Resource defense behavior is often explained by the spatial and temporal distribution of resources. However, factors such as competition, habitat complexity, and individual space use may also affect the capacity of individuals to defend and monopolize resources. Yet, studies frequently focus on one or two factors, overlooking the complexity found in natural settings. Here, we addressed defense and monopolization of nectar feeders in a population of free-ranging ruby-throated hummingbirds marked with passive integrated transponder (PIT tags). Our study system consisted of a 44 ha systematic grid of 45 feeders equipped with PIT tag detectors recording every visit made at feeders. We modeled the number of visits by competitors (NVC) at feeders in response to space use by a focal individual potentially defending a feeder, number of competitors, nectar sucrose concentration, and habitat visibility. Individuals who were more concentrated at certain feeders on a given day and who were more stable in their use of the grid throughout the season gained higher exclusivity in the use of those feeders on that day, especially for males competing against males. The level of spatial concentration at feeders and its negative effect on NVC was, however, highly variable among individuals, suggesting a continuum in resource defense strategies. Although the apparent capacity to defend feeders was not affected by competition or nectar sucrose concentration, the level of monopolization decreased with increasing number of competitors and higher nectar quality. Defense was enhanced by visibility near feeders, but only in forested habitats. The reverse effect of visibility in open habitats was more difficult to interpret as it was probably confounded by perch availability, from which a bird can defend its feeder. Our study is among the first to quantify the joint use of food resource by overlapping individuals unconstrained in their use of space. Our results show the importance of accounting for variation in space use among individuals as it translated into varying levels of defense and monopolization of feeders regardless of food resource distribution. Resource defense behaviors, such as territoriality and temporary defense of food patches, are often explained by the spatial and temporal distribution of food resources Brown . When foThe ability of individuals to defend resources or space containing them can be influenced by many other factors (Maher and Lott Except for certain studies on fishes (Hamilton and Dill Archilochus colubris) to quantify how resource monopolization and the capacity of individuals to defend food resources are influenced by competition, habitat structure, and the use of space by individuals. Our study system consists of a systematic grid of artificial feeders setup in the wild where feeders are equipped with radio-frequency identification detectors (RFID) and individuals marked with passive integrated transponders (PIT tags).In this study, we took advantage of a new technique to mark ruby-throated hummingbirds  at a given feeder in response to the number of competitors, feeder visibility, and space use by the focal individual. NVC is a measure of competitor access to food and can thus be used to quantify the level of monopolization experienced by a focal individual at a given feeder, with a higher NVC meaning a lower level of monopolization. Furthermore, the extent by which the presence of a focal individual lowers the NVC can be interpreted as a measure of its capacity or motivation to exclude competitors. Therefore, the monopolization and the capacity to defend feeders can be differentiated. For instance, if habitat preferences in hummingbirds cause a disproportionate use of feeders in open habitats compared to feeders in forest habitats, a higher NVC could be observed in open habitats strictly because of habitat preferences, resulting in lower feeder monopolization. Yet, through its defense or differential use of feeders, a focal individual could be excluding a greater number or proportion of intruders in these habitats, indicating a better defense capacity, even though the resulting level of monopolization by the individual might be lower compared to an individual defending a feeder located in a poorer habitat. We thus differentiate between monopolization and capacity to defend feeders to test four hypotheses of resource defense theory: (1) the number of competitors reduces the capacity of individuals to defend feeders and increases NVC; (2) higher visibility improves defense capacity, but also causes a higher NVC linked to habitat preferences; (3) higher spatial concentration and stability of focal individuals in their use of feeders lead to a lower NVC. We also manipulated sugar concentration in feeders to assess the influence of resource quality on monopolization and defense of feeders. Specifically, we tested the hypothesis that (4) high food quality leads to greater defense of feeders, but results in lower monopolization because of higher competition for high-quality feeders.We conducted field work during the breeding seasons (20 May–30 August) of 2007–2009 in Cleveland County, Quebec, Canada . Our study system consisted of a grid of 45 feeders distributed systematically over 44 ha Fig. . FeedersFeeders  were red and included a single opening mimicking a yellow flower. They were installed on metal poles at a height of 1.3–1.7 m and covered by an aluminum plate to prevent evaporation and excessive heating from direct sunlight. We changed feeders once a week by sterilized ones filled with a solution of 20% (W/V of solvent) of sucrose, a concentration similar to the nectar of natural flowers Baker . Each feThe ruby-throated hummingbird is known to aggressively defend natural and artificial food sources, especially in the case of adult males  or Hall traps , although all adults were included in the calculation of the number of competitors  and NVC.Competitors should not be restricted in their access to a feeder unless it is defended. We thus expected that spatial concentration would have no effect on NVC if an individual did not defend feeders. On the other hand, we expected a negative effect of spatial concentration on NVC for an individual that aggressively defends feeders. Assuming that spatial concentration reflects the importance of a feeder to an individual, the more a defending individual is concentrated at a given feeder, the greater should be its negative impact on NVC at that feeder. The effect of spatial concentration on NVC thus reflects, at least partially, the ability and/or the willingness to defend of an individual, and the strength of this effect represents the degree or the effectiveness of its defense. Therefore, looking at the effect of different variables on the ability of individuals to efficiently defend feeders required that we include interactions between spatial concentration and variables thought to affect this ability in our models. For instance, if the ability of individuals to defend feeders is reduced by the number of competitors, the negative effect of spatial concentration on NVC should be weaker when the number of competitors is high. We did not categorize individuals as either territorial or nonterritorial because of the difficulty of identifying and tracking individuals in the field by sight, and more importantly, because we feel the dichotomy between these two extremes is too rigid. Indeed, it is well known that hummingbirds can adjust their intensity of defense in response to the quality of their territories or the quality of their defended resources . It was defined as the distance (m) at which ≥90% of a banner (width = 30 cm) located at the feeder was visible by an approaching observer. This measure was evaluated by the same observer at all feeders and averaged over the four cardinal directions. We assumed that feeders located in openings were in high-visibility environments, independently of lateral visibility, while lateral visibility in closed environments was a better indicator of general visibility for a hummingbird at the feeder. This assumption was made because hummingbirds usually perch high in open habitats and thereby have an overview of feeders unobstructed by the low shrubby vegetation that may occur in these habitats. In order to model the influence of visibility on the relationship between spatial concentration and NVC, we thus had to include a three-way interaction among openness, lateral visibility, and spatial concentration. Indeed, we expected the negative effect of spatial concentration on NVC to be independent of lateral visibility in open habitats, but to increase with lateral visibility in closed habitats.We assessed visibility around feeders using two habitat variables. First, each feeder was categorized as being in an opening or not. An opening was defined as a >50 mSelasphorous rufus) and broad-tailed hummingbirds (S. platycercus) at feeders filled with a 30% (W/V) sucrose nectar compared to feeders containing a 20% sucrose nectar. We are therefore confident that a 35% solution represents higher quality nectar compared to a 20% solution. Our manipulation was divided into three blocks of 3 weeks each, lasting from 10 June to 11 August. Each week, we randomly assigned the high concentration to 15 of the 45 feeders with the constraint that every feeder had to be of high concentration exactly once throughout a block, ensuring a complete coverage of feeders within 3 weeks. As the low-concentration treatment corresponded to the standard sucrose concentration found in the first 2 years, we ran analyses using observations from all 3 years  while assigning the low-concentration treatment to all feeders of 2007 and 2008; analyses performed exclusively on the 2009 data gave very similar effect sizes, yet with larger standard errors as expected from the lower number of observations.We manipulated feeder quality in 2009 by increasing the sucrose concentration of some feeders from 20 to 35%. Previous studies showed that hummingbirds preferred nectar of high sucrose concentration and that this preference peaked somewhere between 40 and 65% across studies , suggesting that feeder attraction was maintained through time. We restricted analyses to adults as juveniles were detected only at the beginning of August and most stayed on the grid only for a few days. We were not able to consider the age of individuals, which might affect territorial behavior or dominance  following Burnham and Anderson  in relation to a focal individual, with feeder ID and focal individual ID as random effects. Competitors were defined in relation to a focal individual at a given feeder. Hence, every individual that visited a given feeder on a given day was in turn considered as a focal individual potentially defending a feeder while other visitors were considered as its competitors in the calculation of NVC. Although unmarked individuals could occasionally be seen at feeders, the correlation between the yearly mean number of visits at feeders detected through PIT tags and the yearly mean number of visits