Long-lasting insecticidal nets are an effective tool for malaria prevention, and "universal coverage" with such nets is increasingly the goal of national malaria control programmes. However, national level campaigns in several countries have run out of nets in the course of distribution, indicating a problem in the method used to estimate the quantity needed.A major reason for the shortfall in estimation is the mismatch between the quantification factor used to plan procurement and the allocation algorithm used at community level, in particular the effect of needing to add an additional net to households with an odd number of inhabitants. To solve this problem a revised quantification factor is suggested.Based on data from a broad range of household surveys across Africa, the effect of odd-numbered households on numbers of nets distributed is estimated via two frequently used allocation methods. The impact of these algorithms on the proportion of households reaching a person to net ratio of 2:1, a frequently used marker of universal coverage is then calculated.In order to avoid stock-outs of nets during national coverage campaigns, it is recommended to use a quantification factor of 1.78 people per net, with an additional allocation factor suggested to account for other common problems at the community level resulting in a final recommended ratio of 1.60 people per net. It is also recommend that community level allocation procedures be aligned with procurement estimates to reduce shortages of nets during campaign distributions. These analyses should enable programme managers to make evidence-based decisions and support a more efficient and effective use of LLIN distribution campaign resources. Insecticide treated nets (ITN) are an effective tool for preventing the transmission of malaria . This isCentralized mass distribution campaigns have served as the cornerstone of efforts to achieve universal coverage . Recent ). A similar campaign in Uganda noted that the actual number of nets required for universal coverage was 30% greater than the estimate obtained using the WHO calculation .Inadequate procurements may be one reason that these campaigns fail to provide households with a sufficient number of nets. The World Health Organization/Global Malaria Programme (WHO/GMP) recommends dividing the estimated total population by a factor of two when calculating the total number of nets needed to achieve the desired ratio of 1 net per every two people . In pracInefficient household allocation strategies may be a second factor limiting the ability of these campaigns to achieve universal coverage. Countries use a variety of approaches for allocating nets to households during their distribution campaigns. In some countries, campaigns distribute a fixed number of nets, typically two or three nets, to each household. In other countries, the number of nets allocated to a household varies by the size of the household or by the number of sleeping places in the household. This variation in approaches illustrates the lack of clarity in the current understanding regarding how best to allocate nets in support of universal coverage objectives.Drawing on existing household survey data, this paper provides empirical support for an improved algorithm for estimating the number of LLIN needed to achieve universal coverage within a given population and evaluates the various approaches for allocating LLIN to specific households. These analyses should provide guidance to programme managers to make evidence-based decisions and support a more efficient and effective use of LLIN distribution campaign resources.The current algorithm for calculating the number of LLIN necessary to provide one net for every two people in a population simply divides the total population size by a factor of two . Howeverde jure members  received a quantity of nets equal to half of the household size. The number of nets allocated to households with an uneven number of de jure members differed between the two scenarios. In Scenario A, households received a quantity of nets equal to half of the household size minus 1; while in Scenario B, households received a quantity of nets equal to half of the household size plus 1. Table A more accurate algorithm would use a population-level divisor that accounts for the one additional LLIN required by households with an uneven number of members. To identify this divisor and assess the additional coverage that would result from its use, two scenarios were used to simulate the allocation of LLIN to households sampled in 12 Demographic and Health Surveys (DHS)  and six Once households are allocated a quantity of nets according to the rules described in Table Strategies to allocate nets to households are guided by two primary questions. First, should the campaign allocate a fixed or varying number of nets to each household? And, if households should receive a varying number of nets, should the number of nets allocated to the household be based on the number of people or the number of sleeping places within the household? At present, there is little empirical data upon which program managers can base their decisions to these questions.The decision to allocate a fixed number of nets to each household seems based on the assumption that a single quantity can be identified that provides a substantial number of households with the correct number of nets, while minimizing the proportion of households that receive too many nets or too few nets. To test this assumption, the results of two fixed allocation campaigns, one providing two nets to each household and the other providing three nets, were assessed using the distribution of household sizes in the 18 datasets. Households were classified as receiving the correct number of nets if they received one net for every two people in the household . They were classified as receiving too many nets if they had fewer household members and were classified as receiving too few nets if they had more household members.When the decision is made to vary the number of nets allocated to a household, the number of sleeping places within the household appears to be the logical metric to use to ensure that a mosquito net covers every person sleeping in the household. In practice, this approach presents some concerns. First, where individuals sleep on a variety of surfaces, the definition of a sleeping place may include areas in which several people sleep and that may be too large to be enclosed by a single net. Second, households from lower wealth quintiles may be more likely to have more individuals sharing fewer sleeping places. Use of sleeping places to allocate nets then may result in an inequitable distribution of nets favouring households from higher wealth quintiles. To investigate these concerns, data from five surveys that measured both the number of household members and the number of sleeping places were analysed.Recent experience suggests that dividing the total population by two underestimates the total number of LLIN required to achieve universal coverage, defined as at least one net for every two people in a household. To test the hypothesis that this underestimate is attributed to the additional net required by households with an uneven number of members, simulated allocations of LLIN to households either ignored this additional need (Scenario A) or met this additional need (Scenario B). It was expected that Scenario B would be more likely than Scenario A to reach the household threshold of universal coverage and that the mean number of persons per net in Scenario B would serve as a more effective population divisor for calculating the total number of nets needed to reach universal coverage.de jure members received a quantity of nets equal to half of the household size minus 1. Following this allocation rule, only 50 to 60% of households would receive at least one net for every two people and meet the threshold required for universal coverage. The mean number of persons per net ranged from 2.02 to 2.27, with a median score of 2.19. This "LLIN allocation factor" is slightly higher than the number (2.0) currently recommended by WHO, suggesting that the current approach should result in approximately 60-70% of households reaching the threshold of one net for every two people that is required for universal coverage.Table de jure members are allocated an additional net to accommodate the extra person, all households meet the threshold of one net for every two people that is required for universal coverage. In this scenario, the mean number of persons per net ranged from 1.64 to 1.85, with a median score of 1.78. This indicates that dividing the total population by 1.78 would provide enough nets for a campaign to provide each household with at least one net for every two people in the household. The mean number of persons per net in Scenario B remains remarkably consistent across countries, regardless of the level of urbanization and the mean household size in each country (Figure The results from the Scenario B simulation are presented in Table These results are nearly identical to those obtained from intensive micro-census investigations in Mozambique and Cambodia. In Mozambique, information on the number, gender, and age of household members was used to determine the likely sleeping arrangements of the household members and identified that 1.75 persons per net was necessary to achieve universal coverage . A similThe second hypothesis examined the relative effectiveness of using a fixed allocation of nets to achieve universal coverage. It was expected that the variation in household sizes would limit the effectiveness or efficiency of this approach, suggesting the use of tailored allocations as the recommended approach for determining the number of nets to be given to households.As expected, allocating a fixed number of LLIN to each household does not appear to be an effective approach for achieving universal net coverage or an efficient way to allocate nets to households Table . When twSince this suggests allocating a varied, rather than fixed, number of nets to households, the remaining question concerns the use of household members or sleeping places to determine the amount of nets required by each household. It was hypothesized that the subjective definition of a "sleeping place" may result in an identification of spaces in which multiple individuals sleep and which may be too large to be covered by a single LLIN. It was further hypothesized that the number of sleeping places in a household would be positively related to household wealth and that allocating LLIN by sleeping places would disproportionately favour the distribution of nets to wealthier individuals.Figure de-jure residents did not, resulting in a decreasing mean number of persons per sleeping place with increasing wealth (Table Data from Northern Bahr el Ghazal in Southern Sudan (unpublished data: Malaria Consortium, see annex for details on survey and methodology [Additional file th Table . Using tth Table . In contThese results recommend revising the current approach for calculating the number of LLIN necessary for achieving universal coverage through a centralized mass distribution campaign. Rather than dividing the total population by a factor of 2, a new divisor of 1.78 is necessary to account for the additional LLIN needed in households with an uneven number of members. This revised divisor is very similar to that suggested by "bottom up" approaches of micro-planning at the household level.It should be noted, however, that these simulations are unable to account for some additional logistical hurdles experienced by distribution campaigns. First, campaigns often need to pre-position stocks of LLIN to adjust for inconsistencies between the census estimates used for planning and the actual numbers obtained during registration exercises. Second, since LLIN are packaged in bales of 50 or 100 and are difficult to transport once opened, campaigns typically round-up the number of LLIN sent to the distribution points to the nearest bale. Both practices result in a certain number of LLIN being "stuck" in the distribution chain, resulting in an additional demand of 5% to 10% of initial estimates.The obvious solution is to include a buffer in the quantification that will compensate for these logistical "losses." While there is little empirical information regarding the extent of this margin, reasonable expectations suggest it is between 5% and 15% of the total need. Applying these rates to the recommended population divisor of 1.78 results in the corrected factors presented in Table These results also recommend allocating LLIN to households based on their household size, without an upper limit for larger households, rather than using a fixed number of nets per household. Using the number of household members, rather than sleeping places, appears more likely to provide households with a sufficient number of nets to achieve universal coverage and to result in a more equitable distribution of LLIN across all wealth quintiles. However, if sleeping places are to be used to determine the number of nets required by a household, efforts should be made to confirm that the mean number of people per sleeping place is close to 2.0 and the definition of a sleeping place needs to be clearly articulated and standardized. One possible approach would be to define sleeping places as "a place where people sleep and that can be covered by a single net."Whether household size or sleeping places are used to allocate nets to households, a similar metric should be used to quantify the total number of nets needed for the campaign. If the method of quantification differs from that used for actual net allocation during the campaign, the allocation of nets may require a greater quantity than were estimated by the quantification method. In Kano, Nigeria, LLIN needs were quantified at two nets per household using the standard definition of "people eating from the same pot," but were allocated using the household definition of "wife with her dependents" in polygamous families. Instead of delivering two nets per household, the outcome of the post-campaign survey showed that only 1.7 nets were delivered among those who attended the distribution, with 28% of households receiving only one net .The authors declare that they have no competing interests.AK drafted the manuscript, provided data form the sub-national studies and did part of the statistical analysis. MB provided the statistical analysis of the DHS data and contributed to the manuscript development. HK provided input on structure and campaign logistics, and ML helped draft and edit the manuscript. All authors read and approved the final manuscript.AK is director monitoring & evaluation for the Malaria Consortium; MB is Assistant Professor at the Department of Health, Behavior & Society, Johns Hopkins Bloomberg School of Public Health; HK is senior program officer at the Center for Communication Programs, Johns Hopkins Bloomberg School of Public Health; ML is Director of the Global Program on Malaria at the Center for Communication Programs, Johns Hopkins Bloomberg School of Public HealthNet tracking survey Northern Bahr el Ghazal, Southern Sudan. Details of the study design, methodology and analysisClick here for file


The accumulation of mutant protein is a common feature of neurodegenerative disease. In Huntington's disease, a polyglutamine expansion in the huntingtin protein triggers neuronal toxicity. Accompanying neuronal death, mutant huntingtin aggregates in large macromolecular structures called inclusion bodies. The function of the machinery for intracellular protein degradation is linked to huntingtin toxicity and components of this machinery colocalize with inclusion bodies. An increasing body of evidence implicates the ubiquitin-proteasome pathway in the failure of cells to degrade mutant huntingtin. A number of potential mechanisms that link compromised ubiquitin-proteasome pathway function and neurodegeneration have been proposed and may offer opportunities for therapeutic intervention.


