Stimulating the receptors of a single cell generates stochastic intracellular signaling. The fluctuating response has been attributed to the low abundance of signaling molecules and the spatio-temporal effects of diffusion and crowding. At population level, however, cells are able to execute well-defined deterministic biological processes such as growth, division, differentiation and immune response. These data reflect biology as a system possessing microscopic and macroscopic dynamics. This commentary discusses the average population response of the Toll-like receptor (TLR) 3 and 4 signaling. Without requiring detailed experimental data, linear response equations together with the fundamental law of information conservation have been used to decipher novel network features such as unknown intermediates, processes and cross-talk mechanisms. For single cell response, however, such simplicity seems far from reality. Thus, as observed in any other complex systems, biology can be considered to possess order and disorder, inheriting a mixture of predictable population level and unpredictable single cell outcomes. The innate immune cell, e.g. macrophage, upon recognition of external stimuli, such as a pathogen, invokes a sequence of molecular events, from receptor activation to gene expressions in the nucleus. This results in the induction of various proinflammatory cytokines that subsequently eliminate the intruders, usually through the adaptive immunity . The welA cell, within a population, possesses varying amounts of individual molecular constituents ,5, in a Over the past few years, our research has focused on the population level, well-characterized and co-ordinated dynamic signaling response of macrophages to invading pathogens based on the TLRs 3 and 4. Briefly, in TLR4 signaling, upon bacterial component lipopolysaccharide (LPS) recognition, MyD88 and TRAM molecules bind to TLR4 and trigger their respective pathways Figure  1]. Not. Not1]. 1 X→ 2 X→ 3 X→ ···) at steady-state condition. If the concentration of 1 Xis pulse perturbed, the concentrations of 2X, 3X, etc., will increase, go through a maximum, and then decrease back to its steady-state value in sequential order  generates downstream response waves of output species (e.g. p38 and JNK) that is conserved in terms of information propagation. The changes to an output specie's activations or concentrations, from steady-state or baseline levels, can be represented by the sum of formation and depletion terms:jXj Fand kXi Frepresent each formation (activation) and depletion (deactivation) term for the ith molecule, respectively, and they can be any linear or non-linear function. Note that the number of formation and depletion terms, p and q for each ith molecule, are variables obtained from the network topology of interest.where X = . Our previous works have used the approximation and have shown that a linear superposition of propagation response waves, or first-order mass-action response equations, can sufficiently be used to model the reaction chains of the TLR signaling [Given small perturbation to the generalized Eq.1, higher-order terms become negligible ,16. Hencignaling -21. Thisignaling ,22.Unlike typical kinetic models, which often use similar equations or sometimes with non-linear expressions to model the dynamics of biological networks, our perturbation-response approach considers the network as a sequence of events rather than molecules. As signaling networks are largely not fully understood, this difference is crucial as it prevents rigidly fixing the network topologies, and allows it to be modified according to experimental data so as to prevent overfitting problems and to identify novel features of signaling networks. In addition, as signaling process involves large number (thousands) of intracellular molecular activations, it is currently not plausible to model the dynamics of all possible reactions with the generally limited data. To overcome such difficulties, our approach permits the lumping of several molecules into a signaling specie in the model network. In this way, although the model does not become a comprehensive representation of an entire signaling process, however, it still allows the identification of overtly missing key features.Tnf, Il6, Ifit1 and Cxcl10) for LPS-stimulated murine macrophages [To successfully identify novel features of signaling networks, we set a target that the computational model should be able to simulate not just one experimental condition (like most models do), but in as many conditions as available. For the TLR4 signaling, we developed a wildtype model and compared the simulations with MyD88 and TRAF6 knock-outs (KOs), a total of three conditions. The initial wildtype model's parameter values for formation and depletion terms were determined directly by fitting simulations with quantified time-course activation experimental profiles of proinflammatory molecules , where we observed increased activation of several alternative pathway molecules [In spite of the limitations, we predicted i) the presence of novel signaling intermediates along the TRAM-dependent pathways , ii) croSFR . The preSFR ,24. The y Figure . That isolecules .Similarly to the TLR4 signaling, we also investigated the TLR3 innate immune response against viral attacks by polyinosinic-polycytidylic acid (poly (I:C)). This