and pursuits detected during standardized bi-weekly focal observations is 0.68, indicating that there is a good relation between hummingbird activity at feeders and what is detected through PIT tags. We also expected individuals to vary in their will and ability to defend feeders and thereby allowed the slope characterizing the effect of spatial concentration on the reduction in NVC to vary as a random parameter across focal individuals. We log-transformed NVC to meet assumptions of normality and homoscedasticity. Cases where only one individual was detected at a feeder on a given day were excluded as there is no variation in NVC in such cases and the lack of a competitor more likely represents a feeder less attractive to hummingbirds than a feeder that is perfectly defended, assuming that a perfect exclusion of competitors is unlikely. The time spent at feeders by competitors was also used as a response variable and results were highly similar. Only analyses based on NVC are thus presented here. We selected models and performed multimodel inference based on the Akaike Information Criterion corrected for small sample sizes (AICAnderson  and VaidAnderson . All modAnderson . Model (P < 0.01). Moreover, most if not all individuals made a disproportionately higher proportion of their visits at a single feeder . In spite of considerable variation among individuals, those results suggest that feeders are used more or less exclusively, that individuals are moderately to highly concentrated in space, and that in most cases, every individual can be linked to a “primary” feeder.Over the 3 breeding seasons, we followed 88 focal males, representing 3326 bird-days and 15 037 bird-feeder-days. Individuals that made the most visits to a given feeder on a given day made a disproportionately higher number of visits than other individuals . Variability among focal males was particularly high, with some individuals showing no, or even a positive effect of spatial concentration on NVC, while others showed a strong reduction in NVC with increasing spatial concentration   when food was very abundant  Ewald  and a smThe variability among male ruby-throated hummingbirds in their spatial concentration and the negative effect of spatial concentration on the number of visits by male competitors show that space use must be taken into account to assess how individuals overlap in their use of resources and/or space. Furthermore, the high individual variation in spatial concentration and its effect and the interaction between spatial concentration and stability suggest that males adopt different strategies with respect to the defense of nectar sources. Indeed, high spatial concentration and high spatial stability in the use of feeders might be indicative of individuals adopting a more territorial strategy while low concentration and low stability could be linked to more sporadic use of feeders or to floating individuals unable to successfully defend a specific area  and found that food monopolization increased in habitats perceived as riskier compared to safer habitats of similar visibility. However, males in our study area often used fully exposed perches and chased competitors high in the air and far from cover. Moreover, males made a disproportionate use of feeders in open habitats (Table An alternative explanation for the positive effect of habitat visibility on defense efficiency could be that predation risk may be higher in high-visibility environments and that few individuals are willing to defend food sources or challenge defenders under such a risk. Hamilton and Dill  tested tts Table , suggestts Table , susceptts Table .Impatiens capensis (Bertin Sphyrapicus varius; Southwick and Southwick As nectar sucrose concentration was maintained constant, at least in the first 2 years of the study, differential use of feeders was ultimately determined by surrounding habitat structure. One variable not necessarily related to habitat structure that could have influenced feeder use is the availability of natural food sources. Negative correlations between flower abundance and feeder use have been observed in other hummingbird species (Inouye et al. s Bertin , was in Individuals are often constrained to feed from a single food patch in spatially confined, experimental laboratory setups to facilitate the measurement of resource defense and monopolization (e.g., Chapman and Kramer Our study shows the importance of studying territorial and resource defense behaviors within a multivariate context as many variables will influence the level to which animals can monopolize space or food resources. Moreover, our study highlights the importance of considering the space use of individuals as it strongly affected feeder monopolization by male ruby-throated hummingbirds in our system. Territoriality is often viewed as a static behavior whereby individuals rarely leave their territories. However, studies show that territorial animals often have a more complex space use than what is assumed by such a rigid view of territoriality and that factors other than food resource distribution can affect this use (e.g., Sikkel and Kramer


In contrast with human T-cell leukemia virus type 1 (HTLV-1) that causes ATL (adult T-cell leukemia), HTLV-2 has not been causally linked to malignant disease. The minus strand of the HTLV genomes encode the regulatory proteins HTLV-1 bZIP factor (HBZ) for HTLV-1 and antisense protein of HTLV-2 (APH-2) for HTLV-2. Unlike the viral proteins Tax1 and Tax2, both HBZ and APH-2 are constitutively expressed in infected cells suggesting that they may play important roles in the pathogenesis of these viruses. To date, very little is known about the function of APH-2 except that it inhibits Tax2-mediated transcription of HTLV-2 genes. In the present study, we investigated the role of APH-2 in basal and Tax2B-mediated activation of the AP-1 pathway.in vivo and in vitro and APH-2 acts as an inhibitor of Tax2-mediated activation of AP-1 transcription.We demonstrate that, unlike HBZ, APH-2 stimulates basal AP-1 transcription by interacting with c-Jun and JunB through its non-conventional bZIP domain. In addition, when Tax2 and APH-2 are co-expressed, they physically interact This report is the first to document that HTLV-2 can modulate the AP-1 pathway. Altogether our results reveal that, in contrast with HBZ, APH-2 regulates AP-1 activity in a Tax2-dependant manner. As the AP-1 pathway is involved in numerous cellular functions susceptible to affect the life cycle of the virus, these distinct biological properties between HBZ and APH-2 may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2. Thirty years after the discovery of the first human oncogenic virus, the human T-cell leukemia virus (HTLV) family of retroviruses is now composed of four members: the well documented HTLV-1 and HTLV-2 and the recently discovered HTLV-3 and HTLV-4 ,6. The rIn addition to the structural and enzymatic proteins common to all retroviruses, HTLV-1 also encodes regulatory proteins such as Tax1. The HTLV-1 Tax protein is a transcriptional activator that regulates HTLV-1 gene expression but also modulates the expression of numerous cellular genes through activation of cellular transcription factors including NF-κB , CREB 1-16, SRF AP-1 consists of a myriad of homo- or hetero- dimers that belong to the Jun, Fos, Maf and ATF subfamilies. All AP-1 family members harbour a basic leucine zipper (bZIP) motif, which consists of a DNA binding domain rich in basic amino acids adjacent to a leucine zipper structure required for protein-protein dimerization . AP-1 diTranscription from the 3’ Long Terminal Repeat (LTR) of the HTLV genomes governs the expression of antisense regulatory proteins named HTLV-1 bZIP factor (HBZ) for HTLV-1 , antisenThe role of APH-2 in the pathogenesis of HTLV-2 infection is less defined. To date, only one study reveals that APH-2 does not promote lymphocytosis . APH-2 hIn the present report, we investigated the role of HTLV-2 proteins APH-2 and Tax2B on AP-1 activity. We demonstrated that APH-2 interacts with c-Jun and JunB through its non-canonical bZIP domain and enhances their ability to activate AP-1 transcription. Surprisingly, when APH-2 and Tax2B are co-expressed, APH-2 binds Tax2B and acts as a repressor of Tax2B-mediated activation of AP-1 transcription.Taken together, our results reveal that both APH-2 and Tax2 act as transcription factors that subtly regulate AP-1 transcription. These findings strongly suggest that APH-2 and Tax2 are involved in the regulation of many biological processes involving AP-1 and therefore indirectly help the virus to replicate and/or counteract the host’s immune system.cis-reporter plasmid that contains the luciferase reporter gene driven by a basic promoter element plus seven repeats of AP-1 binding sites. We transfected the luciferase reporter construct together with increasing amounts of HBZ or APH-2 expression vectors into 293T cells  and the LXXLL domain (178–186). In order to further study the interaction between APH-2 and Tax2B, we constructed a FLAG-tagged mutant of APH-2 lacking the C-terminal part of the protein: APH-2 (1–102) Figure . We thenOur report highlights the role of the viral proteins APH-2 and Tax2B in the regulation of AP-1 activity using a minimal promoter harbouring AP-1 binding sites as a model. To examine the physiological relevance of these findings, we investigated the effect of APH-2 and Tax2B on the human collagenase promoter, which contains a TRE ,44. We pThese findings demonstrate that APH-2 acts synergistically with c-Jun and JunB to activate transcription of the collagenase promoter. Taken together our data suggest that the collagenase promoter well illustrates how APH-2 and Tax2B regulate AP-1 activity.HTLV-1 and HTLV-2 differ both in their specific epidemiologic and pathogenic properties. In contrast to HTLV-1 that causes ATL and HAM/TSP, HTLV-2 has not been clearly linked to any disease but has been associated with lymphocyte proliferation and high platelet counts as well as rare cases of chronic neuromyelopathy -9. This The distinct clinical manifestations of HTLV-1 and HTLV-2 can be attributed in part to distinct biological functions of Tax1 and Tax2 . For instax gene is frequently genetically and epigenetically inactivated in ATL cells, suggesting that Tax is not required for the maintenance of the leukemic stage in ATL [Although Tax plays a pivotal role in HTLV-associated transformation of T-cells, the e in ATL ,51. Recee in ATL -26. HBZ,e in ATL . HBZ inte in ATL ,34.In the current study, we reveal that the antisense protein of HTLV-2 (APH-2) also regulates AP-1 activity. However, we postulate that HBZ and APH-2 display opposite effects on AP-1 basal transcription. While HBZ inhibits AP-1-mediated transcription, APH-2 acts as an activator of AP-1 basal activity. Moreover, whereas HBZ has been described as an inhibitor of c-Jun and JunB-mediated transcription ,38, we dWe focused our studies on elucidating the molecular mechanisms involved in the regulation of AP-1 activity by APH-2 and