The human genome encodes several ubiquitin-like (UBL) domain proteins (UDPs). Members of this protein family are involved in a variety of cellular functions and many are connected to the ubiquitin proteasome system, an essential pathway for protein degradation in eukaryotic cells. Despite their structural similarity, the UBL domains appear to have a range of different targets, resulting in a considerable diversity with respect to UDP function. Here, we give a short summary of the biochemical and physiological roles of the UDPs, which have been linked to human diseases including neurodegeneration and cancer.Publication history: Republished from Current BioData's Targeted Proteins database .   Ubiquitin is a small and phylogenetically conserved eukaryotic protein known to covalently modify proteins and thereby mark them for destruction by the 26S proteasome . This prUbiquitin ligation is accomplished in multiple steps , the firThe formation of polyubiquitin chains is a reversible process and several deubiquitylating enzymes (DUBs) play important roles in trimming the chains on target proteins .Different ubiquitin lysine residues are used in the formation of polyubiquitin chains, with various outcomes. For example, whereas Lys48-linked polyubiquitin chains target proteins for degradation by the 2.5 MDa 26S proteasome in an ATP-dependent manner .Ubiquitin is a stable and compact protein consisting of two α-helices and five β-strands arranged in the order ββαββαβ to form the ubiquitin superfold . RecentlThe UDPs are responsible for recruitment of ubiquitylated substrates to the proteasome  and bindUDPs that contain one or more ubiquitin binding UBA (ubiquitin-associated) domain figure  in additin vitro[in vitro by biochemical experiments, which showed that UBL/UBA proteins are essential for degradation of a proteasome substrate [Precipitation experiments have shown that the UBL domain of both Rad23 and its human homologue (HHR23) interacts directly with the 26S proteasome in vitro, while tin vitro. UBL/UBAin vitro. This shubstrate . Since mubstrate , are proDespite the fact that none of the UBL/UBA proteins are essential in yeast, combined loss of Rad23, Dsk2 and the proteasome's ubiquitin receptor Rpn10/S5a results in mitotic arrest . This inIn precipitation experiments the UDPs Rad23, Dsk2 and their human orthologues HHR23 and ubiquilin-1 interact with the 26S proteasome in a UBL domain-dependent manner ,18,19. Hin vitro[Both Ufd2 (an E4) and the 26S proteasome associate with the Rad23 UBL domain in a mutually exclusive manner in vitro. One migin vitro, a protein involved in the development of the neurodegenerative Machado-Joseph disease [In addition to the 26S proteasome and ubiquitylated substrate, HHR23 also interacts with ataxin-3  disease . Similar disease ,25. Sinc disease  and perh disease ,23. Therubiquilin-1 gene are proposed to substantially increase the risk of developing Alzheimer's disease [presenilin-1 and presenilin-2 lead to an increased ratio of β-APP42 to β-APP40  from amyloid β-precursor protein [in vitro translated presenilins, SDS-PAGE of the precipitates revealed a slowly migrating smear. It is therefore likely that only ubiquitylated presenilins can interact with ubiquilin-1. It is also possible that ubiquilin-1 only interacts with ubiquitylated γ-aminobutyric acid A (GABAA) receptors [Ubiquilins 1–4 are human homologues of yeast Dsk2. Ubiquilin-1 and ubiquilin-2 both bind the proteasome  and, int disease . The gen protein . Accumul protein . The pre protein . Howevereceptors .A receptor at inhibitory synapses is tightly regulated [A receptors and facilitates their membrane insertion by increasing the stability of the intracellular pool [A receptors. As proteasomal degradation of the receptors was not observed, ubiquilin-1 may therefore also function in endocytosis [In order to efficiently transmit synaptic signals, the amount of GABAegulated ,33. Thisegulated . Ubiquillar pool . Based oocytosis . Accordiocytosis . Eps15 cocytosis . Althougocytosis . This isocytosis . It is pocytosis . These socytosis . Once thocytosis .Ubiquilin-4 is linked to spinocerebellar ataxia type 1 (SCA1) , an inhePARK2 gene encoding PRKN2  [PARK2 impair its catalytic activity [Autosomal recessive juvenile Parkinsonism (AR-JP) is an early onset and slowly progressing disease, the most common cause for which is mutations in the  parkin) -43. PRKN parkin) . Accordiactivity -47. Seveactivity ,48-51. Tactivity . Moreoveactivity .in vitro precipitation assays failed to confirm the interaction [in vivo[Although most of the pathogenic missense mutations cluster to the RING domain-containing C-terminus of PRKN2, a few mutations have also been identified in the UBL region. NMR interaction studies have identified an interaction between the PRKN2 UBL domain and the proteasome subunit Rpn10/S5a, an observation not made with the pathogenic PRKN2 mutant R42P . Howevereraction , its sig [in vivo. Though  [in vivo,55, this [in vivo, indicat [in vivo. Upon tr [in vivo.PRKN2 can also be destabilized by pathogenic mutations within its UBL domain . HoweverVHL (Cullin–Elongin-BC–VHL) [Von Hippel-Lindau (VHL) disease is an autosomal dominant cancer, which manifests as angiomas of the retina, hemangioblastomas of the CNS and renal clear cell carcinomas . Patient-BC–VHL) . In addi-BC–VHL) .VHL catalyses the ubiquitylation of hypoxia-inducible transcription factor α , the murine orthologue of RAD23B (HHR23B) [Yeast has proved to be an excellent model system for the study of the UBL/UBA proteins. However, developing transgenic mouse models will further reveal important functional aspects of their molecular functions. A knockout is already available in (HHR23B) , which d(HHR23B) .Park2 knockout mouse models have been reported [Park2 knockout, which additionally lacks the PRKN2 co-regulated gene Pacrg[quaking mutants are characterised by dysmyelination in the CNS, resulting in phenotypes including movement disorders, tremors and seizures [To date, two reported ,67, bothene Pacrg. Homozygseizures .Drosophila, the loss of park (homologue of PARK2) results in reduced cell size and number, infertility, reduced lifespan, movement and flying disorders, and increased sensitivity to oxidative stress [park mutant flies do not display age-dependent neuron loss in the brain.In e stress . HoweverDrosophila PRKN2 models will serve as an invaluable tool for understanding the biological role of PRKN2 and may provide important clues to the molecular mechanisms of Parkinson's disease.Collectively, the mouse and Inhibitors of the 26S proteasome, such as bortezomib, are successfully being used in the treatment of certain cancers . HoweverXenopus extracts performed by Verma et al. at Caltech have identified a group of relatively small molecules, called ubistatins, which inhibit the turnover of cyclin-B and the cdk-inhibitor Sic1 in vitro by inhibiting the binding of ubiquitylated proteins to substrate shuttles [Devising efficient inhibitors for non-enzymatic proteins such as the UBL/UBA proteins is more difficult than for proteins with enzyme activity, which can be assayed in high-throughput screens. Nonetheless, recent chemical genetic screens in shuttles . AccordiPARK2 gene with a wild-type copy could perhaps provide a cure in the future.As for many loss-of-function mutations in disease-connected E3 enzymes, it is difficult to envision small molecules that could reactivate the mutant PRKN2 and thereby promote neuronal survival in patients suffering from AR-JP. However, a gene therapeutic approach to replace the dysfunctional In the case of von Hippel-Lindau disease, small molecule activators of mutant VHL may be able to sustain HIFα degradation and prevent tumour angiogenesis. However, it was found that the molecular chaperone Hsp90 protects certain VHL substrates from proteasomal degradation  and cyclIt is evident that the UBL/UBA proteins and PRKN2 appear to be directly involved in the etiology of cancer and neurodegenerative diseases. Although this is by no means a general feature of the UDPs, it highlights the importance of the ubiquitin system in maintaining an appropriate intracellular protein milieu.With respect to the UBL/UBA proteins, one major issue that remains unresolved relates to their specificity. Though some details of their substrate selectivity have emerged , a more In relation to PRKN2, identifying novel substrates of its E3 activity and resolving its proteasome-dependent and/or -independent functions  may shed some light on the molecular mechanism of Parkinson's disease and hopefully lead to novel therapeutic approaches.A, γ-aminobutyric acid A; HIFα, hypoxia-inducible transcription factor α; SCA1, spinocerebellar ataxia type 1; UBA, ubiquitin-associated domain; UBL, ubiquitin-like; UDP, UBL domain protein; UIM, ubiquitin-interacting motif; VHL, von Hippel-Lindau.Aβ, amyloid β-protein; AR-JP, autosomal recessive juvenile parkinsonism; CBC, Cullin–Elongin-BC; DUB, deubiquitylating enzyme; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; GABAThe authors declare that they have no competing interests.).    Republished from Current BioData's Targeted Proteins database (TPdb;


