There is growing pressure for clinicians to prescribe chelation therapy at only slightly elevated blood lead levels. However, very few studies have evaluated whether chelation improves cognitive outcomes in Pb-exposed children, or whether these agents have adverse effects that may affect brain development in the absence of Pb exposure.The present study was designed to answer these questions, using a rodent model of early childhood Pb exposure and treatment with succimer, a widely used chelating agent for the treatment of Pb poisoning.Pb exposure produced lasting impairments in learning, attention, inhibitory control, and arousal regulation, paralleling the areas of dysfunction seen in Pb-exposed children. Succimer treatment of the Pb-exposed rats significantly improved learning, attention, and arousal regulation, although the efficacy of the treatment varied as a function of the Pb exposure level and the specific functional deficit. In contrast, succimer treatment of rats not previously exposed to Pb produced lasting and pervasive cognitive and affective dysfunction comparable in magnitude to that produced by the higher Pb exposure regimen.These are the first data, to our knowledge, to show that treatment with any chelating agent can alleviate cognitive deficits due to Pb exposure. These findings suggest that it may be possible to identify a succimer treatment protocol that improves cognitive outcomes in Pb-exposed children. However, they also suggest that succimer treatment should be strongly discouraged for children who do not have elevated tissue levels of Pb or other heavy metals. This study did not detect a benefit of chelation, relative to placebo, in children with blood Pb levels between 20 and 44 μg/dL . Howevera) determine whether treatment with a succimer regimen shown to produce significant reductions in blood and brain Pb levels (b) determine whether succimer produces lasting cognitive and/or affective impairment when administered in the absence of Pb exposure. Findings from the latter group are needed to gauge the safety of prolonged regimens when treating Pb-exposed children, as well as the safety of the drug for treating autistic children, as currently advocated on numerous websites by physicians, autism parent groups, and organizations such as the American College for Advancement in Medicine.Using a rodent model of early childhood Pb exposure, the present study was designed to b levels  also lesIn this article, we describe a subset of the administered tests—namely, a series of visual attention tests designed to tap several functions reported to be affected in Pb-exposed children, including sustained and selective attention, inhibitory control, learning/associative ability, and regulation of arousal or emotion. Two of these tasks, the sustained attention and selective attention tasks, are similar to ones commonly used to assess attention in human subjects, such as the Continuous Performance Test and Leonard’s 5-choice Serial Reaction Task , described below. The sample size for the behavioral testing was 120 animals, 20 per group. These 120 animals were the female offspring of 60 litters, one succimer-treated and one vehicle-treated animal per litter. All procedures were approved by the Cornell Institutional Animal Care and Use Committee, and the housing facilities were accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.http://www.ehponline.org/docs/2006/9263/suppl.pdf).Nulliparous Long-Evans rats  were mated in the laboratory. They were treated humanely and with regard for alleviation of suffering. Twenty-four hours after birth, each litter was culled to 10 pups and assigned to one of three Pb-exposure conditions . The dams in the no-Pb group received drinking water (deionized distilled) adulterated with 300 ppm sodium acetate from postnatal day (PND)1 to PND30. Both Pb-exposed groups received water adulterated with 300 ppm Pb acetate from PND1 to PND17. On PND17, around the age when pups begin consuming water directly from the drinking water bottle, the water Pb concentration was lowered to 20 ppm for the Mod-Pb group for the remainder of the Pb exposure period (until PND30) in order to maintain the moderate level of Pb intake experienced prior to PND17 when milk provided the sole source of Pb. The High-Pb group was maintained on the 300-ppm Pb acetate drinking water until PND30. At PND30, the pups were weaned and Pb exposure was terminated. The period of Pb exposure, PND1–30, corresponds roughly, in terms of neural development, to the period spanning the sixth month of pregnancy until late childhood/early adolescence in humans . AdditioOn PND30, the pups in each Pb treatment group were divided into succimer  and vehicle (apple juice) subgroups. This assignment was made at the level of each litter to provide littermate controls for the vehicle–succimer comparisons. Succimer or apple juice (vehicle) was administered twice daily via oral gavage from PND31 to PND52. The daily succimer dose was 50 mg/kg body weight/day during the first week of chelation, followed by 25 mg/kg/day for the second and third weeks, similar to the regimen used clinically .http://www.ehponline.org/docs/2006/9263/suppl.pdf).On PND52 (end of chelation therapy) one animal from each litter was euthanized for analysis of blood and whole brain Pb levels by inductively coupled plasma-high resolution mass spectrometry, as previously described  was mounted above each port; the illumination of one of these LEDs served as the discriminative cue in all of the tasks described here. For the selective attention task, compressed, scented air (which served as the olfactory distractors) was projected into the response ports via polyethylene tubes (see http://www.ehponline.org/docs/2006/9263/suppl.pdf) with ad libitum access to water. Testing on the four tasks described in this report was initiated, on average, at 9, 10, 13, and 15 weeks of age, respectively.Behavioral testing began on PND62. All animals were weighed and tested 6 days/week. For all tasks, a daily testing session consisted of 200 trials or 2 hr, whichever came first. All behavioral testing was conducted by individuals blind to the treatment condition of the subjects. Throughout behavioral testing, animals were maintained on a restricted food intake regimen  a delay of varying duration  was imposed between trial onset  and presentation of the cue (the illuminated LED); and b) the duration of cue illumination was shortened to 700 msec. Animals were tested on this task for 20 sessions.This task was a variation of the visual discrimination task with two modifications: The sustained attention task was identical to the previous task, except that the duration of the cue illumination varied between 200, 400, and 700 msec. Animals were tested for 10 sessions on this task.The animals were then tested for 10 sessions on a selective attention task, similar to the preceding task but with modifications of the stimulus parameters and the addition of unpredictable olfactory distractors on one-third or the trials in each daily test session. This task was preceded by three sessions on a baseline task, which had stimulus parameters identical to those in the selective attention task, but without olfactory distractors. In both tasks, the stimulus delay varied between 2 and 3 sec, and the stimulus duration varied between 300 and 700 msec.p < 0.05; p-values between 0.05 and 0.10 were considered to be trends and are discussed if they aid in clarifying the nature of the Pb and succimer effects. All statistical analyses were conducted using SAS 9.0  for Windows 2000. Additional information on the various dependent measures and statistical analyses is provided in the http://www.ehponline.org/docs/2006/9263/suppl.pdf).We analyzed the various performance measures using a generalized linear mixed model (GLMM), which correctly handled the repeated measures on each animal . DiffereBecause of space constraints, the results of behavioral testing presented here include only those instances where effects of Pb and/or succimer were seen. = 8.54; p = 0.0004] and a significant interaction of treatment and session number . The controls (no lead exposure and no succimer treatment) performed significantly better than the High-Pb (p = 0.017) and High-Pb–succimer (p < 0.0001) groups. Chelation offered no benefit; the performance of the High-Pb and High-Pb–succimer groups did not differ significantly. The High-Pb and control groups did not differ significantly by the sixth session, indicating that their inferior performance earlier in testing reflected impaired learning ability.Analysis of average percent errors for the first six sessions on the visual discrimination task revealed a significant main effect of treatment group ; and significant interactions involving treatment and delay , and treatment, session block, and trial block . Rats in the High-Pb group committed a significantly higher percentage of premature responses than controls for trials with the 3 and 6 sec pre-cue delays (averaged across the 20 sessions on the task)  revealed a borderline main effect of treatment , no ben = 3.24; p = 0.009]. As seen in p = 0.001), suggesting some hesitation to enter the testing alcove during this early portion of the task when all groups were performing very poorly. Succimer treatment was beneficial in that the High-Pb–succimer rats did not exhibit this hesitancy in the first block of sessions: they did not differ from controls (p = 0.38) and differed significantly from the High-Pb group (p = 0.01).Alcove latency (AL) is the time elapsed between the raising of the alcove door at trial onset and entry of the animal into the testing alcove. Because the data were highly skewed, with a high proportion of identical values (the minimum latency), the dependent measures used for each animal were the percentages of ALs that fell into each of four bins of latency values: < 0.1, 0.1–0.5, 0.6–1.0, and > 1 sec. A significant treatment × session block interaction was found in the analysis of very short ALS , and treatment, delay, and session block . The increased response latency seen early in the task for all animals (when error rate was very high) was more pronounced for the High-Pb rats than for the control and High-Pb–succimer groups. This Pb effect was normalized by succimer treatment: the High-Pb–succimer group was significantly faster than the High-Pb group during these early sessions and did not differ from controls .We defined response latency as the time between cue onset and a response. The data were log-transformed before analysis. Analysis of the High-Pb, High-Pb–succimer, and control groups revealed significant interactions of treatment and delay  and treatment × delay . The High-Pb group committed a higher percentage of premature responses than the controls for trials with a 3-sec (p = 0.001) or 6-sec (p = 0.05) pre-cue delay (p = 0.001) and 6-sec (p = 0.02) delays and was not different from the High-Pb group for either.Analysis of premature response rate for the High-Pb, High-Pb–succimer, and control groups revealed significant effects of treatment , treatment × trial block × cue duration , and treatment × trial block × previous trial outcome (Prev) . For all groups, we found an increase in percent omission errors across each session, an effect that was most pronounced for trials that followed an error. As shown in An omission error was tallied when a rat entered the testing alcove but failed to respond within 15 sec of trial onset, indicative of missing the cue. The analysis of percent omission errors for the High-Pb, High-Pb–succimer, and control groups revealed the following significant treatment-related effects: treatment × trial block  reflected that a) the High-Pb rats committed a borderline greater percent omission errors than controls in the first block of trials in each session (p = 0.06), and b) during this first block of trials in each session , the High-Pb group benefited less than controls by the lengthening of the cue from 200 to 700 msec (p = 0.009), such that group differences were greatest on trials with the longest cue duration .Additional details of the omission error analyses are provided in the  = 4.88; p = 0.01) and