demonstrated that APH-2 physically interacted with c-Jun and JunB through its non-conventional bZIP domain. HBZ harbours a bZIP domain that is involved in numerous biological functions and especially in its interaction with c-Jun and JunB . Interesin vivo. We postulate that APH-2 and JunD might involve a different mechanism of action than APH-2 and c-Jun/JunB to stimulate AP-1 transcription. Further investigation is needed to better understand how APH-2 indirectly stimulates JunD transcriptional activity on AP-1 transcription.Interestingly, as previously described for HBZ , APH-2 cThe development of ATL in HTLV-1 infected patients has been associated with the deregulation of cellular gene transcription. Among all HTLV-1 proteins, Tax1 is known to play a critical role in ATL development by disrupting major cellular signal transduction pathways such as NF-κB and AP-1 ,47. In tHere, we report that APH-2, Tax2B and Jun can form a ternary complex, as there is no competition between Tax2B and c-Jun/JunB for APH-2 binding. Indeed, unlike c-Jun/JunB, APH-2 and Tax2B, interaction does not involve the non-canonical bZIP domain of APH-2. However, this domain, and consequently the interaction between APH-2 and c-Jun/JunB, is crucial to repress Tax function on AP-1 transcription.According to a recent study, the LXXLL motif of APH-2 is involved in its interaction with CREB and in the repression of Tax function on viral genes . SurprisThe human collagenase promoter is an extensively studied AP-1 responsive promoter ,44. HereAPH2 was expressed in vivo in the majority of HTLV-2 carriers, Tax expression was not detected among HTLV-2 carriers with low PVL [A recent study reported that both APH-2 and Tax2 mRNA expression are correlated with HTLV-2 proviral loads (PVL). However, whereas  low PVL . With thThe present study is the first to demonstrate that HTLV-2 deregulates AP-1 activity. Moreover, together with a previous report , we confThe 293T cells (obtained from the ATCC) were cultured under standard tissue culture conditions. The pAP1-Luc and pRL-TK-Renilla plasmids are commercially available at Stratagene and Promega, respectively. The pCAGGS-Tax2B-His was previously described . The pFL293T cells were transiently transfected with the pAP1-Luc or pcollagenase reporter plasmids, different combinations of expression vectors and the pRL-TK-Renilla vector as an internal control using Lipofectamine™ 2000 (Invitrogen). DNA amounts were normalized across samples using the respective empty vectors. Cells were harvested 48 hours post-transfection and processed for luciferase assays  or Western blot.293T cells were transiently transfected with different combinations of expression vectors using Lipofectamine™ 2000 (Invitrogen). DNA amounts were normalized across samples using the respective empty vectors. Protein extracts were prepared 48 hours after transfection  and 400ug of protein extracts were immunoprecipitated with 5ug of anti-FLAG® M2 , anti-Myc , anti-6xHis , anti-c-Jun (Abcam ab31419), anti-JunB (Abcam ab31421) or rabbit IgG (Millipore) overnight at 4°C. Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology) were then added to the samples and incubated for 2 hours at 4°C. Beads were washed 3 times with IPLS buffer , 3 times with IPHS buffer  and twice with IPLS buffer. Samples were then subjected to Western blot.Cell lysates were subjected to SDS-PAGE and analyzed by Western blot using standard procedures. Membranes were probed using the SNAP i.d. system (Millipore). The antibodies used for Western blot are as follows: anti-FLAG® M2 , anti-α-tubulin , anti-Myc , anti-6xHis  and anti-HA .Purified GST or GST-APH-2 fusion proteins were immobilized on Glutathione Sepharose™ 4 Fast Flow beads  and incubated with purified Tax2B-His overnight at 4°C. After extensive washing with GST Wash Buffer (0.5% Triton® X-100 in PBS supplemented with protease inhibitors), bound proteins were eluted using GST Elution Buffer  and separated by Western blot. Tax2B-His was detected with anti-6xHis antibodies .COS-7 cells were plated onto chamber slides and co-transfected with the indicated expression vectors using FuGENE® HD (Promega). One day post-transfection, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.2% Tween/PBS for 10 minutes at room temperature and incubated with a blocking reagent  for 1 hour at room temperature. Tax2B was detected with an anti-Tax2B antibody (Fusion Antibodies) followed by anti-mouse IgG-HRP and Alexa Fluor® 594 tyramide staining (Molecular Probes). c-Jun-Myc was detected using an anti-Myc-HRP antibody  followed by Alexa Fluor® 488 tyramide staining (Molecular Probes). DAPI (Sigma-Aldrich) was used to stain the nuclei. Slides were mounted using the ProLong® Gold Antifade reagent (Invitrogen). Images were acquired with a Zeiss Axio Imager microscope.The authors declare that they have no competing interests.Conceived and design the study: CM, WWH, NS. Performed the experiments: CM. Carried out the immunofluorescence experiments: AMC. Contributed to cloning: TNB. Wrote the paper: CM. Helped to draft the manuscript: WWH, NS. All authors read and approved the final manuscript.APH-2 associates with c-Jun and JunB but not JunD. 293T cells were transiently transfected with the indicated expression plasmids. Two days after transfection, nuclear extracts were immunoprecipitated with the indicated antibodies (IP). The presence of proteins of interest in the immunoprecipitates was visualized by Western blot using the indicated antibodies (WB). (A) APH-2 interacts with c-Jun. (B) APH-2 binds JunB. (C) APH-2 does not interact with JunD.Click here for fileAPH-2ΔbZIP fails to stimulate JunD-mediated transcriptional activity. (A) APH-2ΔbZIP does not bind to JunD. Nuclear extracts from 293T cells transfected with the indicated vectors were prepared 48 hours post-transfection. Co-immunoprecipitations were then performed with His antibodies, followed by Western blotting with the indicated antibodies (WB). (B) APH-2ΔbZIP does not stimulate the transcriptional activity of JunD. The pAP-1 luciferase reporter construct was transiently co-transfected with the indicated expression vectors. Cells were lysed 48 hours post-transfection and processed for luciferase assays and Western blot analysis. The luciferase values represent an average of at least three independent experiments.Click here for fileSubcellular localization of APH-2 and c-Jun in the presence of Tax2B. COS-7 cells were transfected with the indicated plasmids. Cells were fixed and permealized 24 hours post-transfection. The proteins of interest were immunodetected and stained as indicated. Nuclei were stained with DAPI. Immunofluorescence images were obtained with a Zeiss Axio Imager microscope. Representative images of the entire cell population are shown. (A) Tax2B relocates APH-2 to the nuclear periphery. (B) c-Jun relocates Tax2B in the cell nuclei.Click here for filec-Jun and JunB do not compete with Tax2B in its interaction with APH-2. Competition-binding assays were performed with nuclear extracts from 293 T cells overexpressing the indicated tagged-proteins. Co-immunoprecipitations were carried out using the indicated antibodies and the co-immunoprecipitated proteins were detected by Western blot using the indicated antibodies (WB). (A and B) c-Jun and JunB do not affect the interaction between APH-2 and Tax2B.Click here for fileAPH-2 and c-Jun/JunB interaction is independent of Tax2A. (A and B) Tax2A does not affect the interaction between APH-2 and c-Jun/JunB. Competition-binding assays were performed with nuclear extracts from 293 T cells overexpressing the indicated tagged-proteins. Co-immunoprecipitations were carried out using FLAG antibodies and the co-immunoprecipitated proteins were detected by Western blot using the indicated antibodies.Click here for file


Studies evidences that high proviral loads (PVL) are crucial for the leukemogenesis in the development of ATLL. PVL seems to be stable for years in each individual, but varies substantially among subjects. Previous reports suggest that co-infection of HTLV-1 carriers with S. stercoralis may shorten ATLL onset, thus generating a hypothetical “intermediate state”. We explored this hypothesis by comparing the PVL of ATLL patients and HTLV-1/Strongyloides co-infected individuals.4 PBMC. Non-parametric tests were used for the statistical analysis.We determined PVL in three groups: (1) HTLV-1 asymptomatic carriers without a history of Strongyloidiasis [AC] (n=8); (2) virus carriers with previous episodes of Strongyloidiasis [SS] (n=8); and (3) patients with ATLL [AT] (n=8). History of Strongyloidiasis was defined by the patient’s report and a positive result with a ELISA. Peripheral blood mononuclear cells (PBMC) were isolated to measure PVL by Sybr-Green quantitative PCR. PVL was expressed as HTLV-1 tax copy number/10The PVL in AT was significantly higher than that in AC [7375±4492SD vs.1698±106SD][p=0.01]. PVL was also higher in SS than in AC [4746±1712SD vs.1698±106SD][p=0.002]. Interestingly, no differences were found between AT and SS [7375±4492SD vs.4746± 1712SD][p=0.16].Our results support the hypothesis that HTLV-1/S. stercoralis co-infected subjects are at higher risk of developing ATLL as they seem to share with ATLL patients impaired immunological mechanisms to control virus propagation. Further studies are needed to fully characterize the co-infected condition.


In contrast to HTLV-1 which causes adult T-cell leukemia/lymphoma (ATL/ATLL) and HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP), the role of HTLV-2 in human disease is less clearly defined but infection is associated with rare lympho-proliferative and neurological disorders. Transcription from the 3’ LTR of the HTLV-1 and HTLV-2 genomes governs the expression of two antisense regulatory proteins named HBZ (HTLV-1 basic leucine zipper) and APH-2 (antisense protein of HTLV-2), respectively. HBZ possesses a bZIP motif that facilitates its interaction with several cellular bZIP proteins including CREB2 and members of the AP-1 family such as c-Jun, JunB and JunD. These interactions inhibit Tax dependent LTR activation via CREB and AP-1 leading to the suggestion that HBZ counteracts the function of Tax 1 resulting in reduced viral gene expression and the enhancement of persistence in infected cells. Similarly, APH-2 inhibits Tax 2 mediated activation of the HTLV-2 LTR by interacting with CREB despite the absence of a bZIP motif. In the present study we investigated the transcriptional effects of APH-2 on the AP-1 pathway compared to HBZ. We clearly show that APH-2 stimulates basal AP-1-mediated transcription. In contrast to HBZ, we found that APH-2 enhances the ability of c-Jun to activate AP-1 activity. Co-immunoprecipitation assays demonstrate that APH-2 interacts with c-Jun in 293T cells. Furthermore as shown for HBZ, APH-2 down regulates Tax 2B mediated activation of AP-1 activity. These preliminary results suggest that HBZ and APH-2 may have distinct biological properties, which may contribute to the differential pathogenic potential of HTLV-1 and HTLV-2.