In late 2006, the seaside community in Esperance, Western Australia, was alerted to thousands of native bird species dying. The source of the lead was thought to derive from the handling of Pb carbonate concentrate from the Magellan mine through the port of Esperance, begun in July 2005. Concern was expressed for the impact of this process on the community.This study was designed to evaluate the source of Pb in blood of a random sample of the community using Pb isotope ratios.The cohort comprised 49 children (48 < 5 years of age) along with 18 adults (> 20 years of age) with a bias toward higher blood lead (PbB) values to facilitate source identification.n = 49; geometric mean, 6.6 μg/dL), with four children whose PbB was > 12 μg/dL. The isotopic data for blood samples lay around two distinct arrays. The blood of all children analyzed for Pb isotopes contained a contribution of Pb from the Magellan mine, which for young children ranged from 27% up to 93% . Subtraction of the ore component gave a mean background PbB of 2.3 μg/dL. Several children whose PbB was > 9 μg/dL and most of the older subjects have complex sources of Pb.Mean PbB level of the children was 7.5 μg/dL (range, 1.5–25.7 μg/dL; The death of the birds acted as a sentinel event; otherwise, the exposure of the community, arising from such a toxic form of Pb, could have been tragic. Isotopic data and mineralogic and particle size analyses indicate that, apart from the recognized pathway of Pb exposure by hand-to-mouth activity in children, the inhalation pathway could have been a significant contributor to PbB for some of the very young children and in some parents. In late 2006, the community of about 14,000 people in the town of Esperance in Western Australia  was alerThe source of the Pb ore concentrate was thought to derive from the handling of Pb concentrate at the port that began in July 2005. The Pb concentrate originated at the Magellan mine some 600 km north of Esperance and was transported by road and rail to the port . Other pAfter the birds began dying, state and local government authorities began several investigations, although shipping of the ore concentrate continued for several months. Rainwater from household tanks is one of the main sources of drinking water in Esperance. The local Shire Council and Western Australia Department of Health (DoH) undertook measurements of Pb and Ni in the rainwater tanks and initially found that 10% of residences had Pb levels above the World Health Organization guidelines of 10 μg/L and 30% had Ni levels above the guidelines (20 μg/L). The DoH then undertook a blood Pb (PbB) survey of any resident who wished to be tested, and the Department of Environment and Conservation (DEC) undertook environmental investigations. After results from these investigations were obtained, shipping of the concentrate through the port was suspended.The Pb concentrate being shipped through the port was Pb carbonate, which is an unusual mining product; the most common Pb ore is galena [lead sulfide (PbS)], from which Pb carbonate is derived by oxidation over geologic time. Unfortunately, Pb carbonate is more toxic than PbS: A study by When the first author heard about the problem in Esperance, he alerted the DoH and DEC to the highly unusual Pb isotope signature in the ore concentrate, measured before mining by colleagues in the Commonwealth Scientific and Industrial Organisation (CSIRO), and suggested that the Pb isotope method would be a powerful tool in source apportionment investigations. Some questions that could be, and were, addressed using the Pb isotope method were as follows: Is the Pb in the bird livers derived from the Magellan concentrate? If so, what is the pathway from the concentrate to the birds, especially because they were dominantly nectar feeders? Was the Pb adsorbed on the vegetation? Are the elevated Pb levels in rainwater derived from the Magellan concentrate? What are the sources of Pb in soil in parks and residences? What is the contribution of Magellan Pb to PbB of children and adults in Esperance? In this article, we report the results of the isotopic investigation of blood in children and adults. Because the environmental investigations are the subject of litigation, they will be reported at a later date, although the The DoH undertook opportunistic sampling, taking blood from any resident who wished to have his or her PbB measured. All subjects lived within 3 km of the port. None of the subjects were occupationally exposed to Pb, although some of the adults may have undertaken activities in which they were exposed to Pb, such as renovating older houses with Pb paint or making fishing sinkers (Esperance is a seaside town). DoH took most of the samples in a clinic especially set up for the purpose, following standard protocols; a few samples were taken by general medical practitioners in the town. They collected the samples mostly in March through May 2007, after loading of the Pb concentrate had been suspended. Most were venous samples taken with Vacutainer devices, but sometimes by finger prick or heel prick if the parent expressed a preference or they could not obtain a venous sample. Samples we analyzed for Pb isotope ratios were generally for subjects with PbB values > 5 μg/dL, because other studies have pointed out difficulties in assigning sources when PbB values were < 5 μg/dL . For exa206Pb is derived by radioactive decay from 238U, with a half-life of about 4,500 million years; the decay of 207Pb from 235U is more rapid, with a half-life of about 700 million years, whereas the decay of 208Pb from 232Th is much slower, at about 14,000 million years. The other low-abundance isotope, 204Pb (~ 1%), has no known radioactive parent and is thought to represent Pb present at the time of formation of the earth, some 4,550 million years ago ; it is used as a reference isotope. Hence, Pb mineral accumulations  of different geologic ages have different isotopic abundances. The simplest explanation for the different abundances is that when the deposit formed, the Pb was separated from its parent Th and U isotopes, so no further radioactive decay or changes in the isotope abundances occurred. In this way, ore formed, say, 1,800 million years ago had more primordial Pb and relatively fewer decay products compared with ores formed only 400 million years ago; this also includes Pb formed in the intervening 1,400 million years. Isotopic investigations present data as ratios of abundance of one isotope to the other, such as 208Pb/204Pb, 207Pb/204Pb, and 206Pb/204Pb, or any combination of these. In the earlier days of use of Pb isotopes, because of the difficulty in measurement of the low-abundance 204Pb isotope, studies reported data as 206Pb/207Pb ratios. As an example of the differences in isotopes from different ore deposits, the geologically ancient so-called massive sulfide Pb–zinc–silver Broken Hill and Mt. Isa deposits in Australia formed about 1,700–1,800 million years ago and have a 206Pb/204Pb ratio of 16.0 or 16.1, respectively, whereas geologically younger deposits of similar composition in eastern Australia that formed 400–500 million years ago have a 206Pb/204Pb ratio of about 18.1. The Pb isotope method makes use of the variations, arising from radioactive decay throughout geologic time, in abundances of three of four Pb isotopes, the relative concentrations of Pb, thorium, and uranium, and the time when the ore was formed. Pb has four naturally occurring isotopes, three of which are the stable end products of radioactive decay of U and Th. For example, However, other types of Pb deposits formed by a different mechanism, called Mississippi Valley deposits (found in the Tri-State District of the United States) or sandstone-type deposits . Their isotope ratios are very different from those for the massive sulfide deposits, and their data plot away from the growth curves. The Magellan mine is an example of the sandstone type and is thought to have formed about 1,650 million years ago . To accoThermal ionization mass spectrometry requires good separation of Pb from other elements that may inhibit the ionization of Pb in the mass spectrometer and to avoid interference from ions with the same mass/charge ratio as some of the Pb isotopes. Although Pb levels in the environment have decreased since Pb was removed from gasoline, the processing blank is still a major concern in the design of chemical separation procedures. All reagents we used were ultra-pure, and containers were either Teflon or polypropylene. Under best conditions, we did all processing in class 350 clean rooms with further handling in class 100 laminar flow workbench stations.202Pb-enriched tracer to determine the Pb concentration in the sample (the isotope dilution method). The procedural blank is about 50 pg (50 × 10−12 g), which has an insignificant effect on the measured ratios.Approximately 0.3–0.5 g of blood was digested with concentrated nitric acid/hydrogen peroxide and the Pb separated from other elements on a column of Pb-selective resin . We adde206Pb/204Pb ratios can be obtained, and much better precision can be obtained for the more abundant ratios 208Pb/206Pb and 207Pb/206Pb. To enable comparisons of data across laboratories, we normalized the isotope ratios to the accepted values of the NIST standard SRM 981.We dissolved the sample in water and evaporated a small volume onto a cleaned rhenium filament along with silica gel/phosphoric acid to enhance ionization. We placed the filaments in a VG Sector 354 multicollector thermal ionization mass spectrometer  and measured 250 Pb isotope