a significant interaction of treatment and session . The Mod-Pb group learned significantly more slowly than controls . Chelation offered a significant benefit in that the Mod-Pb–succimer group learned significantly faster than the Mod-Pb group  and did not differ from controls for any session. The three groups performed similarly by the sixth session, indicating that the observed group differences in performance reflected variations in learning ability  = 2.89; p = 0.016]. As depicted in p = 0.05), and succimer treatment offered a significant benefit . The Mod-Pb–succimer and control groups did not differ for any session. The performance of the three groups did not differ during the final 10 sessions on the task (blocks 3 and 4), indicating that the observed performance differences reflected differing learning ability.The analysis of percent premature responses revealed a significant interaction of treatment and session block .Analysis of average percent errors for the first six sessions on the task revealed that the succimer-only group (treated with succimer in the absence of lead exposure) learned significantly more slowly than the controls . The groups did not differ significantly for this type of error during session block 1 (sessions 1–5), an early point in the learning process when most responses were made before cue onset (premature responses).The percentage of inaccurate responses  was significantly higher for the succimer-only group than for controls during session blocks 2, 3, and 4  and of treatment, cue duration, and trial block . As shown in p = 0.004). This pattern was seen only during the beginning and end of each daily session .The analysis of omission errors for the control and succimer-only groups revealed significant interactions of treatment and cue duration .The succimer-only group benefited less than controls from the lengthening of the cue, resulting in increasing group differences in percent inaccurate responses as the cue duration increased , an interaction of treatment and cue duration , and a trend toward a three-way interaction of treatment, distraction condition, and the outcome of the previous trial . The succimer-only group benefited less by the lengthening of the cue than did controls (p = 0.017), group differences were greatest for trials that both included a distractor and that followed an error , whereas for controls, the rates were similar for these two conditions , providing evidence that motivational differences did not account for the observed differences in performance .Motivation was assessed by analyzing the average rate of response trials [number of response trials per session divided by session length (minutes)]. No significant group differences were detected , measured on PND52 after cessation of succimer treatment. None of the other groups differed from controls. The slightly smaller size of the High-Pb rats likely reflects a decrease in growth hormone during the Pb exposure period . Low-level Pb exposure in children has also been associated with small but statistically significant reductions in height, in the absence of any other signs of overt toxicity  reductions in brain Pb levels lag significantly behind reductions in blood Pb following chelation or cessation of Pb exposure (b) blood and brain Pb levels were still quite elevated in the High-Pb group at the end of chelation ; and c) in a previous study from our laboratory, a second 3-week succimer regimen offered significant benefit over one regimen in terms of both blood and brain Pb reductions  blooductions . TherefoFor the four tasks presented here, the rate of all types of errors was significantly greater on trials that followed an error than on trials following a correct response. Similarly, the latency to enter the testing alcove at trial onset was significantly longer on trials following an error than on trials that followed a correct response, a pattern also seen for the latency to make a response after cue onset. The disruption produced by committing an error was significantly greater for the High-Pb animals than for controls for several dependent measures: In the sustained attention task, the percentage of omission errors was significantly higher for the High-Pb group than for controls during mid- to late-session trials following an error—but not following a correct response—a pattern that indicates the combined influences of the disruptive effects of committing an error and the changing motivational and attentional state of the animals across each session. Similarly, in attention task 1, the High-Pb rats took significantly longer to enter the testing alcove and to make a response than controls, but only early in the task when error rate was very high and the rats had not yet learned the task rules. Finally, the drop in performance on trials following an error was also more pronounced for the High-Pb rats than for controls in a conditional olfactory discrimination task with periodic reward omission, an additional task administered subsequently to these rats .The interpretation of the heightened sensitivity to errors of the High-Pb rats is informed by previous studies that examined performance changes as a function of an error on the previous trial, all involving human subjects. In some of these studies, the error rate on post-error trials was exceptionally low e.g., , indicatImportantly, this heightened disruption following an error was very responsive to succimer treatment: In all cases, the High-Pb–succimer group was indistinguishable from controls . These rThe sustained attention task revealed two types of attentional dysfunction in the High-Pb group. First, the early-session increase in omission errors (relative to mid-session), seen for all groups across all sessions on this task, was more pronounced for the High-Pb group than for the controls, with group differences being largest for trials with the longest cue duration (700 msec). This pattern of results may indicate that lapses in attention were more common for the High-Pb rats than for controls during this early part of the session. More frequent attentional lapses in the High-Pb group would have the consequence of flattening the slope across cue duration and making group differences largest on trials for which performance of the controls was best . One interpretation of this pattern is that early Pb exposure may impair the ability to rapidly engage in a new task when transitioning between activities, manifested here as an increased tendency to miss the cue .A second type of attentional dysfunction observed in the High-Pb rats was evident later in each testing session. The incidence of omission errors increased for all groups across each daily testing session, the classic pattern seen when sustained attention is taxed . HoweverThe effectiveness of succimer chelation varied for these different types of attentional deficits. As discussed above, the heightened attentional disruption seen in the High-Pb rats following an error was completely normalized by succimer treatment, as the chelated High-Pb rats differed significantly from the High-Pb group and did not differ from controls. In contrast, succimer treatment only partially alleviated the attentional lapses seen early in each test session; the High-Pb–succimer group was intermediate to the other two, not differing from either.In the sustained attention task, the percentage of premature responses was significantly higher for the High-Pb rats than for controls, indicating deficient inhibitory control. Converging evidence for this area of dysfunction was provided by the higher percentage of premature responses committed by the High-Pb rats in the final block of trials of attention task 1, a point at which the basic rules of the task had already been learned. Succimer treatment was ineffective in alleviating this area of dysfunction: In both instances, the percentage of premature responses for the High-Pb–succimer rats was significantly greater than that of the control rats, and not different from the High-Pb group.The efficacy of the succimer treatment varied as a function of both the level of Pb exposure and the specific functional domain tested. Interestingly, the pattern seen for succimer efficacy parallels the apparent sensitivity of specific functional domains to the Pb exposure. Learning was impaired by both Pb exposure regimens, whereas impaired regulation of arousal or emotion was seen only in the High-Pb group, suggesting that disruption in learning occurs at lower exposures than dysregulation of arousal or emotion. The efficacy of succimer across these domains corresponded to the degree to which chelation reduced brain Pb levels in the two Pb exposure groups. For the Mod-Pb group, succimer treatment almost completely removed Pb from the brain and effectively alleviated the learning dysfunction. In contrast, brain Pb levels were still moderately elevated in the High-Pb group following chelation; these chelated High-Pb rats exhibited the same pattern of dysfunction as the unchelated Mod-Pb group, which also had moderately elevated brain Pb levels on PND52. Both groups also exhibited impaired learning but not affective dysfunction. This constellation of findings suggests that succimer treatment of the High-Pb rats reduced tissue  Pb levels below that which produces lasting impairment in emotion regulation, but not enough to alleviate the learning dysfunction.The mechanistic basis for this pattern of effects is unknown, but likely reflects complex interactions between the timing of the Pb exposure and succimer treatment relative to the ontogenetic stage of specific neural systems, the extent to which succimer treatment reduced Pb levels in specific neural systems , and theThe present study also revealed the unexpected finding that a single 3-week course of succimer treatment during early development produced lasting dysfunction in cognition and arousal regulation in rats not previously exposed to Pb. Note that these four behavioral tests were administered across a 7-month period following cessation of succimer treatment, suggesting lasting brain changes. These results corroborate preliminary results from a similar study in nonhuman primates . The sucThe mechanism(s) responsible for these adverse succimer effects is not known. One possibility is that succimer, a metal-chelating agent, altered essential metal homeostasis and increased metal diuresis, as has been suggested by a number of clinical and nonhuman primate studies .http://www.ehponline.org/docs/2006/9263/suppl.pdf), direct comparison of blood Pb levels across species should be cautioned. The available evidence indicates that the blood Pb levels required to produce overt toxicity  or lasting neurobehavioral effects  are higher in both rats and nonhuman primates than in humans (Both Pb-exposed groups exhibited low blood Pb levels (≈25–35 μg/dL) during the first 3 weeks of the 4-week exposure period. During the fourth and final week of exposure, both groups experienced an increase in tissue Pb levels because of the direct ingestion of Pb-adulterated water. Although the blood Pb levels produced during this final week of exposure were higher than commonly reported for children  succimer-induced reductions in blood Pb overestimate reductions in brain lead (b) the succimer treatment protocol used in the TLC study achieved relatively modest group differences in blood Pb levels (relative to placebo), averaging only 4.5 μg/dL over the 6 months following treatment  study, the one clinical trial in Pb-exposed children that included cognitive outcomes . There aain lead  and b) treatment . In compreatment . Second,In conclusion, this study demonstrates that it is possible for chelation therapy to alleviate certain types of Pb-induced behavioral/cognitive dysfunction in rats. This is an important demonstration because Pb-induced behavioral dysfunction is generally considered to be irreversible e.g., . The preIn the original manuscript published online, the symbols for the control and succimer-only groups were reversed in


In the Funding section, the number of the US National Institutes of Health grant to SST was listed incorrectly. The correct number is: GM34921.