Because endosomal crosstalk may be an important systems-level regulator of pathway communication, scaffold proteins that mediate this crosstalk could be potential targets for pharmacological intervention and synthetic engineering.Endocytosis participates in downregulating incoming signals, but ‘signaling endosomes’ may also serve as physical platforms for crosstalk between signaling pathways. Here, we briefly review the role of endosomes in signaling crosstalk and suggest that endosome-associated scaffold proteins mediate this crosstalk. In addition, using a proteome-wide  Xenopus, because this step prevents the phosphorylation and subsequent proteasomal degradation of β-catenin, a key component of the pathway Endocytosis is a cellular process by which cell surface components and extracellular molecules are internalized into lipid vesicles called endosomes . Endocytp14/−− mouse embryonic fibroblasts identified 31 proteins whose regulation was p14 dependent A signaling endosome also serves as a ‘meeting ground’ for signaling components in silico approach, we propose additional scaffolds that have the potential to mediate crosstalk on endosomes, and suggest that these endosomes can serve as a physical platform specifically to mediate signaling crosstalk  distinct signaling pathways. However, we note that, in genetics, the term crosstalk is sometimes used to denote transcriptional connections between genes functioning in different pathways. Although historically pathways have been viewed as discretely linear, with the advent of network biology it became evident that these pathways are densely interconnected via signaling crosstalk. The importance of crosstalk is apparent if we consider that, in contrast to the wide variety of signaling functions and the macroscopic and microscopic diversity of living forms, the number of signaling pathway types is relatively low (a few dozen) Previously, we examined eight biochemically and evolutionarily defined signaling pathways important in development  and found that crosstalk can occur between any two of these pathways Considering the known dynamic and spatial role of endosomes in signaling, we argue that those endosomes that possess scaffolds capable of connecting proteins from different pathways can localize, isolate, and modulate pathway interactions. Accordingly, we term these endosomes ‘crosstalk endosomes’. Scaffold proteins can be recruited to these endosomes by binding to endosome-specific phosphoinositides (PtdIns) and endosome-related proteins  [42]. DiXenopus laevis embryos The earliest group of early endosomes is characterized by the scaffold protein APPL. Numerous studies in recent years have provided striking examples of how endosomally localized APPL regulates the crosstalk specificity of AKT. For example, APPL is important in crosstalk between the insulin and WNT signaling pathways because it interacts directly with AKT (an insulin pathway member) and GSK3-β (a WNT pathway member) on endosomes In canonical EEA1 early endosomes, a novel function of EEA1 is as a scaffold in angiotensin-II induced AKT activation, which induces hypertrophy of vascular smooth muscle cells The HGS (HRS) scaffold protein has recently been shown to play a role in bone morphogenetic protein (BMP) signaling (a specific subtype of the TGF-β pathway) during mouse embryogenesis Given these examples and the findings presented above, we propose that the major mediators of crosstalk on endosomes are the endosome-associated scaffold proteins. Are there more crosstalk-mediating scaffolds on endosomes awaiting discovery?Based on the presented examples, we collected information on domain compositions, protein interactions, and pathway memberships  to identNext, we examined the pathway memberships of signaling proteins interacting with endosome-associated scaffolds to select those that connect interacting proteins from more than one pathway  and idenWe briefly present two scaffolds, AXIN and SARA, which were found in our analysis and could potentially be mediators of crosstalk on endosomes. Both are known scaffolds, but a crosstalk-mediating function is known only for AXIN, whereas endosomal localization is known only for SARA. Based on our compilation, we hypothesize that both scaffolds can be localized on endosomes and may mediate crosstalk by interacting directly with components of multiple pathways.SH3GL1), an SH3-domain-containing endosomal protein. In addition to the role of Endophilin family members in the formation of endocytic vesicles, they may also interact with signaling proteins AXIN was previously described as a master scaffold for multiple signaling pathways , but the functional role of this interaction has not been identified. β-catenin had already been found in endosomal fractions in human embryonic kidney cells and was associated with another endosomal scaffold, APPL1 As described above, SARA is an endosome-specific scaffold important in TGF-β signaling because it binds SMAD2 and SMAD3 [54]It well established that, for a systems-level understanding of signaling network behaviors, one needs to address the dynamic and spatial aspect of cellular signaling to understand how information is dissipated from the cell membrane across the cytoplasm towards the nucleus The reconstruction-based approaches of synthetic biology could be used, for example, to modify natural scaffolds to enable them to form novel protein–protein or protein–lipid interactions and observe whether this changes their crosstalk activity The EGF receptor is an example of a signaling component known to yield different outcomes based on the spatial localization of its activity. Signals emanating from internalized EGF receptors located on the endosome membrane en route to an internal destination can have different outcomes compared with signals initiated by activated EGF receptors at the plasma membrane Drosophila Toll pathway In vivo genetic experiments in Drosophila have confirmed that endocytic trafficking modulates the strength of the transcriptional signal More specifically, transcription factors, which are normally the terminal players in signaling pathways, may utilize distance-based control to modulate the strength and duration of the signal and communication with other pathways; transcription factors activated far from the nucleus  may be inactivated before reaching the nucleus, whereas those that are activated in close proximity to the nucleus  have a better chance of entering the nucleus in their active form. Thus, an important function of endosomes may be the spatiotemporal regulation of transcription factor activation. For instance, on interleukin (IL)-6 induction, endosome-mediated signaling crosstalk can occur between ERK (EGF/MAPK pathway) and STAT3 (JAK/STAT pathway), which facilitates STAT3 interaction with its transcriptional co-activators CBP and p300 p14 gene resulted in decreased expression of the p14 late endosomal scaffold Specificity, strength, and localization are key properties in interpathway communication. Their precise regulation can be maintained by signaling endosomes serving as physical platforms for signaling pathway crosstalk. Based on this, we coined the term ‘crosstalk endosome’ and assume that endosome-associated scaffold proteins may be the principle components mediating the interaction of different pathway components. Regulating the expression of endosomal scaffolds is likely to be an important mechanism for cells to control signal transduction pathways. Therefore, mutation or changes in the expression of endosomal scaffolds may have pathological effects, as demonstrated in a newly identified primary immunodeficiency syndrome in which a point mutation in the 3′ untranslated region (UTR) of the Future studies will reveal whether endosomal crosstalk is a widespread phenomenon or applies to only a limited number of signaling systems. It is intriguing to speculate that ‘crosstalk endosomes’ may constitute a specialized population of endosomes; however, detecting the dynamics and signaling effects of crosstalk endosomes faces several technical challenges . Enginee


MHC-II expression is primarily regulated at the transcriptional level by the AIR-1 gene product CIITA (class II transactivator). Thus, CIITA plays a pivotal role in the triggering of the adaptive immune response against pathogens. Besides this well known function, we recently found that CIITA acts as an endogenous restriction factor against HTLV-1 (human T cell lymphotropic virus type 1) and HTLV-2 oncogenic retroviruses by targeting their viral transactivators Tax-1 and Tax-2, respectively. Here we review our findings on CIITA-mediated inhibition of viral replication and discuss similarities and differences in the molecular mechanisms by which CIITA specifically counteracts the function of Tax-1 and Tax-2 molecules. The dual function of CIITA as a key regulator of adaptive and intrinsic immunity represents a rather unique example of adaptation of host-derived factors against pathogen infections during evolution.The activation of CD4 The gene encoding CIITA and the elucidation of its function as the master regulator of MHC-II gene transcription and, thus, of antigen presentation to CD4boratory . Upon anboratory . This prboratory . CIITA iboratory . The intboratory . CIITA iboratory , 2003. Bboratory .Several years ago, we discovered that CIITA restricts HIV-1 infection by acting at the level of viral replication. The molecular mechanism at the basis of this inhibition is the competition between CIITA and HIV-1 Tat transactivator for the cyclin T1 subunit of the elongation complex P-TEFb , demonstrating that CIITA is a major restriction factor for HTLV-1. As far as HTLV-2 infection, we found that cells of both the T- and B-lineage are less permissive to HTLV-2 replication in the presence of CIITA. In particular, by using the isogenic B cell system, consisting of CIITA-positive Raji cells and its CIITA-negative derivative RJ.2.2.5 .Beside inhibiting HIV-1 transcriptional elongation, CIITA inhibits also the replication of HTLV-1 and HTLV-2. As far as HTLV-1, we demonstrated that exogenously expressed CIITA in 293T cells transfected with the HTLV-1 molecular clone pACH resulted in strong inhibition of HTLV-1 virus production. More importantly, in promonocytic cells endogenously expressed CIITA produced the same effect . Two pheectively , were stRJ.2.2.5 , it was in vivo the virus may replicate preferentially in cells lacking CIITA. In this regard, it is interesting that during dendritic cell (DC) maturation induced by different stimuli , the expression of MHC-II molecules is increased due to an enhanced transport of preformed molecules to the cell surface. In contrast, de novo biosynthesis of MHC-II mRNA is shut off because of the epigenetic silencing of CIITA gene .In searching for the molecular mechanisms through which CIITA inhibits HTLV-1 and HTLV-2 viral replication, we found that CIITA targets the viral transactivators Tax-1 and Tax-2. Indeed, we showed that exogenous CIITA could inhibit the Tax-1- and Tax-2-mediated HTLV LTR transactivation in LTR-driven luciferase gene reporter assays . By usinAccordingly, HTLV-2 replication was found to be strongly suppressed in RJ.2.2.5 B cells stably transfected with the N-terminal 1–321 fragment of CIITA, which localizes mostly in the nucleus. In contrast, the cytoplasmic mutant CIITA 322–1130, which does not contain the minimal inhibitory domain, did not significantly inhibit HTLV-2 expression .Similarly, cytoplasmic CIITA mutants containing the region 64–144 and partially and /or temporarily accumulating in the nucleus, inhibited Tax-1 transactivation. This was observed with a mutant having the internal deletion of the region 253–410 mediating dimerization of CIITA  and accuThis will be particularly relevant for Tax-2, which, differently from Tax-1, exhibits a predominant cytoplasmic distribution with some accumulation in nuclear bodies .in vivo molecular interaction between CIITA and Tax-1/Tax-2 . The region 1–252 mediates both the binding to the transactivators and their functional inhibition, whereas the region 253–410 binds to, but does not inhibit Tax proteins (in vitro (data not shown) indicating that a bridging cell factor might play a role in this interaction or that PTM, that do not occur in vitro, are crucial to promote the binding. Many studies have shown that sumoylation, ubiquitination, acetylation, and phosphorylation play a critical role in the subcellular localization, protein–protein interaction and function of both Tax-1 and Tax-2, revealing similarities and differences between the two transactivators . If this were case, two hypotheses are equally plausible. Tax bound to CIITA is not assembled on the viral promoter and this correlates with the inhibition of Tax-mediated LTR activation . Alternatively, the binding of CIITA to Tax could still permit its recruitment on the LTR, but not its transcription function. CIITA might prevent the interaction of Tax with components of the transcriptional machinery required for HTLV LTR transactivation . In this context, it is intriguing that, as discussed below, the direct interaction of Tax-1 with PCAF (P300/CBP-associated factor), which cooperates