribute to the limited knowledge base in this area, we investigated this relationship in a 4.5 km road tunnel in Brisbane, Australia. On-road concentrations of ultrafine particles (<100 nm, UFPs), fine particles (PM2.5), CO, CO2 and particle associated reactive oxygen species (ROS) were measured using vehicle-based mobile sampling. UFPs were measured using a condensation particle counter and PM2.5 with a DustTrak aerosol photometer. A new profluorescent nitroxide probe, BPEAnit, was used to determine ROS levels. Comparative measurements were also performed on an above-ground road to assess the role of emission dilution on the parameters measured. The profile of UFP and PM2.5 concentration with distance through the tunnel was determined, and demonstrated relationships with both road gradient and tunnel ventilation. ROS levels in the tunnel were found to be high compared to an open road with similar traffic characteristics, which was attributed to the substantial difference in estimated emission dilution ratios on the two roadways. Principal component analysis (PCA) revealed that the levels of pollutants and ROS were generally better correlated with total traffic count, rather than the traffic composition (i.e. diesel and gasoline-powered vehicles). A possible reason for the lack of correlation with HDV, which has previously been shown to be strongly associated with UFPs especially, was the low absolute numbers encountered during the sampling. This may have made their contribution to in-tunnel pollution largely indistinguishable from the total vehicle volume. For ROS, the stronger association observed with HDV and gasoline vehicles when combined (total traffic count) compared to when considered individually may signal a role for the interaction of their emissions as a determinant of on-road ROS in this pilot study. If further validated, this should not be overlooked in studies of on- or near-road particle exposure and its potential health effects.

A two-metal ion mechanism operates in the hammerhead ribozyme-mediated cleavage of an RNA substrate

Evidence for a two-metal ion mechanism for cleavage of the HH16 hammerhead ribozyme is provided by monitoring the rate of cleavage of the RNA substrate as a function of La3+ concentration in the presence of a constant concentration of Mg2+. We show that a bell-shaped curve of cleavage activation is obtained as La3+ is added in micromolar concentrations in the presence of 8 mM Mg2+, with a maximal rate of cleavage being attained in the presence of 3 microM La3+. These results show that two-metal ion binding sites on the ribozyme regulate the rate of the cleavage reaction and, on the basis of earlier estimates of the Kd values for Mg2+ of 3.5 mM and > 50 mM, that these sites bind La3+ with estimated Kd values of 0.9 and > 37.5 microM, respectively. Furthermore, given the very different effects of these metal ions at the two binding sites, with displacement of Mg2+ by La3+ at the stronger (relative to Mg2+) binding site activating catalysis and displacement of Mg2+ by La3+ at the weaker (relative to Mg2+) (relative to Mg2+) binding site inhibiting catalysis, we show that the metal ions at these two sites play very different roles. We argue that the metal ion at binding site 1 coordinates the attacking 2'-oxygen species in the reaction and lowers the pKa of the attached proton, thereby increasing the concentration of the attacking alkoxide nucleophile in an equilibrium process. In contrast, the role of the metal ion at binding site 2 is to catalyze the reaction by absorbing the negative charge that accumulates at the leaving 5'-oxygen in the transition state. We suggest structural reasons why the Mg(2+)-La3+ ion combination is particularly suited to demonstrating these different roles of the two-metal ions in the ribozyme cleavage reaction.