Occupational and environmental Pb-exposure is associated with protein aggregation diseases which typically present in elderly populations (Parkinsons and cataract). Post-translational processing of crystallins, the major structural proteins of the lens, is altered with short-term Pb-exposure in Fisher 344 rats. In addition, lenses from aged rats become opaque upon long-term exposure to Pb in organ culture. To explore the route to lens opacification in the presence of Pb, cultured lenses from young rats which exhibit higher metabolic activity in lens culture and are more susceptible to experimental cataract in vivo and in vitro were exposed to Pb and evaluated for morphological and biochemical alterations.Following culture in Pb (as lead nitrate) for four days , lenses were examined for clarity, integrity of epithelial layer, and molecular stability including crystallin post-translational modification and choline transport. Clarity of lenses cultured with/without Pb for up to 8 days was assessed to determine if Pb exposure would accelerate opacification.Lenses cultured in Pb for four days exhibited epithelial abnormalities including epithelial cell multilayering and nuclei abnormalities with extension of the nucleated epithelial cells past the bow region. Alterations in crystallin post-translational modifications and decreased membrane transport of choline were noted without corresponding lens opacification or altered α-crystallin chaperone activity. Lenses treated with Pb according to the same exposure protocol with subsequent challenge by hydrogen peroxide became opaque while the contralateral control lenses did not. Lenses which were cultured in the presence of Pb for longer periods with no subsequent oxidative insult exhibited lens failure at earlier time points than did the controls.These data indicate that Pb-exposure can accelerate the degradation of the cultured lens through induction of epithelial cell abnormalities, induce structural protein modifications before opacity, and predispose the lens to opacification with subsequent oxidant challenge. The banning of Pb, a potent environmental toxin, as a pigment, pesticide, and fuel additive has led to decreases in the Pb burden of the general population and of children specifically -3. HowevThe mechanisms related to lenticular opacity associated with Pb-exposure have been explored. Pb accumulation in normal healthy lenses during organ culture or in vivo after oral dosing may be directly related to Pb binding to the lens capsule ,14. Pb hWith the association of Pb exposure and decades later cataract formation, it seems likely that Pb exposure acts in conjunction with later systemic stressors to induce opacification. The current study examines whether Pb acts as a predisposing effector for lens opacity in conjunction with a secondary oxidative challenge in cultured lenses from young rats (approximately 4–6 weeks of age). Additionally, this study explores the effect of Pb exposure on epithelial transport/cellular structure and crystallin protein homeostasis as possible mechanisms through which Pb may induce opacity.Pb nitrate, sodium nitrate, urea, dithiothreitol (DTT), depleted α-lactalbumin, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), trichloroacetic acid, trifluoroacetic acid, α-cyano-4-hydroxycinnamic acid and sequencing grade trypsin were purchased from Sigma Chemical Company . Immobilized pH gradient (IPG) strips were purchased from BioRad . Bis-Tris gels, MOPS running buffer, SilverQuest and Colloidal Blue staining kits were purchased from Invitrogen .2) for 2 h; lenses that were damaged during dissection were identified and discarded during this period by measuring protein leakage into the medium. After the equilibration period, surviving “competent” lenses were cultured with 1 µM Pb(NO3)2 (~20 μg/dl) or unmodified media. The media was changed every other day for a period of 3–9 days. An additional group, 2 µM NaNO3 (n=3), was initially included as a counter ion control exposure grouping to assess the impact of nitrate concentration on lens competence. As TC-199 media contains ferric nitrate, the additional nitrate added through addition of the Pb salt doubled the media nitrate content. The lenses that were cultured in NaNO3 did not vary from the lenses cultured in unmodified media in hydrogen peroxide clearance, choline uptake, protein disulfide formation or in morphology. The data from the NaNO3 (n=3) and unmodified media (n=6) for these assays were combined and represented as the Control group. Subsequent analyses, including the 2D gels and α-crystallin chaperoning assay, used lenses cultured in unmodified media as controls and without the inclusion of a NaNO3 group since the added nitrate salts did not affect lens competence.Eyes were obtained from Sprague-Dawley  rats (4–6 weeks of age) immediately after euthanasia following the NIH Animal Research Advisory Committee guidelines. The competent lenses were cultured in modified TC-199 media as previously reported . The osm3)2 , the Pb containing media was replaced after 3 days with modified TC-199 supplemented with 250 µM hydrogen peroxide and changed daily. One hundred microliter aliquots of the medium were removed to assay for hydrogen peroxide clearance. H2O2 concentration was measured after 2 h using a Model 2700 Biochemistry Analyzer . After 2 days of hydrogen peroxide exposure the lenses were collected and photographed to document alterations in lens clarity. The experiment was repeated 3 times for a total of 12 lenses per group.For the hydrogen peroxide challenge of lenses cultured in Pb. After 4 h, the lenses were removed from culture, rinsed with PBS, blotted, and weighed. The lenses were homogenized in 1 ml of cold 10% trichloroacetic acid and the soluble fraction was collected. One hundred microliters of the lens supernatant and 100 ul of the media were counted on a scintillation counter. The lens water (L) to medium (M) concentration ratios (L/M) were calculated as previously published [Tracer levels (0.2 µCi per well) of [ublished . In brieFollowing 4 days of culture in the presence or absence of various concentrations of Pb nitrate, lenses were collected and homogenized in 500 µl cold 10% TCA. The protein pellets were washed three times with 10% TCA and then resuspended in 10 mM Tris pH 7.4. One hundred microliters of resuspended pellet was mixed with 10 µl of 2.5% sodium borohydride in 0.01 N NaOH. The mixture was incubated for 1 h at 37 °C. Ten percent TCA was added to a final concentration of 1% to stop the liberation of thiols. The freed thiol levels (nmol/mg) were measured by the Ellman’s assay as previously described . A totalThree lenses per group were cultured for 3 days in the presence or absence of Pb nitrate as above, fixed in 2.5% glutaraldehyde in cacodylate buffer for 4 h, and transferred to 10% buffered formalin. The fixed samples were embedded in methylmethacrylate, sectioned and stained with hematoxylin and eosin (H&E). The experiment was repeated twice for a total of 9 lenses per group. Three sections per lens were examined for histological alterations.3. Following centrifugation at 10,000× g for 30 min at 4 °C, the sample was loaded on a Superose-12 column and eluted with the same buffer with 1 ml fractions collected. The α-crystallin peaks were identified by SDS–PAGE and pooled for reinjection on the same HPLC system [3).Three to four lenses per group (control and 1 µM Pb nitrate) were homogenized in 0.5 M Tris buffer (pH 7.4) with 0.1 M KCl, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 0.2% NaNC system . The finThe first dimension isoelectric focusing was performed using a BioRad Protean IEF Cell with 7 cm 3–10NL immobilized pH gradient strips. Aliquots of the purified α-crystallin or whole lens samples  were homogenized on ice with 8 M urea containing 4% CHAPS and 100 mM DTT (rehydration buffer). No insoluble material was visible. Four micrograms of lens protein was loaded onto the IPG strips with rehydration buffer containing 2% ampholytes and bromophenol blue for a total volume of 125 μl. IEF strips were rehydrated for 12 h and then a three step program was used including Step 1: initial removal of salts (500 V for 500 V-h); Step2: an intermediate step (1000 V for 2000 V-h); and Step 3: the final protein separation according to pI . Following protein IEF focusing, the IPG strips were equilibrated with 100 mg/10 ml DTT and 400 mg/ml iodoacetamide and the second dimension SDS–PAGE was performed using 12% pre-cast Bis-Tris gels and MOPS as the running buffer. The gels were fixed and stained with either silver or colloidal Coomassie blue G-250. The gels were scanned with a Molecular Dynamics Personal Densitometer and analyzed using Phoretix image analysis software . Six samples from the 1 µM Pb and 6 samples from the control group were analyzed in duplicate.Protein Prospector algorithm.Protein spots of interest were cut from the gel, destained in 50% methanol in 20 mM ammonium bicarbonate buffer, and digested overnight with 10 ng/ul trypsin . Peptideα-Crystallin and apo-α-lactalbumin (1:3 weight ratio) were dissolved in 50 mM phosphate buffer (pH 6.82) containing 0.1 M NaCl and 2 mM EDTA. DTT at a final concentration of 50 mM was added to initiate denaturation of the lactalbumin. Aggregation was monitored as a function of lactalbumin aggregation as previously described .t-tests were calculated  to determine statistical significance of the mean ±standard deviation of the Pb groups versus the control groups for all assays.Student’s To examine whether longer term culture of young lenses in the presence of Pb would lead to lens opacity formation (as does long-term culture of older lenses) without subsequent oxidative challenge, lenses were cultured up to 9 days in the presence or absence of Pb nitrate. The lens clarity was monitored and graded daily according to the chart shown in For control lenses, at culture day 5, roughly 80% of the lenses were at Stage 1, 10% at Stage 2, and 5% at Stages 3 and 4. By culture day 8, 50% of the lenses had progressed to Stage 2, with approximately 7% at Stages 3 and 4. This indicates that 8 days in culture is approaching the outer limits of lens viability under the conditions employed. In contrast, by day 5 in culture with Pb only 30% of the lenses remained at Stage 1. At culture day 8, the Pb exposed lenses have all advanced to Stage 3 and 4 indicating that lenses cultured in the presence of Pb  fail at an accelerated rate as compared to the control lenses.To examine whether lenses cultured in the presence of Pb are predisposed to failure, lenses were cultured for 3 days in the presence or absence of Pb nitrate and then challenged for 2 days with 250 µM hydrogen peroxide. Lenses cultured with Pb exhibit a higher clearance of hydrogen peroxide from the media than the control lenses  at 2 h aThe degree