with Tax-1 to activate transcription from the LTR .Tax interacts with a multitude of cellular factors, forming the so called Tax interactome, to modulate the expression of viral and host genes . Most ofIn addition, Tax and CIITA share several functional features. Neither are classical DNA-binding transcription factors, but instead interact with a platform of DNA-bound proteins to be recruited to the target promoters. Among them, CREB plays a major role promoting the formation of a multiprotein complex on DNA required for the full transcriptional activation . In partSeveral families of proteins binding to the Y-box sequence have been previously identified . BesidesTable 1). While it is well established that the recruitment of HATs to the 21-bp repeats by Tax plays a critical role in transactivation, it is not clear whether the ATP-dependent chromatin remodeling factor BRG1, also participates in Tax-mediated transactivation. Controversial reports have been published in the past on Tax-1–BRG1 functional interplay. In one study BRG1 was shown to interact with Tax-1 physically and functionally and to enhance its capacity to transactivate LTR promoter  and histone acetyltransferases (HATs) are commonly used by Tax and CIITA  itpromoter . Thus thThe HATs p300, CBP, and PCAF participate with CIITA to the formation of an active MHC-II enhanceosome . MoreoveOverall, the findings discussed above highlight the central role of both Tax and CIITA in transcription through the coordination of enhanceosome complex assembly and the control of transcription initiation and elongation.Figure 4A). Nevertheless, another possibility is that the intrinsic ability of CIITA to interact with Tax-1, could impair Tax-1–PCAF association . Indeed, CIITA decreased the in vivo binding of PCAF to Tax-1 .Besides PCAF, the over-expression of CREB and ATF1 transcription factors restored CIITA-inhibited Tax-1 transactivation. Because the N-terminal region of Tax-1 interacting with CIITA includes CREB-binding site , it is nFigure 4B). It has been reported that NF-Y interacts with PCAF .In this review we provided an update on the anti-viral features that make CIITA a fundamental link between adaptive and intrinsic immunity against HTLV infections. By inducing MHC-II expression and, thus, antigen presentation, CIITA triggers the activation of TH cells, which, in turn, orchestrate the adaptive immune responses against pathogens. In addition, CIITA has a direct inhibitory effect on the replication of HTLV-1 and HTLV-2 retroviruses by suppressing the transcriptional function of their viral transactivators. CIITA exploits different ways to exert this latter inhibitory function: it binds Tax-1 and Tax-2; it specifically modulates the interaction of Tax-1 and Tax-2 with relevant cellular factors; and it affects the subcellular localization of the two viral transactivators. Thus, CIITA seems to have evolved multiple strategies to be more effective in the inhibition of Tax function. It is known that other host restriction factors may target different steps of viral life cycle to block HIV-1 infection. Nevertheless, the restriction is more effective in species other than the human species, because HIV-1 has developed countermeasures against these innate defenses. In contrast, CIITA is a peculiar restriction factor because restricts HIV-1, HTLV-1, and HTLV-2 in their natural host . So far,HTLV-1 and HTLV-2 viruses preferentially infect and replicate in human T lymphocytes, which express MHC-II molecules upon activation. It must be stressed, however, that whereas the expression of MHC-II molecules on the cell surface last for several days, CIITA expression is time-limited because of its very short half-life . Thus, sBesides its role as transcriptional activator of HLTV genome transcription, Tax plays a major role in viral pathogenesis and T cell immortalization . Tax derThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Human T-lymphotropic viruses type 1 (HTLV-1) and type 2 (HTLV-2) present very similar genomic structures but HTLV-1 is more pathogenic than HTLV-2. Is this difference due to their transactivating Tax proteins, Tax-1 and Tax-2, which are responsible for viral and cellular gene activation? Do Tax-1 and Tax-2 differ in their cellular localization and in their interaction pattern with cellular factors? In this review, we summarize Tax-1 and Tax-2 structural and phenotypic properties, their interaction with factors involved in signal transduction and their localization-related behavior within the cell. Special attention will be given to the distinctions between Tax-1 and Tax-2 that likely play an important role in their transactivation activity. HTLV-1 is tropic for CD4 T-cells . In addie genome ,6.in vivo [HTLV-1 and HTLV-2 infectivity and transformation capacity appears to differ from that exerted by other animal retroviruses in that tumor development is not mediated by the presence of a viral oncogene or by the activation of a cellular proto-oncogene. Since HTLV is transmitted essentially through cell to cell contact and both viruses have the capability of inducing T-cell transformation ,8, the ain vivo ,15. Althin vivo ,12,16–19in vivo .+ cells [et al. [Tax-1 and Tax-2 share an amino acid similarity of 85%. However, they present distinct phenotypic differences consistent with a more robust transformation capacity of Tax-1 in comparison to Tax-2 . Compare+ cells , suppres+ cells  and regu+ cells ,25. Tax-+ cells  and to l+ cells . We have+ cells . More re [et al.  have sho [et al.  found th [et al. . Tax-1 e [et al. .2.The protein structure of Tax-1 and Tax-2 is presented as a scheme in The central portion of Tax-1 contains two leucine zipper-like motifs at aa position 116–145 and 213–248 , necessaThis region also presents binding domains for proteins involved in histone methylation, cell cycle progression and in cell signaling transduction ,34,39–42Secretory signals are present at the C-terminal part of Tax-1 that confers to the protein the capability to egress from the endoplasmic reticulum and to migrate to the Golgi apparatus ,54.The presence of a class I PDZ binding motif (PBM) at the C-terminal end of Tax-1 and not in Tax-2 is of particular interest. It allows Tax-1 but not Tax-2B to interact with many cellular factors, mostly of them being scaffolding proteins (reviewed in ).3.Protein-protein interactions represent a relevant issue for deciphering Tax protein functions and its capability to perturb the regulation of cellular pathways. Tax-1 interacts with several factors that are components of the cell signaling system, that control activities strictly linked to cell transformation, including proliferation, intracellular protein distribution, cell migration and virological synapses ,19,55,56The activation of NF-κB pathway is essential for Tax-mediated transformation of human T-cell lines by HTLV-1 and HTLV-2. Both Tax-1 and Tax-2 activate gene expression via NF-κB –72. Of t4.4.1.So far, six Tax-1 residues were identified as phosphorylation targets: Thr-48, Thr-184, Thr-215, Ser-300, Ser-301 and Ser-336 ,82. Seriet al. [Recently, Jeong et al.  providedet al. ,86.in vitro [in vitro.The phosphorylation state of Tax-2B is still unknown, even though it presents 85% homology to Tax-1 for the sequences surrounding the aforementioned phosphorylated residues, which are conserved with the exception of Ser-336. We have recently found that CK2 phosphorylates Tax-1 Ser-336, Ser-344 and Thr-351 in vitro . CompareTax-1 C-terminus, which contains the class I PDZ binding motif  is completely different from that of Tax-2B. Thus it allows Tax-1, but not Tax-2B, to interact with many cellular factors, mostly scaffolding proteins, such as human disc large (hDlg), human homolog Scrib (hScrib), membrane-associated guanylate kinase-3 (MAGI-3), pro-IL16 and Erbin (reviewed in ). DisrupRecently, other PDZ containing proteins that interact with Tax-1 PBM were reported . Of part4.2.Ubiquitination and sumoylation have been shown to play a critical role in the cellular localization, function and protein-protein interactions of both Tax-1 and Tax-2 ,72,94–96Five of ten lysine residues contained in Tax-1 sequence were found to be the major targets for these modifications see . Lys189 The role of ubiquitination and sumoylation in Tax-1 transactivating activity is clearly different. Tax-1 ubiquitination is involved in the activation of NF-κB pathway through the binding with key factors such as IKKγ and TAK-1 binding protein 2 (TAB2), with the subsequent RelA nuclear translocation ,98.et al. [Whether the poly-ubiquitination targets Tax to the proteasome or enhances its activity is still not clear, although Yan et al.  demonstret al. ,100.Other cellular factors have been demonstrated to be important in the post-translational regulation of Tax, such as peptidyl prolyl isomerase (Pin1) . Pin1 prOn the other hand, Tax-1 sumoylation is required to recruit RelA and IKKγ in Tax-1-related nuclear bodies, where Tax-driven transcription is promoted ,94,101.These data suggest that Tax-1 ubiquitination is a key event for controlling Tax-1 cytoplasmic/nuclear functions, whereas Tax-1 sumoylation is essential for its nuclear activity. Nonetheless, the predominance of ubiquitinated forms of Tax in the cytoplasm and of sumoylated forms in nuclear bodies is not a static event and can be affected by various signals. For example, it has been hypothesized that DNA damage regulates post-translational modifications of Tax and, consequently, its protein-protein interaction and localization .Much less information has so far been obtained regarding Tax-2 post-translational modifications. We have recently demonstrated that Tax-2B is ubiquitinated and sumoylated similarly to Tax-1, even though with lower levels of mono-sumoylated, di-sumoylated and tri-sumoylated forms . At vari4.3.Tax-1 acetylation is known to be crucial for activating its functional properties, together with other post-translational modifications . In partTax-1 acetylation results in the promotion of ATF/CREB and NF-κB pathways, probably by the activation of promoters integrated in the chromatin . Tax-1 K5.The pleiotropic activity of Tax proteins is characterized by specific interactions with cellular factors in the nucleus and in the cytoplasm. Consequently, there are specific mechanisms that modulate the cellular trafficking of these proteins. A number of studies have reported nucleo-cytoplasmic shuttling of Tax-1 and functional effects of subcellular localization of Tax, although the regulatory mechanisms of its intracellular distribution remain to be clarified . In a reNuclear import-export of Tax-1 is both carrier and energy independent and relies on the interaction between Tax-1 and the p62 nucleoporin. This interaction is mediated by the amino terminal zinc-finger motif of Tax-1. Several mutations within this motif abolishes Tax1 interaction with p62 and nuclear import .Initial studies on the intracellular distribution of Tax-1 indicated that it was localized essentially in the nucleus ,86,105. However, other studies have reported Tax-1 cytoplasmic localization in a number of cell lines including Tax-1-transfected and HTLV-1-infected cells –108. In cis-Golgi compartment [Tax-1 is accumulated in close association with the Microtubule Organizing Center (MTOC) and is found in the cell-cell contact regions that organize virological synapses formed between an infected donor cell and a target cell ,111. In partment .Tax-2 intracellular localization has been studied by several authors and it was initially reported to be predominantly localized in the cytoplasm ,51,113 wFrom the functional standpoint, it is important to note that the Tax proteins are targeted to native TAB2-containing cytoplasmic structures, which are reorganized to include RelA and calreticulin following expression of Tax proteins . In addi6.Much attention has been dedicated to the understanding of the functional and structural properties of Tax proteins and in particular of Tax-1. All the different facets of its structural domains, its interaction with cellular factors and corresponding deregulation of transduction pathways, its intracellular distribution as well as its post-translational modifications, have been critically and minutely dissected . Much lIn Central to the question of subcellular localization is the issue of post-translational modifications of both proteins. The recent findings that Tax-2 is also modified by ubiquitination and sumoylation  and thatPossible other ways leading to the Golgi, to the MTOC and to secretion, still await further confirmation, especially for Tax-2. The sumoylation process is key to nuclear entry and maintenance, and a prerequisite for Tax localization within the Tax-related nuclear bodies. This post-translational modification appears to represent a subtle difference between the two proteins, inasmuch as only poly-sumoylated forms are clearly evident for Tax-2 . FurtherMore insights and efforts are therefore needed to single out possible differences between Tax-1 and Tax-2 in the post-translational processing and in obtaining a more precise identification of the amino acid residues directly involved in the modifications of the two proteins.