ratios. From replicates of the U.S. National Institute of Standards and Technology  standard reference material SRM 981 and natural samples, a precision of better than ± 0.05% (2σ) for the We present our results in 207Pb/204Pb versus 206Pb/204Pb graph , and Mt. Isa and Cannington in Queensland, have 206Pb/204Pb ratios almost identical with Magellan because they formed at much the same geologic time. However, these other deposits have 207Pb/204Pb and 208Pb/204Pb ratios different from Magellan’s, because the U, Th, and Pb were partially mobilized at Magellan before the ore body was formed. Using the ratios 207Pb/204Pb versus 206Pb/204Pb on a graph allows us to discriminate Magellan deposits from these other deposits, and this combination provides better visual resolution than does a graph of 208Pb/204Pb versus 206Pb/204Pb or even of the more abundant ratios 208Pb/206Pb versus 207Pb/206Pb.The isotopic signature of the Magellan ore is shown by the black cross on the left side of It is possible to calculate the contributions of PbB from different sources using well-established methods from isotope geochemistry assuming two-component mixing . More coa) most of the PbB data that intersects the Magellan ore value and includes the subjects with the lowest PbB of < 1.5 μg/dL  another array  end point is uncertain because no attempt so far has been made to identify and isotopically characterize other Pb sources such as paint, hobbies, and auto repairs in Esperance. The low-Magellan end point has been arbitrarily assigned a value of 17.8 for the n = 49) and geometric mean of 6.6 μg/dL. This compares with the mean value of 3.0 μg/dL obtained for 404 children < 5 years of age in the wider DoH survey , or myriad others. Unfortunately, no environmental samples from individual houses were available for isotopic analysis.The isotopic data for three children lie between the Magellan and older arrays. PbB levels in these three children ranged from 12 to 25 μg/dL, which suggests that there is another source (or sources) of Pb that has an isotopic composition probably lying on the older array with a R2 = 0.12, p = 0.018) of increasing PbB and proportion of Magellan Pb . The data for two subjects, however, could possibly lie on the Magellan array. The isotopic compositions suggest that another source(s) of Pb is present, such as Pb from maternal bone, paint, roof flashing, tank water, hobbies, do-it-yourself activities, unusual diet, medicines, cosmetics, or occupational activities. No environmental samples from individual houses were available for isotopic analysis.The data for 10 subjects, whose PbB ranged from 1.8 to 14.2 μg/dL, lie on the Magellan array and indicate that in some cases they have been exposed to Magellan Pb; the contribution of Magellan Pb to PbB ranged from 29% to 77%. The data, however, for two of these subjects, with low Magellan contributions of 29% and 40%, lie equally well on the older array, along with the data for subjects W725 and W747.The data for the cord blood samples are consistent with those found in other studies; that is, the isotope ratios in the cord and mothers’ blood are similar . DespiteIt is possible to estimate a background PbB value expected in Esperance if there had been no significant Pb exposure from a Magellan source. Excluding the four children . Twenty-two wipe samples were taken from a variety of locations on or outside dwellings, including windows, doors, beams, cubbyhouses, and other outdoor structures. Pb content in these wipes ranged from 0.16 to 34 μg/cm2 . There are no guidelines for dust wipes apart from those promulgated by U.S. Department of Housing and Urban Development (HUD)  and strong ubiquitous winds have resulted in widespread contamination, although only limited environmental sampling of houses has been undertaken. Limited soil sampling around residential parks and school playgrounds undertaken by the Western Australia DEC showed a highest reading of 88 mg/kg, and most were < 10 mg/kg . The DoHnt (HUD)  for clea2/month were reported from August 2005 to August 2006 (2/30 days (geometric mean).The high percentage of Magellan Pb in one 6-month-old child (93%) and in both parents (~ 76%) would suggest that the inhalation pathway of Pb may have been a significant contributor to the overall PbB level. Furthermore, per unit body weight, ventilation rates are much higher in infants than among children or adults. The family lived within 200 m and downwind of the Pb loading shed. The child was not crawling and exhibited limited if any hand-to-mouth activity, according to the parents. The inhalation pathway is unusual these days, apart from occupational exposure, in contrast to the 1970s and 1980s when leaded gasoline was in use. The hypothesis of an inhalation pathway to complement hand-to-mouth activity in many of the children is reinforced by the nature of the Magellan ore concentrate and the high levels of Pb in air measured by the Port Authority using deposition gauges, which measure the particle deposition from the air over a 30-day period. These gauges showed very high levels of Pb, especially during periods of loading the concentrate onto ships. Values ranging from 14 to 42 mg/must 2006 . No guidThe Pb concentrate from the Magellan mine was originally supposed to be transported by rail as pellets or agglomerates, but the company argued against this because of problems with handling on the wharf . We subjElevated PbB levels are commonly found in mining and smelting communities, although those producing Pb carbonate and/or subject to transportation are less common and isotopic studies even more so. One example where Pb carbonate was produced is in the Pb–zinc–silver mines of Broken Hill, NSW, Australia. Mining began in the 1880s with open pit methods, initially of carbonate ore, which later changed to underground mining of sulfide ore. An inquiry into the prevalence of Pb poisoning and deaths among the Broken Hill community was reported in 1893 . A PbB pp = 0.02). Some of the “control” children had higher PbB than the children of mine employees, probably from exposure to leaded paint, because six of eight houses for the control children were > 50 years of age. Five of eight children of mine employees had PbB contributions from mine Pb > 20%. However, the other three children of mine employees had contributions to their PbB from sources other than mine Pb . This study showed that houses of employees from a Pb mine can be variably contaminated by mine Pb even if they are not near the mine.In an example of “take-home” Pb dust, Pb isotope and concentration data were obtained from venous blood and environmental samples  for eight children of six adult male employees and the adults who worked in a Pb–zinc–copper mine located approximately 40 km from the town . These dn = 20) whose PbB levels were > 19 μg/dL lived in Callao, a port that stored and shipped mineral concentrate; the isotopic results were compared with another group of 10 children from outside Callao whose PbB levels were < 10 μg/dL. The isotopic results for the two groups of children were very different, and both were different from five samples of gasoline; the results for the children from Callao were almost identical to those in the mineral concentrate.This incidence of avian Pb poisoning in a pristine environment came as a shock to the community of Esperance. Although the deaths of so many native birds is a tragedy, their role as “canaries in the coal mine” is fortunate for the community, which may otherwise have been left with a legacy so common in many mining and smelting environments. This would have been exacerbated by the Pb concentrate being in the form of Pb carbonate and of such fine particulate size, combined with the ubiquitous strong winds coming off the Great Southern Ocean, resulting in wide dispersion of the material.The other fortunate aspect of this event is the unusual Pb isotopic signature of the Pb concentrate, which has allowed unequivocal identification of the source and contribution of the Pb concentrate to the blood of children and adults in the community, to the birds, and to the environmental samples. If the signature were less distinct, it would not be possible to apportion sources of the Pb with any certainty where PbB levels are < 5 μg/dL, a potential problem noted previously by The major limitation of this investigation is the lack of isotopic data for environmental samples within individual houses, which would have allowed identification of the other sources of Pb. This is especially the case for the children with the highest PbB levels of > 15 μg/dL and whose isotopic results showed that the overwhelming contribution of Pb in their blood was from sources other than the Pb concentrate. Another limitation is the lack of knowledge of potential sources of Pb besides from the concentrate. None of the subjects were occupationally exposed to Pb, although some of the adults may have undertaken activities that exposed them to Pb, such as renovating older houses with Pb paint or making fishing sinkers.This study has shown that the isotopic analysis of environmental and blood samples can contribute significantly to the investigation of elevated Pb levels. Investigation of such episodes should include the early development of protocols and procedures to enable systematic and valid sampling of environmental and blood specimens for isotopic analysis.