of protein mixed disulfides present, a measure of protein oxidation and oxidative stress, was increased in Pb-exposed lenses 4 days; . In FiguAlterations in the lens epithelium following culture in 1 μM Pb for 3 days as compared to the control cultured lenses  include ins-crystallin form, which is present in rodents but not humans or other primates [α-Crystallin, a molecular chaperone and major lens structural protein, is believed to maintain lens transparency through prevention of protein aggregation. Post-translational modification of α-crystallin may negatively affect maintenance of lens clarity through decreased chaperone function. To examine the degree of post-translational modification of α-crystallin in Pb exposed lenses, α-crystallin was purified from lenses cultured for 4 days in the presence or absence of Pb and separated by two dimensional gel electrophoresis with the individual protein spot intensities quantified by densitometry. A comparison of the 2D gel spot patterns obtained from 10µg of protein from control and Pb exposed lenses is shown in primates .Six forms of αB-crystallin are visible on the gels shown in ins-crystallin isoform, spots αA8- and αA9-crystallin are increased in abundance in the Pb cultured lenses when normalized against the dominant αA1-crystallin spot. The spot αA7-crystallin does not change significantly in the Pb cultured lenses as compared to the control cultured lenses. This indicates alterations in the normal processing of αA-crystallin do occur in optically transparent lenses which have been cultured in the presence of Pb nitrate. It is also interesting that the total amount of αAins-crystallin appears to increase in lenses cultured with Pb nitrate since there is approximately 5%–10% more of two of the three spots identified as αAins-crystallin for a total change in the amount of αAins-crystallin by 10%–20%. This result was not statistically significant but the trend was evident in nine out of twelve individual lenses examined in the Pb exposed group.Quantitation of the relative spot densities of the different forms of α-crystallin is shown in Three forms of αB-crystallin which are present in both the control and Pb cultured lenses, representing more acidic  and cleaved forms , have significant increases in relative abundance in the Pb cultured lenses when normalized against the dominant spot form of αB-crystallin , is mosThe current study also examined whether lenses from young rats (4–6 weeks) cultured in the presence of Pb for longer periods of time would develop opacities. The significantly increased rate of failure of Pb cultured lenses was associated with development of lenticular opacities, severe abnormalities of the epithelial layer, increased weights, and pronounced acidification and degradation of crystallins (data not shown). Lenses cultured in unmodified media exhibited the same types of abnormalities upon failure, though failure was at a later time point. Thus Pb may accelerate the decline in lens membrane function ultimately leading to cataract formation in both young lenses and in the older, less metabolically active lenses (from 4.5 month old rats) from our prior study . We propThis study has demonstrated the ability of Pb to compromise lens integrity following short-term in vitro challenge and to predispose the lenses from 4 to 6 week old rats to opacities induced by subsequent oxidative challenge. Pb-exposure also negatively impacted lens clarity at longer exposure times which may point to a direct cataractogenic effect of Pb. Coupled with our previous in vivo data demonstrating alterations in lens homeostasis following short-term oral Pb-exposure in young rats, these data support the hypothesis that Pb-exposure is causally related to lens opacification. While there is a lack of knowledge of the type of cataract associated with Pb-exposure in humans, the current study raises the question of whether cortical opacities and lens epithelial cell biochemical abnormalities may be linked to in vivo Pb-exposure in humans.


Lipid peroxidation in chondrocytes was measured by conjugated diene formation. Protein oxidation and aldehydic adduct formation were studied by immunoblot assays. Antioxidant effect of glucosamine was also tested on malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) formation on purified lipoprotein oxidation for comparison. Glucosamine sulfate and glucosamine hydrochloride in millimolar (0.1 to 50) concentrations specifically and significantly inhibited collagen degradation induced by calcium ionophore-activated chondrocytes. Glucosamine hydrochloride did not inhibit lipid peroxidation reaction in either activated chondrocytes or in copper-induced oxidation of purified lipoproteins as measured by conjugated diene formation. Glucosamine hydrochloride, in a dose-dependent manner, inhibited malondialdehyde (TBARS) formation by oxidized lipoproteins. Moreover, we show that glucosamine hydrochloride prevents lipoprotein protein oxidation and inhibits malondialdehyde adduct formation in chondrocyte cell matrix, suggesting that it inhibits advanced lipoxidation reactions. Together, the data suggest that the mechanism of decreasing collagen degradation in this in vitro model system by glucosamine may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.Osteoarthritis (OA) affects a large segment of the aging population and is a major cause of pain and disability. At present, there is no specific treatment available to prevent or retard the cartilage destruction that occurs in OA. Recently, glucosamine sulfate has received attention as a putative agent that may retard cartilage degradation in OA. The precise mechanism of action of glucosamine is not known. We investigated the effect of glucosamine in an  Osteoarthritis (OA) is characterized by the progressive degradation and loss of articular cartilage . OA is tin vitro chondrocyte and explant cultures  with specific activity of 90 curies per millimole was obtained from American Radiolabeled Chemicals, Inc. .NZW rabbits (2.2 to 2.9 kg) of either gender were killed by intravenous injection of Beuthanasia-D special . The chondrocytes were isolated as described previously . The via5 cells per well in 1 ml of complete media. Chondrocytes were allowed to attach for 3 to 5 days, and media were changed every 3 days. Confluent cells in multiwell plates were labeled with 1 to 2 μC/well with [3H]-proline during the last 24 to 48 hours of cell culture. The cell monolayer was washed at least four to five times with warm HBSS by flipping the plates to remove unincorporated proline from the matrix. Albumin- or serum-free EBSS was added to wells. Experiments were carried out in triplicate wells. The test reagents were added, and the total volume was adjusted to 0.5 ml with EBSS. The cultures were incubated at 37°C in a humidified 5% CO2 incubator for 4 to 24 hours. [3H]-proline release was measured in cell supernatant and cell lysates. A 100-μl aliquot was removed and processed for scintillation counting. The plastic-bound [3H]-proline-labeled matrix  was solubilized with 0.5 M NaOH and counted. Percentage release of total [3H]-proline-labeled collagen was calculated.Primary rabbit articular chondrocytes were distributed into 24-well plates at a concentration of 1 to 2 × 102+ (copper ion) or 5 μM Cu2+ and 50, 5, or 0.5 mM glucosamine. Data are expressed as malondialdehyde (thiobarbituric acid-reactive substances [TBARS]) equivalents in nanometers.The very-low-density lipoprotein and low-density lipoprotein (LDL) fractions were isolated from serum by ultracentrifugation at a density of 1.063 g/ml and were kindly provided by Vincent A. Rifici and Avedis K. Khachadurian from the Department of Medicine of our medical school . Lipoprog for 10 minutes at 25°C, and the absorbencies of the supernatants were measured in a spectrophotometer at 532 nm. TBARS are expressed as nanomoles of malondialdehyde equivalents of lipoprotein protein compared with tetramethoxypropane standard -proline-labeled collagen as compared with the background amount of collagen released by unstimulated control chondrocytes. In the presence of 25 mM concentrations of glucosamine hydrochloride or glucosamine sulfate, there was statistically significant inhibition of the release of labeled collagen at 4 hours. In comparison, N-acetyl glucosamine and N-acetyl mannosamine did not result in inhibition of collagen degradation. The data indicate that glucosamine hydrochloride and glucosamine sulfate have specificity and significantly inhibit collagen degradation by activated chondrocytes.We tested the effect of glucosamine hydrochloride and glucosamine sulfate on chondrocyte-dependent collagen degradation in the previously described ro model . For comAs shown in Figure We monitored conjugated diene formation as an indicator of lipid peroxidation in activated chondrocytes and purified lipoprotein oxidation with or without glucosamine hydrochloride . As showWe investigated the effect of glucosamine hydrochloride on TBARS formation in Cu-induced oxidation of lipoproteins. As shown in Figure We tested the effect of glucosamine hydrochloride on aldehyde-protein adduct formation in control and stimulated chondrocytes. Protein gel electrophoresis and immunoblot analysis using MDA2, specific for MDA-modified lysine of chondrocyte extracts, is shown in Figure We tested the effect of glucosamine hydrochloride on lipoprotein protein oxidation using the identification of protein carbonyls as one of the modifications as described in oxidized proteins ,34. The in vitro model of chondrocyte activation-dependent collagen degradation, we show that glucosamine specifically and significantly inhibited collagen degradation. Inhibition of collagen degradation by glucosamine was not mediated by inhibiting the chondrocyte lipid peroxidation process but by inhibiting advanced lipoxidation reactions. Specifically, glucosamine inhibited purified lipoprotein protein oxidation and aldehydic oxidation of chondrocyte matrix.Using this in vitro model, we had previously shown [in vitro model to human OA pathogenesis was demonstrated by detection of in vivo molecular imprints of lipid peroxidation in which OA and normal cartilage tissue sections were studied [in vivo role of lipid peroxidation in the OA pathogenesis [Using this ly shown ,35,36 thly shown ,35. Thisogenesis . CollectWe investigated the effect of glucosamine in our assay system. As shown, only glucosamine hydrochloride or glucosamine sulfate specifically and significantly inhibited collagen degradation by activated chondrocytes and the effect was dose-dependent. Similar effects by both agents (glucosamine hydrochloride and glucosamine sulfate) excluded the possibility that the inhibition observed was mediated by the sulfate moiety in the latter compound. Glucosamine hydrochloride had little or