BMC Biology editorial board and a leader in the field of cell signaling. I first met Tony when I was a graduate student in the 1980s, at one of those summer research meetings where science was followed by beer and conversation. I still have vivid memories of Tony, with his shock of white hair and ready smile, chatting into the night with students and other colleagues about his current passion, an obscure tyrosine kinase called Fps. Tony exemplified the investigators working on oncogenes at the time - smart, collegial, and burning to understand how cell signaling worked and how it could go so terribly wrong in cancer. In a few short years Tony emerged from this group to become one of the most influential biologists of the past two decades, shaping the new field of cell signaling.The scientific community was deeply saddened by news of the untimely death of Tony Pawson, a member of the To understand the significance of Tony’s contributions, we need to think back to how little was known at that time. We knew that unrestrained tyrosine kinase activity could stimulate cells to proliferate, and that the receptors for mitogens such as epidermal growth factor and platelet-derived growth factor also had tyrosine kinase activity. These kinases seemed to work by somehow activating additional signaling proteins such as the G protein Ras and serine/threonine kinases, but we knew almost nothing about the mechanism. How were tyrosine kinases regulated, and how did they transmit downstream signals? How could these mechanisms be subverted in cancer?Tony’s lab at the time was focused on Fps, which like its much more celebrated cousin, Src, was an oncogenic tyrosine kinase first identified in an avian sarcoma virus. Tony’s group was interested in what caused the activity of the oncogenic kinases to be unregulated compared with that of their unmutated cellular counterparts, and he zeroed in on a region N-terminal to the catalytic domain. In a landmark 1986 paper with Ivan Sadowski , he coined the term ‘SH2 domain’ for this region, for Src Homology domain 2 - a second region of sequence similarity between Fps and Src, the first being the tyrosine kinase catalytic domain. Soon such ‘homology domains’ began popping up in more and more signaling proteins as they were cloned and sequenced - Ras-GAP, phospholipase C, phosphatidylinositol 3-kinase, and the small oncogenic protein Crk, which seemed to consist of little else. Clearly these small regions had a powerful influence and were widely used in cell signaling, but how might they work?Taking full advantage of new and improved research tools as they became available , Tony’s group and others quickly demonstrated that SH2 domains bound to specific tyrosine-phosphorylated peptides and thus served to relocalize cytosolic effector proteins containing SH2 domains to the membrane upon activation of membrane-associated tyrosine kinases. Other modular signaling domains were soon found to mediate specific interactions with other peptides or lipids. These discoveries were quite stunning at the time, hinting at an entirely new and unexpected paradigm for signaling. But beyond the mere details of mechanism, Tony was the one who saw the bigger picture most clearly - the fundamental importance of protein-protein interactions and subcellular localization as essential currencies of information transfer. He also saw how modular protein and lipid interaction domains provided a credible means for signal transduction pathways to emerge and gain new functions in the course of evolution. More than anyone else, Tony made sense of the new discoveries and revealed the underlying logic for all to see.It is hard to overstate the excitement of those times. The answers to big, important questions came tumbling out one after the other, as disparate pieces of evidence from the fields of biochemistry, genetics, virology, and developmental biology began to fit together in a profoundly satisfying way. And through it all Tony led the way - through the work of his lab, through his beautifully written and deeply insightful review articles, and through his willingness to share what he knew with his colleagues.In the decades that followed these initial discoveries, most of those involved moved on, or focused on ever-smaller pieces of the puzzle. By contrast, Tony took on the more challenging task of building on those results on a grand scale. In his position as director of the Lunenfeld Research Institute in Toronto, he worked to assemble large interdisciplinary teams that were needed to tackle the questions he wanted to address. He was a pioneer in incorporating emerging disciplines and technologies such as bioinformatics and mass spectrometry into signaling research. In short, he made Toronto the center of the signaling universe. He had a big vision and the skills, energy, and intellect to pursue that vision, probably better than anyone else in the field.While Tony’s scientific accomplishments were immense, they cannot explain why so many of us will miss him so keenly. Above all, Tony was a wonderful colleague and friend. I can personally attest that he was always far kinder to me than I had any reason to expect, given my position as a junior investigator directly competing with his lab. He provided wise advice, shared his lab’s results before publication, collaborated when possible, and gladly acknowledged and appreciated the work of others. He was a genuinely selfless and humble man, terms that are not often associated with scientists of Tony’s stature. Although he was aware of his own accomplishments, he never let it go to his head .I got to know Tony better in recent years as we worked together on a textbook of cell signaling. The clarity of his vision and his comprehensive knowledge of the field were the solid foundation upon which the book is built. Sadly, personal tragedies, particularly the wrenching loss of his beloved wife Maggie to cancer, made it more difficult for Tony to focus on science in the past few years. It is a reminder that despite our talents, accomplishments, and essential goodness, we all live close to the edge of disaster. But although we lost Tony far too soon, we can still take joy in all that he accomplished while he was with us: the many trainees, colleagues and friends whose lives he touched, the new insights and discoveries he brought to light, the institutions and ideas that he fostered. Many of us will feel that the world is a far better place because of Tony Pawson.


Xenoestrogens (XEs) are chemicals derived from a variety of natural and anthropogenic sources that can interfere with endogenous estrogens by either mimicking or blocking their responses via non-genomic and/or genomic signaling mechanisms. Disruption of estrogens’ actions through the less-studied non-genomic pathway can alter such functional end points as cell proliferation, peptide hormone release, catecholamine transport, and apoptosis, among others. Studies of potentially adverse effects due to mixtures and to low doses of endocrine-disrupting chemicals have recently become more feasible, though few so far have included actions via the non-genomic pathway. Physiologic estrogens and XEs evoke non-monotonic dose responses, with different compounds having different patterns of actions dependent on concentration and time, making mixture assessments all the more challenging. In order to understand the spectrum of toxicities and their mechanisms, future work should focus on carefully studying individual and mixture components across a range of concentrations and cellular pathways in a variety of tissue types.  Trace levels of industrial and naturally occurring chemicals have been shown to perturb endocrine systems. These endocrine disrupting chemicals (EDCs) are currently the subject of intense research and regulatory action. A large number of these EDCs act via the estrogen receptor (ER), imperfectly mimicking and interfering with the physiologic actions of endogenous estrogens. Xenoestrogens (XEs) can bind to ERs in the cell nucleus, where the complex recognizes DNA response elements and alters gene expression; in the non-genomic pathway XEs can bind to membrane-bound ERs and rapidly initiate signaling cascades that culminate in kinase and phosphatase activations, ultimately influencing cellular function by post-translational modifications of a variety of proteins . Functional consequences observed at the organismal level include decreased fecundity in aquatic organisms, altered sexual behavior and memory in rats, and malformations and decreased mobility of human sperm ,4,5,6,7.In real-world situations, however, humans and wildlife are not exposed to one chemical at a time, but rather to complex mixtures. The potential health hazard from EDC mixtures is one of the most difficult challenges for a regulatory agency to evaluate. Nevertheless, several legislative mandates (Superfund Amendments and Reauthorization Act 1986); Food Quality and Protection Act (1980); Safe Drinking Water Act (1996)) require the U.S. Environmental Protection Agency to examine mixtures of chemicals in regulatory decision making 86; Food ,24,25,26Estrogens, other steroids, and related compounds were traditionally thought to mediate their actions exclusively via nuclear actions, so the name “nuclear receptor” has become exchangeable with “steroid receptor.” ERs are one category of nuclear receptors in the larger family through which EDCs can act. Others are the receptors for compounds such as androgens, thyroid hormones, aryl hydrocarbons, and pregnane-x compounds ,32,33,34The reason for the existence of different ER localizations and subtypes is not yet clear. We have yet to document enough examples of the serpentine-type GPR30 receptor actions to determine if it fits discernible subcategories of functional responses. Different ERs (membrane and nuclear) could exist to accommodate the wide variety of estrogenic molecules with distinct functions , as mERsMembrane-initiated signaling may be a “first alert system” for the cell to eventually prepare for more permanent make-overs . ActivatOur group and others have explored the similarities between the mERα and the intracellular estrogen receptor (iERα) ,46; a clαi subclass of G proteins at the cell membrane. Interactions of ERα and caveolin-I were also demonstrated by epitope proximity ligation studies which supported the idea that these proteins jointly participate in estrogen-induced signaling in the membrane [Other results have also suggested identity of ERα in both subcellular locations by virtue of the proteins with which they associate. Recently, we used Duolink immunofluorescence imaging to visualize the partial co-localization of mERα and the Gmembrane ,49,50,51The mERα was also deemed identical to its nuclear counterpart in MCF7 breast cancer cells by membrane isolation (affinity chromatography) and mass spectrometry analysis . These r2  [2 in its ability to initiate rapid non-genomic responses from membrane receptors [2, coumesterol, and BPA, we have been able to correlate strong and sustained ERK activity together with weak JNK activation responses to the