Allium sativum L. is a potential plant for absorption and accumulation of heavy metals. In previous investigations the effects of different concentrations (10-5 to 10-3 M) of Pb were investigated in A. sativum, indicating a significant inhibitory effect on shoot and root growth at 10-3 to 10-4 M Pb. In the present study, we used EM and cytochemistry to investigate ultrastructural alterations, identify the synthesis and distribution of cysteine-rich proteins induced by Pb and explain the possible mechanisms of the Pb-induced cellular defense system in A. sativum.Electron microscopy (EM) techniques enable identification of the main accumulations of lead (Pb) in cells and cellular organelles and observations of changes in cell ultrastructure. Although there is extensive literature relating to studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not yet been fully explained. After 1 h of Pb treatment, dictyosomes were accompanied by numerous vesicles within cytoplasm. The endoplasm reticulum (ER) with swollen cisternae was arranged along the cell wall after 2 h. Some flattened cisternae were broken up into small closed vesicles and the nuclear envelope was generally more dilated after 4 h. During 24-36 h, phenomena appeared such as high vacuolization of cytoplasm and electron-dense granules in cell walls, vacuoles, cytoplasm and mitochondrial membranes. Other changes included mitochondrial swelling and loss of cristae, and vacuolization of ER and dictyosomes during 48-72 h. In the Pb-treatment groups, silver grains were observed in cell walls and in cytoplasm, suggesting the Gomori-Swift reaction can indirectly evaluate the Pb effects on plant cells.A. sativum exposed to lower Pb have a rapid and effective defense system, but with the increased level of Pb in the cytosol, cells were seriously injured.Cell walls can immobilize some Pb ions. Cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction. The morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress. Vacuoles are ultimately one of main storage sites of Pb. Root meristematic cells of  Lead (Pb) exists in many forms in natural sources throughout the world. According to the USA Environmental Protection Agency, Pb is one of the most common heavy metal contaminants in aquatic and terrestrial ecosystems and can have adverse effects on growth and metabolism of plants due to direct release into the atmosphere . There hIt is well known that the roots are the main route through which Pb enters plants , and aboAllium sativum L. is a potential plant for absorption and accumulation of heavy metals [-5, 10-4 and 10-3 M) of Pb on growth for 20 d were investigated in hydroponically grown A. sativum. Pb had significant inhibitory effects on shoot growth at high concentrations (10-3 M), on roots at 10-3 and 10-4 M during the entire experiment [A. sativum.Although there is extensive literature relating to cellular levels and physiological studies on the influence of heavy metals on plants, Pb tolerance strategies of plants have not been fully explained yet ,15,17. Ay metals ,23. In aA. sativum grown in control solution and in solutions containing 10-4 M Pb for different durations of time revealed extensive differences. Control cells had typical ultrastructure. Plasma membrane was unfolded with a uniform shape in all parts. Large amounts of rough ER, dictyosomes, mitochondria and ribosomes were immersed in dense cytoplasm. The nuclei with well-stained nucleoplasm and distinct nucleolus were located in the center of cells, whereas vesicles were distributed in root tip cells , indicating that Pb accumulated primarily in roots; the concentration in bulbs and shoots was much lower [Zea mays [Allium cepa [In previous work, the uptake and accumulation of Pb in ch lower . When Pbch lower . The ultZea mays  and Alliium cepa  revealedium cepa .A. sativum. Pb retention in the roots is based on binding of Pb to ion-exchange sites on the cell wall and extracellular precipitation, mainly in the form of Pb carbonate deposited in the cell wall [Our results here indicated that Pb ions were localized and accumulated in cell walls and vacuoles in ell wall . Once exell wall . Plasma ell wall .It is well known that the ER is the principal site of membrane synthesis within the cell. It appears to give rise to vacuolar and microbody membranes, as well as to the cisternae of dictyosomes in at least some plant cells . Our resThe vacuole is the final destination for practically all toxic substances that plants can be exposed to, and the vacuoles of root cells are the major sites of metal sequestration . CytoplaSalvinia minima was a direct response to Pb accumulation, and PCs participate as one of the mechanisms to cope with Pb in this Pb-hyperaccumulator aquatic fern [Tolerance to metal stress relies on the plant's capacity to detoxify metals that have entered the cell. Inside cells, plant protection against metal toxicity involves synthesis of PCs and related peptides, organic acids and their derivatives . Chelatitic fern . PC bindtic fern .Stichococcus bacilaris, PCs were detected after only 30 min of Cd exposure. In the presence of excess metals, PCs are formed and effectively capture metals [Pisum sativum, Vicia faba and Phaseolus vulgaris [P. sativum, despite the fact that this plant had a medium-tolerance index value, while the concentration of PCs in the roots of V. faba was much lower but their induction took place after only 2 h. The results showed that the rapid initiation of this cytoplasmic detoxification system, which consists of PCs, could transport Pb-PC complexes through the cytosol into vacuoles at lower concentrations of heavy metals [The histochemical test by Gomori-Swift reaction is highly sensitive and allows the detection of cysteine-rich proteins where toxic elements were usually detected . Evidence metals . Piechalvulgaris . They foy metals . Thus thy metals .A. sativum exposed to low Pb concentrations have a rapid and effective defense system, but at increased levels of Pb in the cytosol, cells are seriously injured.The results of the present and previous studies strongly suggest that: (1) cell walls, a first barrier against Pb stress, can immobilize some Pb ions. The cysteine-rich proteins in cell walls were confirmed by the Gomori-Swift reaction; (2) the morphological alterations in plasma membrane, dictyosomes and ER reflect the features of detoxification and tolerance under Pb stress; and (3) vacuoles are ultimately one of the main storage sites of Pb. Thus, root meristematic cells of Allium sativum L. were chosen and allowed to form roots in containers of modified Hoagland's nutrient solution [3)2, 5 mM KNO3, 1 mM KH2PO4, 50 μM H3BO3, 1 mM MgSO4, 4.5 μM MnCl2, 3.8 μM ZnSO4, 0.3 μM CuSO4, 0.1 mM (NH4)6Mo7O24 and 10 μM FeEDTA at pH 5.5. Pb was provided as lead nitrate (Pb(NO3)2). The controls were grown on Hoagland solution alone. Seedlings were exposed to 10-4 M Pb for 1, 2, 4, 8, 12, 24, 36, 48 and 72 h.Healthy and equal-sized cloves of solution . Plants The terminal portion (about 2 mm) of each root of the control and the treated groups were cut and fixed in a mixture of 2% formaldehyde and 2.5% glutaraldehyde in 0.2 M phosphate buffer (pH 7.2) for 2 h and then thoroughly washed with the same buffer three times. This was followed by post-fixation with 2% osmium tetroxide in the same buffer for 2 h. They were dehydrated in an acetone series, and embedded in Spurr's ERL resin. For ultrastructural observations, ultrathin sections of 75-nm thickness were cut on an ultramicrotome  with a diamond knife, and were mounted in copper grids with 300 square mesh. The sections were stained with 2% uranyl acetate for 50 min and lead citrate for 15 min. Observation and photography were accomplished by transmission electron microscopy .The Gomori-Swift test was used in the present investigation to detect whether cysteine-rich protein was induced under Pb stress.Sections of 100-nm thickness from fixed material were cut and mounted on gold grids. The Gomori-Swift reaction was performed in the solution obtained by mixing two components just before staining. Solution A containing 5 mL of 5% silver nitrate and 100 mL of 3% hexamethylenetetramine, and solution B consisting of 10 mL of 1 × 44% boric acid and 100 mL of 1 × 9% borax were prepared. The final stain was obtained by mixing 25 mL of A, 5 mL of B and 25 mL of distilled water ,36.The grids were floated in the silver methenamine solution for 90 min at 45°C in the dark, and then washed four times for 2 min. The grids were then floated on 10% sodium thiosulfate solution for 1 h at room temperature to dissolve metallic silver and rinsed in deionized water four times for 2 min. The sections were continuously stained with uranyl acetate and lead citrate.Controls were carried out to block SH and SS groups by the reduction of disulfide bonds in benzylmercaptan, followed by alkylation of SH groups in iodacetate boric acid. The procedures were described by Swift  and Liu WJ carried out the present investigation, participated in sample preparation and observation and drafted the manuscript. DL conceived the study, and participated in its design and coordination and revised the manuscript. All authors read and approved the final manuscript.


The delivery of ubiquitinated proteins to the proteasome for degradation is a key step in the regulation of the ubiquitin-proteasome pathway, yet the mechanisms underlying this step are not understood in detail. The Rad23 family of proteins is known to bind ubiquitinated proteins through its two ubiquitin-associated (UBA) domains, and may participate in the delivery of ubiquitinated proteins to the proteasome through docking via the Rad23 ubiquitin-like (UBL) domain.In this study, we investigate how the interaction between the UBL and UBA domains may modulate ubiquitin recognition and the delivery of ubiquitinated proteins to the proteasome by autoinhibition. We have explored a competitive binding model using specific mutations in the UBL domain. Disrupting the intramolecular UBL-UBA domain interactions in HHR23A indeed potentiates ubiquitin-binding. Additionally, the analogous surface on the Rad23 UBL domain overlaps with that required for interaction with both proteasomes and the ubiquitin ligase Ufd2. We have found that mutation of residues on this surface affects the ability of Rad23 to deliver ubiquitinated proteins to the proteasome.We conclude that the competition of ubiquitin-proteasome pathway components for surfaces on Rad23 is important for the role of the Rad23 family proteins in proteasomal targeting. Targeted protein degradation by the ubiquitin-proteasome pathway is a key means of regulating a wide variety of cellular processes, ranging from cell cycle progression  to antigRecent work indicates that ubiquitin receptors, which bind ubiquitin but are not intrinsic subunits of the proteasome, facilitate the docking of ubiquitinated substrates to the proteasome -8. The bEach Rad23 family member has a ubiquitin-like (UBL) domain that binds proteasomes -13 as weTo determine how UBL and UBA domain interactions contribute to Rad23/HHR23A function in ubiquitin-mediated proteolysis, we identified mutations that disrupt UBL-UBA binding, then tested the ability of the mutant proteins to bind components of the ubiquitin-proteasome pathway