variable effect on hydrogen peroxide-induced collagen degradation, suggesting that it did not inhibit oxygen radical/hydrogen peroxide-mediated collagen degradation (data not shown).in vitro model for studies on lipoxidative modification of proteins [Since the mechanism of collagen degradation in this model appears to involve the activation of lipid peroxidation in chondrocytes, it raises the possibility that glucosamine was acting like a chain-breaking antioxidant similar to vitamin E. However, glucosamine had no discernable effect on conjugated diene formation by activated chondrocytes, suggesting that its mechanism of action was not due to chain-breaking antioxidant activity. As expected, vitamin E inhibited conjugated diene formation by chondrocytes. To further confirm these findings, we tested the effect of glucosamine in a purified lipoprotein oxidation model system, a commonly used proteins . Again, The inhibition of collagen degradation by glucosamine was manifested even when the addition of glucosamine was delayed in activated chondrocyte cultures, indicating that its mechanism of action involved downstream events of chondrocyte activation rather than interfering with or blocking the early events of chondrocyte activation by calcium ionophore. We tested the effect of glucosamine on TBARS formation by Cu-induced oxidation of purified lipoproteins. Glucosamine in a dose-dependent manner inhibited malondialdehyde formation by oxidized lipoprotein. The data suggest that glucosamine either inhibited or scavenged aldehydic products of lipid peroxidation. However, glucosamine did not interfere in the detection of control malondialdehyde in TBARS assay, suggesting that most likely glucosamine inhibited advanced lipoxidation reactions rather than scavenging aldehydic products.in vitro model system.The identification of aldehydic adducts provides a molecular clue of chondrocyte matrix damage mediated by lipid-free radicals . On immuInhibitors of advanced lipoxidation reactions such as aminoguanidine and pyridoxamine have been evaluated in animal models of diseases such as diabetes ,39. Thesin vivo tissue levels of glycosaminoglycans in cartilage are hundreds perhaps thousands of folds higher than in serum or joint fluids, glucosamine, which is a structural component of aggrecan, may locally provide an antioxidant environment that may protect cartilage collagen from oxidative damage.The pharmacokinetics of oral administration of glucosamine sulfate show that plasma levels increase more than 30-fold from baseline and peak at approximately 10 μM with the standard 1,500-mg once-daily dosage . We postin vitro model system may be mediated by the inhibition of advanced lipoxidation reaction, preventing the oxidation and loss of collagen matrix from labeled chondrocyte matrix. Further studies are needed to relate these in vitro findings to the retardation of cartilage degradation reported in OA trials investigating glucosamine.Our data suggest that the decrease in collagen degradation by glucosamine observed in this in vitro model of cartilage collagen degradation in which collagen degradation induced by activated chondrocytes is mediated by lipid peroxidation reaction, glucosamine decreases collagen degradation by inhibiting advanced lipoxidation reaction and thus prevents the oxidation and loss of collagen matrix from labeled chondrocyte matrix.In an AGE = advanced glycation reaction; BSA = bovine serum albumin; Cu = copper; DMEM = Dulbecco's modified Eagle's medium; DNP = dinitrophenyl; EBSS = Earl's balanced salt solution; ECL = enhanced chemiluminescence; FBS = fetal bovine serum; HBSS = Hanks' balanced salt solution; HRP = horseradish peroxidase; IL-1 = interleukin-1; LDL = low-density lipoprotein; OA = osteoarthritis; PBS = phosphate-buffered saline; TBARS = thiobarbituric acid-reactive substances; TBS = Tris-buffered saline.The authors declare that they have no competing interests.MLT developed the study experimental protocol. All authors participated in conducting and analyzing the experiments. All authors were involved in the drafting, review, and final approval of the manuscript.


Candida) in the elderly. The subjects were 64 elderly subjects with fixed prostheses and 49 who wore removable partial dentures aged over 65 years. We used one-way ANOVA to test for overall differences of the number of MT among 5 oral environmental factors. The significant differences were observed in the lactobacilli counts for different number of MT. The number of MT increased with an increase in the lactobacilli counts with removable denture. In conclusion, for the patients wearing removable dentures, increasing number of MT was associated with an increase in the lactobacilli counts in saliva. For the patients with crowns and fixed partial dentures, the number of MT was not significantly affected by salivary mutans streptococci, lactobacilli, and Candida counts.The purpose of this study was to investigate the relationship between the number of missing teeth (MT) and the statuses of oral environmental factors (the stimulated salivary flow rate, buffering capacity, and the counts of mutans streptococci, lactobacilli, and  Caries activity test using saliva is believed to provide useful information for selecting the type of prosthesis. The oral environmental factors considered in the caries activity test include both host and microbial factors associated with caries. We investigated the differences between the statuses of the oral environment factors in elderly individuals with fixed and those with removable prostheses. The results of our study revealed that the amount of cariogenic bacteria in persons with fixed prostheses is different from that of elderly persons with removable dentures . HoweverThe population of the elderly has been increasing worldwide. There is an increase in the number of risk factors of oral diseases and MT in the elderly. The type of prosthesis was decided by the number of MT in most cases. However, the distal extension missing must place removable denture even if the number of MT is little. Therefore, the oral environment should examine not only the statue of the prosthesis, but also the number of missing teeth. To prevent an increase in the number of MT, maintenance of oral environmental factors is valuable and important. The purpose of this study was to investigate the relationship between the statuses of oral environmental factors and the number of MT in elderly persons with different types of prosthesis.This study protocol was screened and approved for its ethical acceptability by the Committee on Experimental Research on Humans of the Osaka Dental University. The volunteers selected included 113 elderly aged over 65 years divided into 2 groups: The volunteers were divided into the crowns and fixed partial denture group (Dentate group), in whom MT were restored only by using fixed partial dentures, and into the removable denture group (Denture group), in whom MT were restored using removable partial and complete dentures.The one group comprised 64 dentate elderly with fixed prosthesis alone, with a mean age and the number of remaining teeth of 69.8 ± 4.4 years and 26.7 ± 2.8 (Dentate group). The other group comprised 49 elderly with removable prostheses, with a mean age and the number of remaining teeth of 69.4 ± 6.1 years and 15.3 ± 6.6 (Denture group). Candida (CA).We examined their oral conditions and assessed the oral environmental factors. We selected the following 5 factors for the evaluation of the oral environment: the stimulated salivary flow rate, buffering capacity, and the counts of mutans streptococci (SM), lactobacilli (LB), and The number of MT and the kind of prosthesis  were determined by oral examination.The saliva test was performed using the Dentocult series , and saliva was collected more than 1 hour after breakfast, between 9:30 am and 11:30 am. The stimulated salivary flow rate was measured by spitting out saliva after chewing a 1 g paraffin pellet for 5 minutes. The stimulated salivary flow rate was the volume of whole stimulated saliva collected per minute. The stimulated saliva buffer capacity was measured by Dentocult buff strip. The amount of bacteria in the saliva was counted using simple culture kits, Dentocult SM, LB, and CA. After culture, the SM, LB, and CA counts were determined by a comparison with model charts attached to the kits –4. The fWe examined the relationship between the number of MT and oral environmental factors in individuals with different types of prosthesis (Dentate and Denture group) using one-way ANOVA. The significance level was set to 0.05.Dentists should select restorations so as to avoid secondary caries and periodontitis, which will in turn prolong the survival of the remaining teeth. Increase in the number of missing teeth necessitates extensive and large number of restorations. Therefore, it is important to compare the number of MT with the bacterial counts with different types of prostheses.In general, the volume of stimulated salivary flow is decreased in the elderly. However, the number of MT did not influence the amount of the stimulated salivary flow rate in each group. The results regarding buffer capacity may have been influenced by the saliva volume. The bicarbonate level in saliva is strongly dependent on the amount of saliva secreted. Bicarbonate is one of the main factors determining the buffer capacity of saliva. A significant effect on the number of missing teeth was not detected for SM and CA counts in each group. The Dentate group had a lot of low risk level for SM, LB, and CA counts. Therefore, we think that there was no difference. In the Denture group, SM and CA counts have been distributed at each level. However, the number of MT was significantly affected only by LB. LB is associated with dental caries (open carious lesions), poorly executed restorations, and poor oral hygiene , 6. ThusSM increase caries activity in the acid stress , whose cCA coexist with SM in denture plaques and readily adhere to resin-base dentures . CA counWe think that in routine clinical practice, significant microbial counts should be considered in designing prosthesis. Fixed partial and removal dentures are the 2 main methods for restoration of MT. Removal of dentures is indispensable in cases of a high number of MT, but it is associated with a risk for developing caries. We think that it is preferable to use a fixed partial denture than a removable denture when fixed restoration is possible.In conclusion, for the patients wearing removable dentures, increasing number of missing teeth was associated with an increase in the lactobacilli counts in saliva. For the patients with crowns and fixed partial dentures, the number of missing teeth was not significantly affected by salivary mutans streptococci, lactobacilli, and Candida counts.