leads to the activation of MAP kinases and NF-κB, which results in the induction of type I interferons and proinflammatory cytokines to combat the viral infection. Here, again analyzing the activation dynamics of the NF-κB, p38 and JNK in wildtype, TRAF6 KO and TRADD KO using a linear response model obeying the law of information conservation, we inferred i) the existence of missing intermediary steps between extracellular poly (I:C) stimulation and intracellular TLR3 binding, and ii) the presence of a novel pathway which is essential for JNK and p38, but not NF-κB, activation .Here I present, with examples from TLR 3 & 4 signaling in wildtype and several mutants, that the law of conservation and first order response equations are sufficient and important to reveal novel features of the complex immune process. This result is surprising as there is a general consensus that we need to fully understand all molecular interactions in the signaling network in order to make useful predictions ,13. FurtSo, instead of losing interest in the population level dynamics and move towards more single cell analyses, I propose biology is a system that possesses both microscopic and macroscopic dynamics, as observed in other physical sciences. For example, in the study of fluid dynamics, at microscopic level we observe the motion of each individual particle to be highly random and unpredictable and at macroscopic level, the velocity of airflow follows the fundamental law of fluid mechanics . Thus, it is necessary to treat the two dynamics distinct.It is also interesting to ponder the origins of averaging effect from stochastic response of a single cell when ensembles of them form a population. I believe that the emergence of average cell deterministic response from single cell stochastic response complement each other. For example, the stochastic fluctuations produced by a single cell are necessary to induce probabilistic differentiation from genetically identical cells -28. ThisOn the other hand, the well-coordinated response of cell populations, such as differentiation or growth, demonstrates that the single cell noise could cancel out when ensembles of cells are formed to generate a stable and robust response. Thus, the search for governing laws arising from single cell dynamics will enable us to better understand the coordinated response of cell populations. Most importantly, finding the connection between microscopic and macroscopic dynamics and the unifying laws are crucial for understanding the origins of evolutionary and developmental robustness of living systems to diverse environmental attacks.MyD88: myeloid differentiation primary response gene (88); TRAM: TRIF-related adaptor molecule; TRIF: TIR-domain-containing adapter-inducing interferon-β; TIR: Toll/Interleukin-1 receptor; NF-κB; nuclear factor-κB; JNK: c-Jun N-terminal kinases; MAP; mitogen-activated protein; TRAF6: TNF receptor associated factor 6 protein; TNF: tumor necrosis factor; TRADD: Tumor necrosis factor receptor type 1-associated DEATH domain protein; IRF-3: interferon regulatory factor-3; IκBα: inhibitor of kappa B-α.The author declares that he has no competing interests.


Objective: Fertility preservation stands before us as an issue of quality of life for cancer patients and their partners and families. Therefore, the object of the present study was to determine the extent of the knowledge that hematologists have about fertility preservation and to understand their attitudes and practices regarding this matter. Materials and Methods: A total of 25 hematologists participated in a survey. The questionnaire included questions on sociodemographic characteristics and awareness concerning the subject of fertility preservation, as well as questions designed to determine the extent of the knowledge that hematologists had on the subject and to understand their attitudes and practices in this context.Results: Of the participants in the study, all expressed their awareness of the adverse effects that the various treatments they were prescribing could have on fertility; 2 (8%) revealed that they had never heard of the concept of fertility preservation. Of the participants, 19 (76%) indicated that they did not have adequate knowledge about fertility preservation, but 22 (88%) fortunately expressed a need for acquiring more knowledge about the subject. Of the respondents, 23 (92%) said that they did not have any brochures or published resources on this subject and stated their belief that if hematologists did have such documents, they would have more opportunity to discuss the various fertility preservation options with patients. All of the participants in the survey supported the idea of the Turkish Society of Hematology publishing a guidebook on this subject and organizing a session on fertility preservation in their regular congress.Conclusion: Meeting the needs of hematologists for training and knowledge in the subject of fertility preservation and ensuring the development of appropriate attitudes and practices in this area is an important issue. The Turkish Society of Hematology may play a significant key role.Conflict of interest:None declared. The advances made in providing effective treatment options and the steadily increasing percentage of cured patients or of patients with 5-year life expectancies in hematological malignancies