induction of cell proliferation in the GH3/B6/F10 pituitary cell line [BPA and other XEs have been found to be “weak” inducers of estrogenic activity via the genomic pathway in comparison to Eference) . Howevereceptors . Non-geneceptors ,59,60,61ell line ,26—the E2+, K+. Na+, H+) are a common signaling responses to steroids and related compounds, often leading to changes in cell mobility, downstream signaling processes, and peptide hormone secretion [2+ levels (PRL release) upon stimulation with low picomolar and sub-picomolar E2 concentrations [2+ influx were monitored in order to establish a correlation between Ca2+ levels and PRL secretion [2+ fluxes were not non-monotonic, and thus did not explain the bi-modal nature of the changes seen with PRL release. Therefore, for this and other reasons, it is likely that regulation of peptide secretion also involves additional signaling pathways [Ion fluxes , hormesis is defined as “a dose-response phenomenon characterized by low-dose stimulation and high-dose inhibition”[in-vivo or in-vitro models exposed to high concentrations of chemicals uncommonly found in human populations or the environments to which they are exposed [According to the hibition”. The occhibition”. Still,  exposed ,70. Past exposed . However exposed . Further exposed ,71,72,73 exposed . i.e., pERK, pJNK, p38) [−15–10−7 M), MAPK responses eventually decrease [Various theories have been offered as explanations for non-monotonic dose responses; these have been previously reviewed ,77,78,79NK, p38) ,81,82,83NK, p38) ; hence, decrease ,25,26,84decrease ,75,85,86decrease ,73,87,88For mixtures toxicology, the significance of non-monotonicity has not been adequately characterized . This is2), estrone (E1), and estriol (E3) play diverse roles in human and wildlife physiology beyond those required just for reproductive success, affecting metabolism, bone integrity, cardiovascular functions, behavior and mood, and other functions [2. E1 is found at elevated levels (~150–200 pM) during post-menopausal stages, while E3 is high during pregnancy (~10–100 nM); males also have lower development stage-specific blood levels of endogenous estrogens [3 have been linked to complications of eclampsia and an increased probability of Down’s syndrome in offspring [2, these other endogenous estrogens have yielded weak genomic responses [Produced primarily in the testes and ovaries, estrogens such as estradiol ; phytoestrogens may prevent the increased risk of cancer that can occur from taking other estrogenic hormone supplements ,116,117.2 [Unless taken individually as dietary supplements, phytoestrogens are typically found in the diet as chemical mixtures. Therefore the health benefits of resveratrol, for example, could be the result of an additive effect with one of the other hundreds of “minor phenols” found in red wine ,125, and2 . We therAnthropogenic or man-made chemicals with estrogenic capabilities have become abundant pollutants in our environment ; many are by-products of plastics, preservatives, industrial surfactants, and pesticides. Agricultural compounds  have been detected in breast milk, urine, maternal blood, and serum in appreciable amounts, with links to low birth weight, fetal death, and childhood cancers ,127,128.The highly controversial XE, BPA, has also been detected in significant levels (in urine samples) in 93% of U.S. residents ≥ 6 years of age. BPA’s prevalence is due to its incorporation into many manufactured goods that we as consumers use on a daily basis; these include: plastic food and water containers, thermal coatings on cashier paper receipts, linings in canned foods, and dental repair materials, among many others ,136,137.Pesticides/herbicides are perhaps the most studied of this class of chemicals in mixtures, as multiple remedies are often applied against different insect and weed categories simultaneously for efficiency ,147, andconcentration or dose addition method, which assumes that mixture components act on a similar target and therefore elicit a common response; and (2) the response addition method, which assumes that components act on different targets  [Various approaches for evaluating chemical mixtures have been proposed by the scientific community ; howeverponents) ,152. Thei.e., estrogenic, anti-androgenic, and thyroid-disrupting agents) [The overall goal of every method is to establish principles of how chemicals behave based on structure and mode of action. Once enough examples are processed and the model optimized, then predictions for unknown mixtures should be possible. In a review, Kortenkamp extensively discussed mixture effects of several classes of EDCs ( agents) , focusinpERK) upon exposure to binary mixtures of endogenous estrogens  at single physiologic (nM) concentrations with increasing (10−15–10−7 M) concentrations of alkylphenol compounds in the GH3/B6/F10 pituitary cell line [2. The composite responses were not additive, and often showed attenuation at the higher concentrations. The degree of attenuation was based on the response magnitude and potency of the paired xenoestrogen. The stronger the XE’s activating response, the more it was able to attenuate the physiologic estrogenic response. Jeng and Watson studied the phospho-activation of MAPK ; hence, when assessing non-genomic pathways one has to take into consideration the variety of signaling responses, and probably the interactive nature of signaling “webs”. Response inhibitions by combinations of estrogens may be governed by cellular protective mechanisms against combined hormone overstimulation. Overstimulation can be wasteful and even dangerous when the enhanced function  can lead to diseases like cancer.The overall pattern observed in these two studies was that when a compound with a weak response is paired with a physiologic estrogen, the response is enhanced. But, when a compound elicits a potent estrogenic effect, then it inhibits the paired physiologic estrogen’s response. This progression is summarized graphically in pERK response when present by itself did not correlate with a strong PRL secretion. However, PRL release did decrease when BPA was paired with either E2 or E1 (but not the weaker E3) in pituitary cells [Interestingly, not all signaling pathways culminating in different functional responses may behave in the same fashion. When another type of response (PRL release) was monitored under the same (binary mixture) circumstances, BPA’s strong ry cells . This meAssessing enough examples of XE mixtures to establish predictive principles is still largely beyond our current datasets. To do so will undoubtedly require a large training dataset of both genomic and non-genomic cellular responses, including recognition of signaling cross-talk, and resulting functional outcomes. The effects of XEs can vary greatly, depending upon cell, tissue, and organ type. Complications due to the potential non-monotonic nature of responses and variations in the makeup of mixtures will also require systematic evaluation. Until now, the vast majority of XE studies focused solely on a single chemical’s effects without appreciation for the more realistic conditions of chemical mixtures of XEs. Furthermore, while assessing XE effects on humans or wildlife alone or in mixtures, few studies have addressed the impact of XE combinations with endogenous hormones ,24,25. E


Gap junctions are membrane specializations that allow the passage of ions and small molecules from one cell to another. In vertebrates, connexins are the protein subunits that assemble to form gap junctional plaques. Connexin-35 (Cx35) is the fish ortholog of mammalian Cx36, which is enriched in the retina and the brain and has been shown to form neuronal gap junctions. As a first step toward understanding the role of neuronal gap junctions in central nervous system (CNS) development, we describe here the distribution of Cx35 in the CNS during zebrafish development. Cx35 expression is first seen at 1 day post fertilization (dpf) along cell boundaries throughout the nervous system. At 2 dpf, Cx35 immunoreactivity appears in commissures and fiber tracts throughout the CNS and along the edges of the tectal neuropil. In the rhombencephalon, the Mauthner neurons and fiber tracts show strong Cx35 immunoreactivity. As the larva develops, the commissures and fiber tracts continue to be immunoreactive for Cx35. In addition, the area of the tectal neuropil stained increases vastly and tectal commissures are visible. Furthermore, at 4–5 dpf, Cx35 is seen in the habenulae, cerebellum and in radial glia lining the rhombencephalic ventricle. This pattern of Cx35 immunoreactivity is stable at least until 15 dpf. To test whether the Cx35 immunoreactivity seen corresponds to functional gap junctional coupling, we documented the number of dye-coupled neurons in the hindbrain. We found several dye-coupled neurons within the reticulospinal network indicating functional gap junctional connectivity in the developing zebrafish brain. Ever since electrical synapses were first described ] did not exhibit major deficits in physiology or behavior  to Cx36 and has been shown to be expressed and enriched in the CNS. It has been previously demonstrated to be present in gap junctions formed by the Mauthner neuron in goldfish and zebrafish  were obtained from a commercial supplier, housed in a recirculating water system  and used for generating embryos. All procedures were approved by the Institutional Animal Ethics Committee, National Centre for Biological Sciences.Adult wild type zebrafish Zebrafish larvae were staged based on external morphological features . After recovery larvae were examined under the Olympus SZX16 epifluorescence stereo-microscope for dye labeling in the hindbrain. Larvae having dye-filled neurons were euthanized in cold MS-222 followed by fixation in 4% w/v PFA for 12–14 h. Brain and spinal cord were then dissected out as described above and processed further with 5μg/ml Streptavidin-Alexafluor-488. Larvae were then imaged under a Zeiss LSM 510 meta NLO confocal microscope using the appropriate excitation wavelengths and filters.Larvae were anaesthetized in ethyl 3-amino benzoate methane sulfonate . A mixture of 25% w/v Neurobiotin  and 25% w/v tetra methyl rhodamine-dextran , dissolved in autoclaved, filtered and deionized water was pressure injected into the spinal cord of the anaesthetized larvae using a Pico-spritzer III . In some larvae, only tetra-methyl rhodamine dextran was injected and these larvae were subsequently processed for whole-mount immunostaining as described above. Larvae were allowed to recover for 24 h in Hank's solution  and figures were produced using Adobe Photoshop. The neurobiotin-filled cells were counted using plug-ins in Fiji and results were tabulated in Microsoft Excel.Images were viewed offline using Fiji  and 15 dpf, the distribution pattern of Cx35 immunoreactivity was similar to that seen at 6 dpf. The regions that stained positive at 6 dpf, continued to stain positive through 15 dpf and no new regions were seen Figure .n = 6), indicating a high degree of gap junctional coupling between