Reversible modification of proteins through the attachment of ubiquitin or ubiquitin-like modifiers is an essential post-translational regulatory mechanism in eukaryotes. The conjugation of ubiquitin or ubiquitin-like proteins has been demonstrated to play roles in growth, adaptation and homeostasis in all eukaryotes, with perturbation of ubiquitin-mediated systems associated with the pathogenesis of many human diseases, including cancer and neurodegenerative disorders.Apicomplexa. For each of the 13 species investigated, a set of proteins predicted to be involved in the ubiquitylation pathway has been identified and demonstrates increasing component members of the ubiquitylation pathway correlating with organism and genome complexity. Sequence homology and domain architecture analyses facilitated prediction of apicomplexan-specific protein function, particularly those involved in regulating cell division during these parasite's complex life cycles.Here we describe the use of an HMM search of functional Pfam domains found in the key components of the ubiquitin-mediated pathway necessary to activate and reversibly modify target proteins in eight apicomplexan parasitic protozoa for which complete or late-stage genome projects exist. In parallel, the same search was conducted on five model organisms, single-celled and metazoans, to generate data to validate both the search parameters employed and aid paralog classification in This study provides a comprehensive analysis of proteins predicted to be involved in the apicomplexan ubiquitin-mediated pathway. Given the importance of such pathway in a wide variety of cellular processes, our data is a key step in elucidating the biological networks that, in part, direct the pathogenicity of these parasites resulting in a massive impact on global health. Moreover, apicomplexan-specific adaptations of the ubiquitylation pathway may represent new therapeutic targets for much needed drugs against apicomplexan parasites. Plasmodium, Toxoplasma and Cryptosporium. Toxoplasma gondii and Cryptosporium parvum are the etiological agents of toxoplasmosis and cryptosporidiosis, respectively, which are predominantly opportunistic infectious agents responsible for severe mortality amongst immuno-suppressed patients such as those infected with HIV. The human malarial parasite Plasmodium falciparum, which is responsible for over a million deaths annually P. falciparum has evolved resistance to many front-line antimalarial drugs P. falciparum but other apicomplexan parasites as well.Apicomplexans are obligate protozoa intracellular parasites responsible for several major human diseases prevalent in the developing world. These include organisms belonging to the genera P. falciparum and the rodent malaria parasites P. yoelii, P. berghei and P. chabaudi have been published P. vivax well underway. In addition, the complete annotated genomes of T. gondii, C. parvum and Cryptosporium hominis have been recently released P. falciparum malaria Genome sequencing projects are available for several apicomplexan parasites, with many of them completed. The full genome sequence of the human malarial parasite via covalent conjugation to ubiquitin (or more often polyubiquitin chains) is a well-established signal for proteosomal destruction P. falciparum has been established Here we describe a comparative analysis of one of the essential post-translational regulatory networks commonly found in eukaryotic cells–the ubiquitin/proteasome system (UPS). Modification of proteins via an activation and transfer cascade via conjugation to the E3) to form an isopeptide bond. Since ubiquitin contains several lysine residues, it can itself be ubiquitinylated, leading to the formation of polyubiquitin chains. Differences in affinity for ubiquitin/UBLp by the component parts of the cascade, as well as a hierarchical increase in the numbers of these proteins , drive the transfer of ubiquitin/UBLp through the cascade with the final target specificity mediated through the E3 complex.Specificity in the conjugation of ubiquitin and ubls to their final target is elegantly achieved Ubiquitin, a highly conserved 76 amino acid peptide, was first described in 1974 via a transesterification reaction relies on additional motifs present in E1. The E2 protein contains a single motif that mediates interaction with both E1 and E3, signifying the “shuttle” status of E2 in the transfer of ubiquitin/UBLps between activation and subsequent ligation to their final target. E2s are present as multiple isoforms, each with distinct roles. E2s exist for each UBL modifier, with multiple E2s capable of accepting ubiquitin. However, even within the ubiquitin E2 isoforms, there is functional divergence in the specific E3s they interact with, and thus the cellular processes they are involved in. For example, Rad6p and Cdc34p E2 isoforms deliver ubiquitin to E3s that ultimately target proteins involved in the regulation of DNA repair and cell cycle progression, respectively Analysis of the E1-activating enzymes indicates that they share sequence homology to MoeB/ThiF domains of prokaryotic biosynthetic proteins involved in sulphur donor systems e.g. MDM2 known to target p53) and U-box. Two sub-classes of RINGs have further been defined: RING in between RING-RING (RIR) and Cullin-RING ligases (CRL), which are multi-protein complex E3s , DNA-repair (ubiquitylation of p53 by the RING finger MDM2) and transcriptional regulation (activation of NF-kB by the RING finger TRAF6).E3 ubiquitin ligases are very diverse. They have been classified into three main classes according to the presence of specific domain motifs: Homologous to E6-associated protein C-terminus (HECT), Really Interesting New Proteins  Conversely, de-ubiquitinylation enzymes (deubiquitinases or DUBs) specifically remove ubiquitin/UBLps. DUBs are a large group of cysteine proteases or zinc-dependent metalloproteases that specifically cleave after the terminal carbonyl of the last residue of ubiquitin adducts. Compared to the proteins involved in the activation, conjugation and ligation of ubiquitin/UBLps relatively little is known about the functional role of DUBs. However, evidence suggests that DUBs are key regulators of the ubiquitin system; DUBs are functionally similar to protein phosphatases in the phosphorylation system. Based on their sequences similarities, structural studies and potential mechanism of action, DUBs fall into at least six distinct subfamilies: the ubiquitin C-terminal hydrolases (UCH-Peptidase_C12), the ubiquitin specific proteases (USP-UCH), otubains (OTU), the ataxin-3/Josephin ubiquitin protease (MJD), the JAMM isopeptidase (Mov34) and the recent Pseudomonas syringae has been shown to induce sensitivity in tomato plants by targeting a host kinase, Fen, to the proteasome, which leads to the inhibition of the Fen-activated immunity-associated programmed cell death Salmonella enterica), which causes gastroenteritis in humans, has similarly been implicated in its virulence The UPS is known to play important roles in modulation of immune and inflammatory responses. Deregulation of the UPS can lead to the development of inflammatory and autoimmune diseases, such as inflammatory arthritis, psoriasis, allergy and asthma . With regards to the five eukaryotic model organisms that were used, the threshold E-value ≤0.5 gave the most consistent results when compared to previously published results. The number of UPS-related proteins in A. thaliana and the other model organisms has previously been analyzed, particularly the number of E2 and E3 enzymes that are found in A. thaliana, H. sapiens, C. elegans and S. cerevisiae . In H. sapiens, 162 DUBs/DUBLs were found although a previous publication only identified 95 putative DUBs/DUBLs from which 79 exhibited conserved catalytic residues H. sapiens and D. melanogaster genomes contain multiple isoforms for some families of DUBs and DUBLs. Furthermore, the Hidden Markov Model that we used to search for UPS components compiled more complete datasets than many other search approaches would do. For example, while our HMM search identified domain OTU-carrying proteins in apicomplexan parasites (OTU is a major sub-class of DUB) none was reported in a recent publication on parasitic protozoa deconjugating enzymes where the authors used a more selective BLASTP homology search Amongst the 13 proteomes investigated in this study, a total of 4453 proteins were identified as carrying one or more of the 24 selected Pfam domains . For exae.g. 43% in A. thaliana) while only few of them were identified in apicomplexan organisms. F-box-containing proteins are adaptor proteins in Cullin-RING-Ligase complexes (CRLs), and are involved in direct and specific substrate recognition. Previous authors have hypothesized that the very high number of F-box proteins in A. thaliana suggests that plants can assemble numerous CRLs, which could control a wide array of substrates With regards to the relative abundance of each domain family, a striking observation is that a high proportion of F-box-carrying proteins are present in multi-cellular organisms  in triangular distance matrices. Results of this analysis are shown in A. thaliana UCHs and deSUMOylases. In the other subclasses, relative divergences exist within and between species. An increased divergence can be observed in the metalloprotease (JAMM/Mov 34). The function of the WLM family, usually found only in plant and fungus, in Plasmodium and Toxoplasma thus deserves to be fully investigated.With regards to the eight subclasses of DUBs analyzed, the Josephins (MJD), UCHs (peptidase_C12), autophagins (peptidase_C54) and deSUMOylases (peptidase_C48) families