and to mediate delivery of a ubiquitinated substrate to the proteasome. Our results show that the interactions of the UBL and UBA domains with each other and with other proteins are interdependent, and that modulating proteasome-binding is important for the role of Rad23/HHR23 in proteasomal targeting.To identify UBL mutations that affect UBA-binding, we established an affinity column chromatography assay using resin-bound UBA domains and mobile ligands. To demonstrate that our assay can distinguish proteins based on their relative affinities for the UBA domains, control experiments were performed with ubiquitin and SUMO. Both proteins are similar in size and structure to the UBL domain but ubiquitin binds the UBA domains whereas SUMO does not ,15,21. ETo abrogate UBL-UBA binding, we mutated residues located on the UBA-binding surface of HHR23A  that areTo determine the affinity of these UBL mutants for UBA domains relative to wild-type UBL, UBL domain constructs were mutated accordingly and tested in our affinity column chromatography assay. As shown in Figure The ubiquitin-binding surface on the UBA domains overlaps significantly with that involved in binding the UBL domain ,20. DisrThe HHR23A UBL mutants were then tested as competitors in the GST-pulldown competition assay. All the bands in the polyubiquitin Western blot are conjugates of free ubiquitin, but rather than quantify a smear, we quantified the specific band corresponding to tetra-ubiquitin, which is the minimum length required for proteasomal targeting . The amoIn contrast, the UBA domain double mutant L198A/L355A did not compete with GST-HHR23A for polyubiquitin binding. The UBL domain mutants L10E/K47E and K47E/T77E, which showed reduced UBA-binding Figure , competeIn addition to regulating ubiquitin-binding, UBL-UBA domain interactions could also affect proteasome-binding such that it is enhanced when the HHR23 proteins are bound to ubiquitinated proteins. For this hypothesis to be true, the proteasome must bind a surface on the UBL domain overlapping with that which binds the UBA domains. Indeed, the UBL domain is necessary and sufficient for interaction with the proteasome  and the Saccharomyces cerevisiae as a model system as it is better-characterized and easier to manipulate genetically.Just as ubiquitin itself binds multiple subunits of the proteasome, including Rpn10/S5a and Rpt6/S6' ,27, so c32P-labelled Rad23 proteins and purified yeast proteasomes in a electrophoretic mobility shift assay. In the presence of proteasomes, the mobility of wild-type Rad23, but not Rad23~ΔUBL, decreased, as indicated by the arrowheads in Figure in vitro. This finding provides strong evidence that Rad23 binds the proteasome via the same surface that is responsible for S5a- and UBA-binding in HHR23A.L10, K47 and T77 in HHR23A correspond to F9, K43 and S73 in Rad23 respectively, as determined by sequence alignment. We mutated these Rad23 residues to glutamic acid as we had done for HHR23A. To test proteasome-binding, we used recombinant, purified In addition to the proteasome, the Rad23/HHR23A UBL domain binds other proteins involved in ubiquitin-mediated degradation, including the ubiquitin ligase Ufd2 ,28. We tin vitro in the presence of a 20S proteolytic inhibitor such as epoxomicin, and can be used as a functional readout for recruitment of model ubiquitinated substrates to purified 26S proteasomes ATP (NEN) and heart muscle kinase according to the manufacturer's instructions (Pharmacia Biotech). The radiolabelled proteins were cleaved from the resin with thrombin or PreScission protease, then quantified by scintillation counting and by Bradford assay. The specific activities of the proteins were normalized and mixed with proteasomes in a 1:50, 1:20, and 1:10 molar excess of proteasome over proteins. After incubation at 30°C for 15 minutes, the mixtures were resolved by 3.5% native PAGE essentially as previously described [Proteasomes were purified from escribed . All Radescribed ,36, thouGlutathione sepharose was saturated with purified GST-Ufd2. Purified Rad23 proteins were then added together with 50 mM HEPES pH 7.5, 150 mM NaCl, 5 mM EDTA, 2% Triton X-100, 0.2 mg/ml BSA, and protease inhibitor cocktail (Roche). After mixing at 4°C for 60 minutes, the resin was washed with PBS and 0.1% Tween-20. The resin-bound Rad23 proteins were detected by Western blotting using an antibody against Rad23 (a gift from Kiran Madura).in vitro deubiquitination assay was performed as previously described [S. cerevisiae and preincubated with 100 μM epoxomicin for 45 minutes at 30°C to inhibit the protease activity of the proteasome. Ubiquitinated MbpSic1 was then added to the proteasome and its deubiquitination was analyzed by Western blotting for Sic1. Rad23 was added to the deubiquitination assay where indicated.The escribed . EssentiS. cerevisiae, Saccharomyces cerevisiae.UBL, ubiquitin-like; UBA, ubiquitin-associated; His, polyhistidine; Ub, ubiquitin; HHR23, Human Homolog of Rad23; in vitro deubiquitination studies and helped to revise the manuscript. RJD and DF participated in the design of the study and helped to revise the manuscript. PMH participated in the design and coordination of the study and helped to revise the manuscript. All authors read and approved the final manuscript.AMG participated in the design of the study, carried out experiments and drafted the manuscript. KJW created the models of the UBL domains, participated in the design of the study and helped to revise the manuscript. SE participated in the design of the study, contributed reagents, and helped to revise the manuscript. RV performed the Retention of proteins on the GST-HHR23A~ΔUBL column corresponds to their ability to interact with UBA domains. (A) Purified GST-HHR23A~ΔUBL protein was used to saturate glutathione-sepharose resin, which was in turn used to pack an HR10/10 column. Equal amounts of HA-tagged SUMO (HA-SUMO), polyhistidine-tagged ubiquitin (Ub-His) and FLAG-tagged UBL (UBL-FLAG) were loaded simultaneously onto the column and the column was resolved in PBS. Fractions were collected and analyzed by Western blotting using epitope-specific antibodies. (B) Purified GST was bound to glutathione-sepharose resin in excess, which was then used to pack an HR10/10 column. Equal amounts of HA-SUMO, Ub-His and UBL-FLAG were mixed, loaded onto the column and analyzed as described above.Click here for file


Creatinine kinase BB (CK-BB) is elevated in many tumours including those of the breast. We have recently described a new, highly sensitive and specific method for measuring CK-BB, based on monoclonal antibodies and time-resolved fluorometry. Using this method, we quantitated CK-BB in 172 breast tumour cytosols and examined the associations between CK-BB and other clinicopathological variables and patient survival. High CK-BB levels were seen more frequently in tumours from patients who were younger (age < 50 years), patients who qualified for chemotherapy and patients with oestrogen receptor-positive tumours. No association was seen between CK-BB and tumour stage, grade, size, histological type or the progesterone receptor. In univariate analysis, the risk of relapse or death was higher in the group with tumours containing high CK-BB levels but the difference did not reach statistical significance. In multivariate analysis, the risk of death was statistically significantly higher in the high-CK-BB group. Analysis of subsets of patients revealed that patients with oestrogen receptor-negative cancer have higher risk of death if their tumours contain high levels of CK-BB. Our data suggest that, in general, CK-BB is associated with more aggressive tumours but its value as a prognostic indicator is limited. CK-BB content of breast tumours may be more useful as an aid in selecting therapy directed at inhibiting this enzyme activity and thus depriving tumour cells of their energy source.


A few recent studies have demonstrated heightened hypothalamic–pituitary–adrenal (HPA) axis reactivity to acute stress in animals exposed to heavy metal contaminants, particularly lead. However, Pb-induced dysregulation of the HPA axis has not yet been studied in humans.In this study, we examined children’s cortisol response to acute stress (the glucocorticoid product of HPA activation) in relation to low-level prenatal and postnatal Pb exposure.Children’s prenatal blood Pb levels were determined from cord blood specimens, and postnatal lead levels were abstracted from pediatrician and state records. Children’s adrenocortical responses to an acute stressor were measured using assays of salivary cortisol before and after administration of a standard cold pressor task.Pb exposure was not associated with initial salivary cortisol levels. After an acute stressor, however, increasing prenatal and postnatal blood Pb levels were independently associated with significantly heightened salivary cortisol responses.Our results suggest that relatively low prenatal and postnatal blood lead levels—notably those below the 10 μg/dL blood lead level identified by the Centers for Disease Control and Prevention for public health purposes—can alter children’s adrenocortical responses to acute stress. The behavioral and health consequences of this Pb-induced HPA dysregulation in children have yet to be determined. Recent research has evaluated the effects of lead exposure on responses of the hypothalamic–pituitary–adrenal (HPA) axis to acute stress. These responses have been considered in animals, through assessment of glucocorticoid blood levels before and after the onset of an acute stressor. Several studies , 2004 haSeveral studies have considered factors that affect children’s adrenocortical responses to acute stress. Such research has generally focused either on intrinsic differences such as temperament  and attaPb-induced increases in HPA reactivity in children are likely to have far-reaching consequences. The association of trace metal concentrations in blood with cardiovascular disease has been assessed in various epidemiologic studies. Such studies suggest positive associations between Pb and blood pressure , left veIn addition to cardiovascular effects of Pb exposure, low-level Pb exposure is associated with cognitive deficits in children . In somen = 25) or had missing salivary cortisol samples (n = 8). Reasons for not being tested included inability to schedule within the testing window (n = 16), technical problems (n = 4), and refusal (n = 5). Prenatal Pb levels were available for 154 children and postnatal Pb levels were available for 120 children. The child’s response to an acute laboratory stressor was assessed within 2 weeks of attaining 9.5 years of age, and the family was paid $60 for participation in the current visit.Participants were recruited in the context of an ongoing longitudinal study of the effects of environmental toxicants on development . Of the p > 0.50). After the cold pressor task, we administered two cognitive stressors, mirror tracing and reaction time tasks . As previously reported  an initial rest period (10 min); b) a cold pressor task (1 min of submerging the dominant arm in ice water followed by a 2-min recovery); c) an intertask rest (8 min); d) a choice reaction time task (3 min); e) an intertask rest (8 min); f ) a mirror-tracing