reticulospinal neurons and other hindbrain neurons.We sought to determine if the high intensity of Cx35 immunoreactivity in the hindbrain of zebrafish larvae indicates a high level of gap junction connectivity between hindbrain neurons. We retrogradely labeled reticulospinal neurons by pressure injecting a 50:50 mixture of neurobiotin and tetra-methyl rhodamine dextran into the spinal cord. These dyes are retrogradely transported by the spinal axons to their respective somata in the hindbrain. While tetra methyl rhodamine dextran stays within the same cell, neurobiotin can cross any gap junctions due to its small mass 287 Da). When the neurobiotin is visualized using fluorophore-coupled streptavidin, while the cells directly labeled retrogradely colocalize both neurobiotin and the higher molecular weight dextran, the dye-coupled cells are only positive for neurobiotin , other structures such as the habenulae and the cerebellum begin to express Cx35 only by 4 dpf. However, on the whole, the Cx35 distribution pattern seems to mature in the early larval stage and remains stable into late larval stages Figure .We used a monoclonal antibody raised against perch Cx35, which shows specificity for Cx35 in perch, zebrafish and gold fish (Pereda et al., We found that Cx35 distribution in the zebrafish CNS recapitulates Cx36 distribution in the mammalian CNS to a large extent. For example, Cx36 is known to be present in the retina, olfactory bulb, cerebellum, habenula, inferior olive and other brain stem nuclei in the adult rodent brain (Condorelli et al., While an earlier study indicated that Cx35 is not present at 1 dpf (McLachlan et al., The optic tectal neuropil showed intense Cx35 immunoreactivity. The extent of staining seen increased with development as the neuropil grew (see Figures In contrast to the optic tectum, Cx35 immunoreactivity in the habenulae and the cerebellum developed later, starting at 4 dpf. Although the habenulae and the habenular commissure can be distinguished by 2 dpf (Hendricks and Jesuthasan, The Mauthner neurons are large neurons in rhombomere 4 that are critical for escape behavior. Electrical synapses on the Mauthner neurons are numerous: they are present in mixed synapses formed by the eighth cranial nerve club endings and in spiral fiber endings on the axon cap. These gap junctions are known to be present by 2 dpf (Kimmel et al., Cx35/36 is a neuronal connexin—it is enriched in the retina and in neurons and is not found in non-neuronal tissues (O'Brien et al., The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Virology Journal 2011, 8:535, Neto et al. described point mutations into Tax-responsive elements (TRE) of the LTR region of HTLV-1 isolates from asymptomatic carriers from Sao Paulo, Brazil, and hypothesized that the presence of the G232A mutation in the TRE-1 increase viral proliferation and consequently the proviral load (PvL), while the A184G mutation in the TRE-2 do not have such effect.In pol) and sequenced LTR region of HTLV-1 isolates from 24 HIV/HTLV-1-coinfected patients without HTLV-1-associated diseases from the same geographic area. These sequences were classified as belonging to the transcontinental subgroup A of the Cosmopolitan subtype a. The frequency of G232A mutation  was high as much as 61.8% reported by Neto’s in HTLV-1 asymptomatic carriers with high PvL. High frequency  of double mutations G232A and A184G was also detected in HIV/HTLV-1-coinfected patients. We did not quantify PvL, but comparative analyses of the cycle threshold (Ct) median values of the group of isolates presenting the mutated-types sequences  versus the group of isolates with the wild-type sequences  showed no statistical difference (p = 0.4220).We performed the real-time PCR assay (The frequencies of mutated-type sequences in the TRE-1 and TRE-2 motifs were high in HIV/HTLV-1-coinfected patients from Sao Paulo, Brazil. If these LTR point mutations have predictive value for the development of HTLV-1-associated diseases or they correspond to the subtype of virus that circulate in this geographic area has to be determined. The real-time PCR (pol) optimization for HTLV-1 was conducted with the cell line C91-PL using 10-fold DNA dilutions, and showed detection limit of 10 ng of DNA input. We did not quantify the proviral load (PvL), and used the PCR as qualitative assay. The coefficient of variation of the cycle threshold (Ct) values for the intra- and inter-assays were less than 1.6% and 1.0%, respectively [A previous study conducted by our group working at the Instituto Adolfo Lutz  analyzed the best algorithm to be employed in the diagnosis of HTLV-1 and HTLV-2 in at-risk individuals of Sao Paulo. We performed confirmatory assay by the real-time PCR (at least in duplicate) to detect pol) were also sequenced for partial LTR region, and by NCBI-Genotyping and REGA-Subtyping tools websites and phylogeny they were classified as belonging to the transcontinental subgroup A of the Cosmopolitan subtype a, the same subgroup A detected by us in a previous study conducted with HIV/HTLV-1-coinfected patients [At present study, DNA samples of 24 HIV/HTLV-1-coinfected individuals of which we have Ethnical Board approval and that scored HTLV-1 positive by the real-time PCR assay  of the TRE-1 motif (the TRE-1 G232A mutation) and the substitution G → A in the third nucleotide (A domain) of the TRE-2 motif (the TRE-2 A184G mutation) was summarized in Table pol) between the two groups. There was no statistical difference in the median of the Ct values between patients with the any mutated-types sequences of 33.5  and patients with the wild-type sequences of 32  .The frequency of the TRE-1 G232A mutation (66.7%) in HIV/HTLV-1-coinfected patients was high as much as 61.8% reported by Neto’s in HTLV-1 asymptomatic carriers with a high PvL [Regardless the limited number of samples of our study we confirmed the frequency of mutated-type sequences in the TRE-1 and TRE-2 was high in HIV-HTLV-1-coinfected patients. We do not know if these mutated-type sequences are the result of a selective advantage of such virus during chronic infection or if they denote the types of HTLV-1 that circulate in this geographic area. Interestingly, in our ongoing study, when we compare LTR sequences from Brazil and sequences from elsewhere, mutated-type sequences in the TRE-1 and TRE-2 regions are detected mostly in Brazil (data not shown). In opposition of what is hypothesized by Neto et al., we suggest that these LTR point mutations could have a protective effect, since no case of HTLV-1-associated diseases was detected among such patients, although a longitudinal study is necessary to confirm this hypothesis. Yet, it is relevant to point that all the individuals from the present study were undergoing antiretroviral therapy, which could interfere in retrovirus selection. Corroborating our hypothesis, Montagne et al. studying the effect of point mutations in the A, B and C domains of TRE-3 observed that the integrity of the B domain was absolutely required to mediate the Tax1 activation of the 21-bp enhancer element, while mutation of the A and C domains reduced the Tax1 effect, and mutation in both A and C domains abolished the Tax1 effect . Thus, tTaking together these data we do not know if the high PvL precede the emergence of the mutated-type sequences or is a consequence of the presence of such mutated strains. Only longitudinal studies could help to answer this question. Finally, it is important to extend these data to clarify the role of the LTR point mutations in TRE, and to identify markers that could have predictive value for the development of HTLV-1-associated diseases, mostly in countries were HTLV-1 is endemic, like Brazil.The Ethical Board of Instituto Adolfo Lutz approved this work. Written informed consent to publish the data in the Table was obtained from all the patients.Tax-responsive element; LTR: Long-terminal repeat; HTLV-1: Human T-cell lymphotropic virus type 1; PvL: Proviral load; PCR: Polymerase chain reaction; Ct: Cycle threshold.TRE: The authors declare that they have no competing interests.MCM conceived the study, conducted the characterization of the LTR sequences and data analysis of the sequences, and wrote the manuscript. EASC did the real-time PCR assays. ACA was responsible of scientific version, discussion and interpretation of data. All authors read and approved the final manuscript.MCM present address: Laboratório de Hepatologia por Vírus, Hospital das Clínicas, Faculdade de Medicina, Universidade de São Paulo, S.P., Brasil.


Early studies mapped the entry receptor for HTLV-1 and HTLV-2 to the long arm of chromosome 17. However, later observations that the titer of HTLV Env-pseudotyped viruses was low on some mouse-human hybrid cell lines containing human chromosome 17q led to the belief that this gene was not involved in HTLV infection. Recent studies showing that efficient HTLV-I infection requires three molecules , prompted us to examine whether a gene on chromosome 17 is related to this receptor complex.Since NRP-1 or GLUT-1 map to other chromosomes, we examined chromosome 17q for genes involved in the synthesis of HSPGs. A gene at 17q21.33, XYLT2, encodes Xylosyltransferase 2 (XT-II), one of two isoforms of an enzyme involved in glycosaminoglycan-chain modifications on proteoglycans. Studies revealed that both the titer of HTLV-1 pseudotyped virus and the level of binding of soluble HTLV-1 SU proteins were dramatically lower on a cell line lacking functional xylosyltransferase (CHOK1-745) than on the parental line (CHOK1). Expression of transgenic XT-II in CHOK1-745 cells increased the levels of HTLV-1 SU binding and the titer of HTLV-1 Env pseudotypes to levels similar to those in wild-type cells. The role of XT-II in HTLV-2 and HTLV-3 Env-mediated entry is being investigated, as well as the role of xylosyltransferases in the infection of T and dendritic cells by HTLVs. These data provide molecular evidence for the previously reported association of a chromosome 17-encoded product in HTLV-1 entry and confirm the critical role of proteoglycans in this process.


To investigate the effect of uric acid (UA) on 6-hydroxydopamine (6-OHDA) -induced injury in SH-SY5Y cells.The cell viability was measured by the MTT reduction assay. The cell apoptosis was assessed by Hoechst 33342 staining with fluorescence microscopy. The phophorylation of Akt and GSK-3β(ser9) was determined by Western blot analysis.Treatment with 6-OHDA at 50µM for 12 h significantly decreased the viability of SH-SY5Y cells. Pretreatment with UA  prior to 6-OHDA treatment markedly increased the cell viability of SH-SY5Y cells, as compared to that of 6-OHDA-treated group. The beneficial effects of UA against 6-OHDA-induced apoptosis were also confirmed by Hoechst 33342 staining assay. Moreover, 6-OHDA decreased the Akt activity and increased the GSK-3β activity, which could be blocked by UA (200-400µM) pretreatment.These data suggest that 6-OHDA-induced cell injury was attenuated by UA. The underlying mechanisms may involve the up-regulation of Akt and the reduction of GSK-3β activity.