are well conserved within and between species with the exception of a clear differential expansion in the http://lerochlab.ucr.edu/UPS_prediction_data). When possible, the complete dataset was used to predict apicomplexan functional homologs of known UPS components from data available from the five model organisms investigated here. For each domain, dendrogram trees were built with all the 13 species that we used in this study. For purpose of clarity, only apicomplexan data are presented here. However, our complete results are available for download on the laboratory website (P. chabaudi and P. yoelii) contain partial ubiquitin sequences; however it was impossible to definitively assign the final ubiquitin gene based on the sequence available. In addition to ubiquitin/UBLps, several genes were identified by the HMM search that contain the highly related ubiquitin-binding domain (UBD). These typically N-terminal located domains are found in proteins that have evolved to adopt the ubiquitin domain in a non-conjugated role in processes such as signal transduction and proteasomal delivery Ubiquitin is a 76 amino acid protein extensively conserved between all eukaryotic sequences, with similarities in excess of 98% between humans, yeast and apicomplexan parasites de novo synthesis of ubiquitin and recycling of ubiquitin following cleavage from their target proteins. During periods of stress, elevated demands for ubiquitin are met, in part, by increased levels of polyubiquitin expression P. falciparum indicates all three ubiquitin genes are expressed throughout the parasite's life cycle As in all eukaryotes, ubiquitin is encoded by one of three types of fusion-protein precursors in the apicomplexans investigated here . AlthougPlasmodium spp., single copies of genes encoding SUMO have been identified across all the apicomplexan organisms investigated here , is most similar to ubiquitin at the primary sequence level. NEDD8 typically accumulates in the nucleus where its only known target, cullin, is found S. cerevisiae, conjugation to proteins involved in mRNA and pre-mRNA splicing have been described, and may more likely reflect the role of HUB 1 in apicomplexans Escherichia coli sulphur carring proteins ThiS and MoaD involved in thiamin and molybdopterin synthesis, respectively S. cerevisiae, URM1 has only been found to conjugate to alkyl hydroperoxide reductase 1 (AHP1), suggesting some role in adaptation to oxidative stress may similarly operate in apicomplexans Single copies of genes encoding the less characterized UBLps URM1 and HUB1 are found throughout the apicomplexan lineages investigated here . Both UBS. cerevisiae identified two UBLps involved in this system, termed ATG8 and ATG12 Leishmania spp. and play a key role in parasite virulence P. falciparum as being an ATG12 paralog .The autophagy system facilitates degradation of the cytoplasm following engulfment in a vesicle followed by fusion to lysosomes, a process necessary for both cell differentiation and response to starvation. Analysis of mutations in autophagy in P. falciparum and T. gondii), suggests that these UBLps are expressed at all the life stages investigated. These data suggest that ubiquitin/UBLps are essential components in controlling cellular processes throughout apicomplexans complex parasitic life cycles.A number of UBLps typical of higher eukaryotes  have not been found in this analysis, nor that previously described by Ponder and Bogyo (2007). Although some UBLps may not be expected based on their predicted roles in immune system regulation in higher eukaryotes, their absence, coupled with that of SUMO variants and ATG12 in apicomplexans suggest a more restricted role for UBLps in apicomplexan cell biology. However, analysis of gene expression data (microarray and proteomics) for SUMO, NEDD8, HUB1, URM1 and ATG8, where available  can deliver activated ubiquitin to several E2 isoforms, the E1s responsible for activating the UBLps SUMO and NEDD8, termed UBA2 and UBA3, respectively, only transfer to a single cognate E2 (see below). UBA1 has two UBA domains on a single polypeptide. UBA2 and UBA3, each only have one UBA domain containing the active site cysteine required for the covalent attachment of activated SUMO and NEDD8, and actually represent one part of a E1 heterodimer complex with AOS1 or APPBp1, respectively P. falciparum of UBA2 and UBA3 with those of the unassigned UBA containing proteins, as well as searching for existing characterized yeast two hybrid interactions, did not provide any additional clues in defining AOS1 or APPBp1 paralogs E1 proteins are characterized by the presence of the ubiquitin activating (UBA) Pfam domain. Additional motifs in E1 are responsible for the correct selection of ubiquitin/UBLp for activation and subsequent E2 to which transfer the activated ubiquitin/UBLp In addition to those described above, three additional E1 proteins are indicated in P. falciparum provide extensive evidence for the ubiquitous expression of E1s throughout the parasite's life cycle T. gondii similarly suggest constitutive expression of E1s throughout apicomplexan life cycles.Proteomic and transcriptomic profiling data available for via a transesterification reaction, is buried in a shallow groove Eukaryotes express a number of E2 isoforms, typically of between 17–22kDa  are only found in Plasmodium spp. and T.gondii in this analysis and are atypical E2s of up to 54kDa with a long N-terminal extension. N- and C-terminal extensions in E2 are thought to play key roles in recognition and association with E3s and their subsequent protein target and thus these atypical E2s may reflect a specific adaptation in the Plasmodium and Toxoplasma lineages.The apicompexan parasites investigated here have eight to fourteen E2 proteins, similar to the 14 described for the only other single cell eukaryote revisiae . The numP. falciparum indicates a clear association of the UBC13 and UEV paralogs in this organism Different isoforms of E2 have distinct roles in regulating downstream functions through specific interaction with distinct E3s . While sP. falciparum E2s suggest a diverse pattern of steady state mRNA accumulation at different stages of intraerythrocytic development. The fact that different E2 isoforms are expressed at distinct stages in the parasite's life cycle suggests that a temporal profile of delivering ubiquitin/UBLps to different E3s exists, which highlights a potential additional level of temporal control in the UPS system during apicomplexan parasite's life cycles.Extensive gene expression data for nine of the fourteen P. falciparum, and their homologs in T. gondii, C. parvum, and yeast. There are three superfamilies of E3 ubiquitin/UBL ligases. HECT ubiquitin ligases have a direct role in catalysis during ubiquitylation, whereas RING  finger and U-box E3s are involved in multi-protein complexes. RING finger E3s are the most abundant ubiquitin/UBL ligases.E3 ubiquitin/UBL ligases are a very diverse group of proteins involved in specifically transferring ubiquitin/UBLps to a given substrate. In all organisms, 48% of the predicted UPS components identified belong to the E3 ubiquitin/UBL ligase family. This high percentage of E3 reflects the specificity that is required for specific substrate recognition. P. falciparum, and other apicomplexans. Three of them have a homolog in S. cerevisiae: TOM1, UFD4, and HUL5 (HUL5 has unknown functions). The fourth HECT-domain protein that we identified in apicomplexans does not match any protein from yeast but is similar to UPL5 in A. thaliana  has been recently described as being involved in cell cycle arrest after DNA damage, mediating CDC6 ubiquitylation, a protein essential to initiation of DNA replication P. falciparum, and 80.m02344 in T. gondii) is involved in the ubiquitin fusion degradation pathway (UFD pathway) in vivo half-life of a protein to the identity of its N-terminal residue; for example, in eukaryotes, a protein with an isoleucine at its N-terminal end will be targeted to the proteasome more rapidly than a protein with a glycine, which is a stabilizing residue; see Our search identified four HECT domain-containing proteins in iana see . UPL5 haS. cerevisiae UFD2  and PRP19  homologs  contains WD40 repeats, which are known to be involved in a wide array of cellular processes ranging from signal transduction and transcription regulation to cell cycle control and apoptosis PRP19 is an oligomeric U-box-containing E3 ligase Plasmodium species  and shares extensive homology with the human protein CHIP . Like UFD2, CHIP has been described as being an E4 ubiquitin ligase in human. A particular feature of CHIP is that its catalytic activity requires its homodimerization through the U-box domain . Interestingly we also identified CHIP homologs in P. berghei, P. chabaudi, P. vivax and P. yoelii  whereas no homolog was found in yeast Cryptosporidium spp. and T. gondii. Thus, it can be hypothesized that CHIP homologs in Plasmodium are involved in the hepatic infection stage, although a precise role remains to be determined.A third U-box containing ubiquitin ligase, that is absent in yeast, has been identified in P. falciparum, and 80.m02207 in T. gondii). The SCF contains at least four subunits: CDC53 (Cullin1) which is stabilized by the ubiquitin-like modifier NEDD8, the RING E3 RBX1, the adaptor protein Skp1 and a F-box protein for substrate recognition , the cullin classically involved in the APC/C (APC2) was not found in apicomplexan parasites. The role of CUL8 is not well known. Previous data suggest that it may be involved in anaphase progression P. falciparum is not entirely unexpected. Cell division during schizogony is apparently