task ; and g) a final recovery/rest period (10 min).On the day of testing, the participant arrived at the laboratory at about 1630 hours  and before beginning read and signed an assent form, while his or her parents read and signed a separate consent form approved by the Institutional Review Board of SUNY Oswego. The laboratory session began with measurements of the child’s height and weight. Each experimental session comprised the following: 2EDTA) evacuated glass tubes. Over the course of the study, both Vacutainer  and Monoject  tubes were used. However, each lot of blood tubes and needles used, regardless of source, was prescreened for Pb contamination and was certified for blood Pb measurements in the study by the analyzing laboratory. Specimens were shipped to the New York State Department of Health’s Wadsworth Center and were analyzed for Pb and erythrocyte protoporphyrin, a biochemical marker of Pb exposure. Each specimen was analyzed for Pb in duplicate, using a method based on electrothermal atomic absorption spectrometry (ETAAS) with longitudinal Zeeman-effect background correction. The method has been fully validated and is described in detail elsewhere  age of 2.62 ± 1.20 years, through the children’s pediatricians and county public health agencies. New York State law requires blood Pb testing for all children before entering kindergarten, and only those laboratories certified by the New York State Department of Health’s Wadsworth Center can accept and analyze blood Pb specimens drawn in New York State. Although more than 75 laboratories are certified to perform blood Pb testing in New York State, blood specimens are typically sent to a variety of clinical laboratories in both the commercial and public health sectors, depending on local screening practices and health insurance coverage. Analytical techniques used for blood Pb are ETAAS and anodic stripping voltammetry (ASV). Specific laboratories used to analyze specimens from children enrolled in the Oswego Children’s Study included Quest Diagnostics Clinical Labs in Norristown, Pennsylvania, formerly SmithKline Beecham ; Wadsworth Center, Albany, New York ; Oswego Hospital Laboratory, Oswego, New York ; A. Lee Memorial Hospital Laboratory, Fulton, New York ; Quest Diagnostics Inc., Teterboro, New Jersey ; and SUNY Upstate Medical University Clinical Pathology Laboratory, Syracuse, New York . These laboratories participate regularly in the New York State proficiency testing program that is organized by the Wadsworth Center and are certified under both New York State and federal regulations . Blood Pn = 76) or two (n = 35) blood Pb tests; however, a few children had three (n = 9) or more (n = 2) tests before 9 years of age. The median value was used for the children with more than one draw. Blood Pb levels in the Oswego cohort ranged from 1.5 to 13.10 μg/dL, with only six children having blood Pb concentrations > 10 μg/dL, the level defined by the Centers for Disease Control and Prevention (CDC) as elevated for public health purposes , and during recovery (60 min after the cold pressor task). Following in vitro diagnostic measure of adrenal function . The test used 25 μL saliva, had a lower limit of sensitivity of 0.007 μg/dL, a range of sensitivity from 0.007 to 1.8 μg/dL, and average intra-and interassay coefficients of variation of < 5% and 10%, respectively. All samples were assayed in duplicate, and with the average of the duplicates used for all test results. Cortisol units are expressed in micrograms per deciliter. To create a more normal distribution, we eliminated a single outlier  and used a logarithmic transformation  to avoid dairy products for 30 min before collections (restriction based on evidence that some bovine hormones cross-react in immunoassay), b) to rinse their mouths with water 10 min before sample collections (no snacks were provided between sample collections), and c) to not brush their teeth within 1 hr of testing, so as to avoid blood contamination in saliva  and were given instructions to have no snacks during the 1 hr preceding the testing. If the child reported having had a recent snack, the session was either rescheduled or briefly delayed, depending on the timing of the snack. In addition, participants were instructed ng 48 hr .2). A complete list of these variables is provided in To strengthen our inferences regarding children’s blood Pb levels, we considered various potential confounding variables, including characteristics measured during pregnancy, at birth, at 7 years of age, and at 9.5 years of age. Measures included paternal height and weight, maternal prepregnancy weight and height, maternal weight gain during pregnancy, maternal reported illness during pregnancy, obstetric complications , head circumference at birth, birth weight, gestational age, maternal substance use during pregnancy , the Home Observation for Measurement of the Environment (HOME), the Clinical Epidemiological Studies–Depression CES-D;  inventorbis(p-chlorophenyl)ethylene , and hexachlorobenzene. We assessed mercury levels from maternal hair cuttings, with position along hair strands used to differentiate mercury levels during the first and second halves of pregnancy. Further details on these other toxicant measures can be found in prior publications , 1,1-dichoro-2,2-ications .We computed change scores for cortisol by subtracting initial levels from the task means. Therefore, our primary outcome variable was change in cortisol at 21, 40, and 60 min after the initiation of the cold pressor task.p < 0.20) to cortisol responses served as covariates in all analyses. Several studies  between exposure and outcome by ≤ 10%, is an extremely effective and rigorous means of controlling residual bias in multivariate correlational data sets  of that analysis. Only relationships that were statistically significant after this two-tiered approach were considered meaningful.Every analysis, therefore, included all covariates related to the outcome at n = 37), 1.1–1.4 μg/dL , 1.5–1.9 μg/dL , and 2.0–6.3 μg/dL . For postnatal Pb exposure, quartiles corresponded to the following blood Pb levels: 1.5–2.8 μg/dL , 2.9–4.1 μg/dL , 4.2–5.4 μg/dL , and 5.5–13.1 μg/dL . In the analyses of quartiles, we used SAS PROC GLM (SAS Institute Inc.) with a linear contrast to test the effects of increasing blood Pb levels controlling for all potential confounds .Although the distribution of pre-natal and postnatal blood Pb levels was nearly normal, regression models can be sensitive to the presence of outliers. In addition, use in the analyses of a value of one-half the MDL for postnatal blood Pb levels reported as below the MDL, resulted in differing Pb values, as a function of the testing laboratory’s MDL (either 3 or 5). Therefore, as was done previously , we focuF  = 4.79, p < 0.005, as well as postnatal blood Pb levels, F  = 7.35, p < 0.0001. The mean values from these analyses are shown in F  = 3.19, p < 0.05] and postnatal  Pb exposure. To further analyze this interaction, we first considered initial adrenocortical levels as a function of Pb exposure.As a preliminary analysis, we conducted a 4 (blood Pb quartile) × 4  mixed factorial analysis of variance, with the saliva sample being a repeated measure. This analysis was initially performed for prenatal and postnatal Pb using a test for the linear contrast across quartiles, but without covariate control. These analyses revealed a significant interaction between the time of the cortisol sample and prenatal blood Pb levels, F  = 0.01, p > 0.25. Similar results were obtained when prenatal Pb levels were analyzed as a continuous variable, F  = 0.24, p > 0.25. After adjustment for covariates, post-natal Pb quartiles were not significantly associated with initial cortisol levels , nor were postnatal Pb levels when analyzed as a continuous variable . Having established that initial levels of salivary cortisol were unaffected by prenatal and postnatal Pb exposure, we next analyzed changes from initial  as a function of Pb exposure.After adjustment for covariates, pre-natal Pb quartiles were not significantly associated with initial cortisol levels, F  = 11.78, p < 0.001 (for linear contrast). This association was equivalent for samples collected at 21, 40, and 60 min . The adjusted means for this analysis are shown in F  = 18.20, p < 0.0001.Prenatal Pb exposure had a significant effect on cortisol responses to acute stress at 9.5 years of age, with increasing Pb associated with an increasing cortisol response, F  = 11.21, p < 0.005. This association was again equivalent for samples collected at 21, 40, and 60 min . The adjusted means for this analysis are shown in F  = 4.06, p < 0.05.Postnatal Pb exposure had a significant effect on cortisol response to acute stress at 9.5 years of age, with increasing Pb associated with an increasing cortisol response, t  = 2.97, p < 0.005], 40 min , and 60 min . Similarly, postnatal Pb exposure was significantly or marginally significantly associated with cortisol reactivity at 21 min , 40 min , and 60 min , as shown in the lower row of p > 0.10).We next performed regression analyses, with covariates entered first, followed by Pb levels as a continuous variable. As shown in the upper row of p > 0.25). In addition, the interaction between Pb exposure timing  was not significant ; however, we do illustrate the additive nature of prenatal and postnatal Pb exposure on mean cortisol reactivity (adjusted for covariates) in Finally, we tested SES and sex as potential effect modifiers, as found in previous research on Pb-induced changes in glucocorticosteroids . NeitherIn the present study we examined the relationship between children’s prenatal and postnatal Pb exposures and subsequent adrenocortical activity at rest as well as in reaction to acute stress, measured at 9.5 years of age. We found that initial cortisol levels were not significantly associated with either prenatal or postnatal Pb exposure. This null association with basal glucocorticosteroid levels is consistent with findings in Pb-exposed birds , althougThe mechanism(s) for Pb effects on the HPA axis are not well understood. Pb exposure has been shown to affect the brain’s mesocorticolimbic dopaminergic system  and coulA few concerns related to the current study should be noted. First, postnatal Pb was measured in various laboratories that used an MDL of either 3 or 5 μg/dL, whereas prenatal Pb was measured in just one laboratory using a single, well-characterized method  with an MDL of 1 μg/dL. Therefore, the effect of prenatal Pb exposure could have been stronger relative to postnatal Pb effects, because a more sensitive and precise measurement method (and corresponding reductions in measurement uncertainty) was used in a single laboratory for the prenatal exposure assessment.The second concern is the consistent effect of Pb exposure across the 21-, 40-, and 60-min cortisol specimens. If Pb exposure affects only adrenocortical reactivity, and if 60 min is a sufficient length of time for recovery, then Pb exposure effects should have become attenuated by 60 min after the acute stress task. However, the effects of prenatal and postnatal Pb exposure on cortisol reactivity were not significantly different for the three cortisol samples. There are two possible explanations for this pattern of findings. Either 60 min was insufficient for the children’s recovery, or else our initial level did not truly represent resting adrenocortical activity. Although it is not uncommon for salivary cortisol changes to persist even after 60 min e.g., , recoverThat our assessment of postnatal Pb occurred approximately 7 years before our assessment of adrenocortical activity is a third concern. However, multiple measures of Pb over time have been shown in prospective studies of children to be highly correlated e.g., . FurtherThe present study demonstrated that prenatal Pb exposure was associated with a significant increase in the child’s cortisol reactivity to acute stress at 9.5 years of age. Comparable but independent effects on cortisol reactivity were observed for postnatal Pb exposure. Although the long-term implications for this association are unclear, they are undoubtedly complex. Increased HPA axis activity is associated with a number of emotional, behavioral, and physical problems in children . However