asynchronous in P. falciparum, that is, several rounds of DNA replication/DNA division occur before final cytokinesis, instead of the paradigm of successive cycles of alternating DNA replication/DNA division/cytokinesis . The most famous CRLs are the Skp1-Cullin1-Fbox (SCF) complex, containing the cullin protein CDC53 in yeast, and the Anaphase Promoting Complex/Cyclosome (APC/C), containing the cullin protein APC2 in yeast. Both the SCF and the APC/C are involved in cell-cycle progression . For example, we have found potential RAD16, RAD5, and TFB3 homologs , which are known to be involved in nucleotide excision repair (NER) P. falciparum) or pre-mRNA maturation  P. falciparume.g. PfBre1 PFF0165c in P. falciparum, 35.m01589 in T. gondii), or chromatin remodeling and silencing . Recent data have increasing shown that epigenetic mechanisms play a key role in the control of gene expression in Apicomplexa. In Plasmodium, chromatin modeling is one of the proposed mechanisms that control allelic exclusion of the var virulence genes T. gondii, acetylation of histone H4 or acetylation on lysine 9 of histone H3 as well as trimethylation of lysine 4 of histone H3 were shown to be located close to the 5′ UTR of the active genes RING finger and RING-like E3 ubiquitin ligases are the largest group of E3s. They do not have a direct catalytic role in linking ubiquitin and ubiquitin-like modifiers. They rather act as adaptor partners: RING E3s interact with both an E2 ubiquitin-conjugating enzyme (that is carrying ubiquitin) and its given specific substrate, bringing the substrate in close proximity with ubiquitin. Since RING and RING-like E3s are involved in substrate recognition, and given the diversity of proteins that are targeted by the UPS, a large diversity of RING and RING-like E3s is expected , RING/helicase, RING/forkhead-associated domain (FHA), and RING/in between RING (IBR). It is interesting to observe that predicted Plasmodium RING E3s with a single RING domain appear more abundant than those of yeast. Several of these proteins carry several coiled-coil regions, e.g. up to six in PFF0165c. Coiled-coil domains are known to be involved in regulation of gene expression and many other biological processes . Membrane bound E3 ubiquitin ligases have been shown implicated in the regulation of immune recognition during virus infections  were recently identified in in vivo has been challenging in other model organisms. It is possible that in vivo protein localization and the presence of adaptors such as E3 ligases can increase specific interactions Determination of target specificity P. falciparum, respectively) and could be potentially involved in the regulation of parasite virulence genes, principally the var gene family in P. falciparum.Increasing evidence suggests that several DUBs are implicated in remodeling chromatin structure, transcriptional regulation and gene silencing. In yeast, UBP8 and UBP10 have been implicated in the dynamic histone monoubiquitination of H2B. The presence of UBP8 correlates with changes in transcriptional regulation Plasmodium and a locus containing a DUB related to USP7 DUBs are also implicated in the endocytic pathway and intracellular traffic P. falciparum life-cycle, providing additional evidence for a functional role in the parasite's life cycle.Expression profiles analysis P. falciparum and other Apicomplexa. All apicomplexans possess the complete machinery that is required to ubiquitylate proteins . Ubiquitin and the common UBL modifiers SUMO, NEDD8, HUB1, URM1, and ATG8 were identified in apicomplexans. However, several UBLps are missing, such as SUMO variants and ATG12, although it has been suggested that autophagy is one of the parasite's death pathway P. falciparum and T. gondii have up to triple the molecular weight of E2s found in other organisms, which could reflect adaptation of Plasmodium and Toxoplasma lineages. Finally, the superfamily of E3 ubiquitin ligases is very diverse, and several of the E3s predicted in apicomplexans do not find homolog in other eukaryotic organisms. Such proteins could have a cellular role directly linked to parasitic processes, such as invasion. The over-representation of RING E3 ligases that contain coiled-coil domains, known to play important role in host-pathogen interactions, supports this hypothesis.The present study allowed for the identification of up to 114 proteins that are predicted to be involved in the UPS of Plasmodium genome. The presence of a modified APC complex in the atypical erythrocytic Plasmodium cycle is not surprising. The cell cycle in Plasmodium can be closely related to the early embryogenesis division observed in D. melanogaster with a depletion of key cell cycle regulators. During the first 13 cell divisions of a D. melanogaster zygote, the divisions are rapid, synchronous and occur in the absence of cytokinesis and detectable gap phase. These divisions result in the formation of a syncytial cell containing a large number of nuclei in a single cytoplasm. In Plasmodium, divisions during schizogony are rapid and asynchronous with up to four or five rounds  of DNA synthesis and mitosis during the trophozoite and early schizont stages. Cytokinesis takes place late in the cycle during the mature schizont stage. This is a major divergence from the classical cell-cycle events that consists of a linear succession of G1/S/G2/M phases. A proposed model for the Plasmodium cell-cycle control is presented in An example of apicomplexan adaptation in the UPS is an apparently modified APC/C . In yeast, SCF and APC are known to play a fundamental role in cell-cycle control. All proteins known to be involved in the SCF machinery are present in the Plasmodium and other apicomplexa is a good estimate when compared to other eukaryotic organisms. Our results demonstrate that E3 ubiquitin/ubl ligases remain one of the most specific components of the UPS. The presence of numerous and diverse E3s, and in particular RING domain-containing proteins, suggests that target-specific ubiquitylation via E3 ligases is a complex and important part of cellular regulation in eukaryotic cells, including apicomplexan parasites. In terms of sequence homology, when counterparts exist, most of the ubiquitin ligases identified in Apicomplexa display divergences from their human-host counterpart. The domain architecture analysis for P. falciparum's RING finger E3s revealed an abundance of coiled-coil domain. Since such domains have been shown to play a major role in signal transduction during the molecular cross-talk that occurs during a viral or a bacterial infection, our hypothesis is that E3 ubiquitin/UBL ligases in P. falciparum and other Apicomplexa are involved in pathogen virulence and/or pathogenicity.The number of genes that our work predicted to encode components of the UPS in via drugs that target the proteasome component of the UPS has been established in P. falciparum. However, this potential to exploit these drugs is limited. The latest generations of drugs that target the UPS have focused more on the specificity of the action of E3 ligases and DUB/DUBLs. Given the apparent apicomplexan diversity in these proteins, opportunities to develop small molecule inhibitors specific against the the apicomplexan-specific E3 ligase and DUB/DUBLs offer some possibility for new, much needed, therapeutics for these devastating global diseases.The potential to treat apicomplexan parasites P. falciparum, P. vivax, P. yoelii, P. berghei, P. chabaudi, C. parvum C. hominis, Toxoplasma gondii, S. cerevisiae, C. elegans, D. melanogaster, A. thaliana and H. sapiens. Additional information regarding data sources and release information are given in http://pfam.janelia.org .Full proteome datasets from the following thirteen organisms were downloaded: http://hmmer.janelia.org. The program hmmsearch was used to analyze domain distribution among all proteome datasets and to extract sequences that carry the Pfam domains described above. HMM searches were run using a series of incrementally increasing threshold E-values, from E-value ≤1 to E-value ≤0.1, and results were checked for false positives. Threshold E-value ≤0.5 gave the best quality results, and thus was used in the present study.Components of the HMMER 2.3.2 package (released in October 2003) were used throughout the study Proteins carrying same domain motifs were pair aligned by BLASTP. Output bits scores were used as protein distance values: the higher the values are, the more proteins are similar and therefore could be evolutionary closed. All scores below 20 were set to 20. Values were scaled in the range  by normalizing using a global transformation (d = minimum score/score). A color-mapping matrix, from red to blue, was built, using MATLAB® 7, from the normalized scale. The red color means “highly divergent”, and blue means “highly conserved”.Protein sequences were aligned using ClustalW P. falciparum and yeast RING and RING-like E3 ubiquitin ligases were searched for functional domains using SMART Our datasets of Table S1Exhaustive list of the proteins identified by the HMM search.(0.37 MB XLS)Click here for additional data file.Table S2http://orthomcl.cbil.upenn.eduComponents of the Cdc48-Npl4-Ufd1 escort pathway in P. falciparum, predicted by BlastP, and their orthologs retrieved from (0.04 MB XLS)Click here for additional data file.Table S3Homologs of Skp1 in apicomplexan parasites.(0.03 MB XLS)Click here for additional data file.Table S4Protein dataset sources and release information.(0.02 MB XLS)Click here for additional data file.Figure S1By-domain dendrogram trees of the predicted apicomplexan proteins(1.64 MB ZIP)Click here for additional data file.