SDF1 gene variation, plasma SDF-1alpha level, and the number and function of circulating EPCs. We also tested whether EPC number and function was related to CXCR4 gene variation.Stromal cell-derived factor-1 (SDF1) and its receptor CXC chemokine receptor 4 (CXCR4) play a critical role in progenitor cell homing, mobilization and differentiation. It would be interesting to assess the predictive value of SDF-1alpha level for EPC number, and to ascertain whether there is a relationship between SDF1 and CXCR4 genes, and measured blood SDF1α level as well as EPC number and function. SDF1α levels were correlated with age, gender, alcohol consumption, circulating reticulocyte numbers, and concentrations of matrix metalloproteinase-9, C-reactive protein, cystatin C, fibrinogen and homocytein. In blood samples taken in 2005, EPC number was inversely associated with SDF1α level (p<0.001). EPC number in 2005 was also inversely associated with SDF1α level in 2000 (p = 0.009), suggesting a predictive value of plasma SDF1α level for EPC number. There was an association between the SDF1 gene rs2297630 SNP A/A genotype, increased SDF1α level (p = 0.002) and lower EPC number (p = 0.006).We genotyped a cohort of individuals who participated in the Bruneck Study for single nucleotide polymorphisms (SNPs) in the Our data indicate that a SDF1 gene variation (rs2297630) has an influence on SDF1α level and circulating EPC number, and that plasma SDF1α level is a predictor of EPC number. Asahara and colleagues in 1997 demonstrated for the first time that purified CD34 positive haematopoietic progenitor cells from peripheral blood could differentiate, ex-vivo, into an endothelial phenotype and were named Endothelial Progenitor Cells (EPCs)SDF1 or CXCR4 gene in mice led to early embryonic lethality due to abnormality in the cerebellar and gastrointestinal vasculature and in hematopoiesis developmentSDF1 gene transfer using the adenovirus infection techniqueAnimal studies have indicated that SDF1  and its receptor CXC chemokine receptor 4 (CXCR4) plays a critical role in progenitor cell homing, mobilization and differentiation. Inactivation of the SDF1 gene can have an influence on SDF1α levels+ cell mobilizationSDF1 gene variation, SDF1α level and circulating EPC number. In addition, we tested whether there was also a relationship between variation in the gene encoding the SDF1α receptor CXCR4 and EPC number.There is emerging evidence indicating that variation in the human SDF1 and CXCR4 genes. Blood samples taken in 2000 (n = 684) and 2005 (n = 574) respectively were used for measurements of SDF1α levels. EPC number and function were assessed in blood samples collected in 2005 (n = 571 for EPC number and n = 542 for EPC colonies number respectively). Subjects with and those without EPC number and EPC function assessments did not differ in age, sex and cardiovascular risk factors. The appropriate ethics committees (Autonome Provinz Bozen-Sanitatsbetrieb Bozen Ethikkomittee) approved the study protocol and all study subjects gave their written informed consent before entering the study.The subjects of this study were residents of the Bruneck area in the Bolzano Province of Italy, who participated in the Bruneck StudySystolic and diastolic blood pressure was taken with a standard mercury sphygmomanometer after at least 10 min of rest (mean of three independent measurements). Hypertension was defined as blood pressure ≥140/90 mm Hg or the use of anti-hypertensive drugs. The average number of cigarettes smoked per day was noted for each smoker and ex-smoker. Diabetes was diagnosed according to ADA criteria. Assessment of regular alcohol consumption was performed with a standardized questionnaireand quantified in terms of grams per day (g/day).EPC numbers in the blood samples were determined as described previouslyThe EPC-CFU assay performed as described previouslyCommercially available SDF1α and matrix metalloproteinase-9 (MMP9) ELISA kits  were used to determine plasma SDF1α and MMP9 levels. All ELISA tests were carried out at room temperature on freshly thawed plasma samples. The concentration was determined by comparison with a standard curve, following manufacturer's instruction. Other laboratory parameters and vascular risk factors were all examined by standard methodsSDF1 and CXCR4 genes from the HapMap database (data release in June 2005). Three SNPs in SDF1  and two in CXCR4 (rs16832740 and rs12691874) to capture SNPs with a minor allele frequency >0.2 at these loci and R2>0.8 were typed in this study. In addition, we studied the rs266085 SNP which had been reported to influence SDF1 expressionWe selected tagging SNPs in the eB-transformed. Non-parametric tests yielded very similar findings (data not presented). Associations of EPC number, EPC-CFU and SDF1α level with SDF1 and CXCR4 genotypes were assessed using generalized linear models adjusted for age and sex. Multivariate models additionally included standard cardiovascular risk factors and the factors associated with SDF1α levels . A Bonferroni correction was performed to account for the multiple comparisons (5 SNPs in SDF1 and 2 in CXCR4) performed.The data were analyzed using the SPSS 15.0 and BMDP software packages. Continuous variables were presented as means±SD or medians (interquartile range), and dichotomous variables as percentages. Correlations between EPC number and EPC-CFU, SDF1α level and other parameters were estimated by calculation of crude and partial (age/sex-corrected) Pearson correlation coefficients. Variables with a skewed distribution were logB. Kolmogorov-Smirnov p>0.05). Mean SDF1α levels in 2000 and 2005 were 2652 pg/ml and 2565 pg/ml, with a range of 880 pg/ml to 5595 pg/ml and 1322 pg/ml to 4174 pg/ml, respectively. SDF1α levels in 2000 and 2005 were highly correlated , indicating that the SDF1α level is relatively consistent over time. As expected, both plasma SDF1α level measured in 2000  and 2005  increased with age , indicating that SDF1α has an influence on EPC number in the general population. Importantly, EPC number in 2005 was also associated with SDF1α level in 2000 (p = 0.009), indicating a long-term predictive value of SDF1α level for circulating EPC number (EPC number and EPC-CFU number (per 1 ml blood) displayed a non-normal distribution, with the majority of subjects (more than 75%) having 0 to 1000 EPC and 0 to 340 EPC-CFU, respectively. SDF1α levels were significantly and inversely associated with EPC number (p<0.001) in samples collected in 2005 1C, indSDF1 and CXCR4 SNPs examined in this study are shown in . The genotype distributions were consistent with Hardy-Weinberg equilibrium.Allele and genotype frequencies of the SDF1 rs2297630 SNP was associated with plasma SDF1α level (p = 0.002) and circulating EPC number (p = 0.006), with the A/A genotype associating with higher SDF1α level and lower EPC number  and the relationships of the SDF1α levels at these different time points with circulating EPC numbers. A key finding from our study is the high degree of correlation the two SDF1α measurements in blood samples taken in 2000 and 2005 respectively, suggesting that although SDF1α levels are correlated with cardiovascular risk factors, the levels in an individual are relatively consistent over time. In addition, the study revealed an association between SDF1α level in 2000 and EPC number in 2005, suggesting a long-term predictive value of SDF1α level for EPC number. Another important, novel finding of this study is that there is an association between variation in the + cells in peripheral blood was lower but the level of SDF1α was much higher at 14 days after ischemia, as compared with control miceSDF1 is a member of the chemokine CXC subfamily originally isolated from murine bone marrow stromal cells and is expressed also by stromal cells of various tissuesSDF1 gene. In particular, we found that SDF1α level and circulating EPC number are associated with the SDF1 gene rs2297630 SNP in the Bruneck study cohort. A potential interpretation for this novel finding is that variation in the SDF1 gene can influence EPC number via an effect on the level of SDF1α. The rs2297630 SNP is located in intron 3 of the SDF1 gene. It is possible that the association of this SNP with SDF1α level and circulating EPC number has arisen from a direct functional effect of this SNP on SDF1 expression or mRNA splicing. Alternatively, the rs2297630 SNP might be a functionally neutral marker that is in linkage disequilibrium with a functional polymorphism located elsewhere at the SDF1 locus.A recent study showed that there is significant correlation in the number of circulating EPCs between parents and their offsprings, leading to the hypothesis that EPC number is, at least in part, genetically regulatedSDF1 SNPs, rs266085 and rs1801157, have been reported to be associated with SDF1 levels+ cell mobilizationSDF1 locus due to linkage disequilibrium. This possibility is consistent with the fact that the SDF1 gene is transcribed into two isoforms, SDF1α and SDF1β, and that the rs1801157 SNP is located in the 3′ untranslated region of the SDF1β transcript but not in the SDF1α transcriptTwo other Some particular attentions should be drawn regarding our findings. Firstly, the consideration of the nature of EPC is important, since there is no a unique criteria for identification of EPC, yet. Immense attention regarding the definition of EPC has recently been drawn due to the fact that the characterization and function of EPC isolated with different methodologies were quite different from each otherIn summary, our data indicate that although the SDF1 level and EPC number may both increase in response to acute ischaemic events, the relationship between blood SDF1 level and EPC number is complex and they are inversely correlated in the normal situation as observed in the Bruneck study cohort which was recruited from the general population. Our data also indicate that circulating EPC number is influenced by variation in the SDF1 gene likely via an effect of the genetic variation on SDF1α expression. These findings help understand the mechanisms underlying the inter-individual variability in EPC number which has an implication in the pathogenesis of atherosclerosis and other cardiovascular diseases.