have brought the subject of such patients’ quality of life into the foreground . In thisWith the development of combination chemotherapies and steady improvements in more effective treatment modalities, today the 5-year survival rate of patients with Hodgkin’s lymphoma has reached a level of 90% . FertiliToday, the subject of fertility preservation is steadily gaining more and more attention. An increasing number of clinicians are reporting that patients and their families and spouses are fearful about loss of fertility and reporting their will and desire to discuss fertility preservation options. Major associations in the United States have published recommendations on this subject ,7. Today, sperm and embryo cryopreservation are the first options that are offered in fertility preservation ,7,8. LivThere are only a limited number of studies in the literature today that have explored the matter of fertility preservation from the standpoint of hematologists. To the best of our knowledge, there has been no study conducted in Turkey on the knowledge, attitudes, and practices of hematologists regarding fertility preservation. This study therefore was carried out to understand how aware hematologists are about the subject of fertility preservation, the extent of their knowledge of this topic, their attitudes toward fertility preservation, and what their practices are in this area as a part of their daily work. The study was conducted as descriptive and cross-sectional research. An attempt was made to reach members of the Turkish Society of Hematology. Hematologists who gave their consent and agreed to participate in the study were asked to fill out the questionnaire. The respondents answered questions about their sociodemographic characteristics and their awareness, knowledge, attitudes, and practices regarding the subject of fertility preservation. The participants were informed that the questionnaire would be implemented with full confidentiality and anonymity. The questions in the survey were prepared in light of previous studies. These questions probed into the participants’ sociodemographic characteristics and their awareness, knowledge, attitudes, and practices regarding the subject of fertility preservation. The questionnaire was initially administered to 3 hematologists in order to test the comprehensibility of the questions. The validity and reliability of the questionnaire was studied and the internal consistency coefficient of the questionnaire was found to be 0.85. The questions were revised on the basis of the feedback received. The study protocol was approved by the local ethics committee. StatisticsThe statistical analysis was performed using version 11.5 of the Statistical Package for Social Sciences . Descriptive characteristics such as frequency and summary characteristics were calculated for variables of interest. A total of 16 male (64%) and 9 female (36%) hematologists responded to the questionnaire. In the present study, Cronbach’s alpha was found as 0.83. The mean age of the participating hematologists was 47.36±4.32 years. Of the responding hematologists, 4 (16%) worked at private hospitals, 2 (8%) at state hospitals and at training-research hospitals, and 19 (76%) at university hospitals. The characteristics of the participants are presented in All respondents stated their awareness of the possible adverse effects on fertility of the treatment modalities that they were using. Twenty-three (92%) revealed that they had heard of the concept of fertility preservation while 2 (8%) asserted that they had not. The responses of the participants are given in Of the participants, 15 (60%) said that they did not inform patients about fertility preservation options as a routine procedure and 11 (44%) stated that they did not obtain informed consent from the patients or their legal guardians regarding the adverse effects of drugs and/or treatment procedures on fertility. Twenty-three (92%) expressed their feeling that patients and their families should be informed about the subject with the obtaining of written informed consent. On the other hand, 14 (56%) of the participants revealed that their patients or patients’ families knew about and asked whether they could discuss the topic of fertility preservation. Of the participants, 23 (92%) said that they did not have a brochure or any other printed materials on the subject of fertility preservation, and they stated that if such documents could be made available, they would have more opportunity to discuss fertility preservation with patients and their partners and families. Of the respondents, 19 (76%) said that they did not feel that they had enough information about fertility preservation and, fortunately, 22 (88%) expressed their desire to learn more. Among the hematologists participating in the survey, 24 (96%) said that they should consider fertility preservation a priority topic when planning a modality of treatment for patients; only 15 (60%), however, did say that they considered the subject when planning the treatment. Of the participants, 17 (68%) professed knowledge about fertility preservation options for male patients and 11 (44%) said that they knew about fertility preservation options for female patients. Of the participants, 22 (88%) said that they did not have enough knowledge about oocyte cryopreservation and 13 (52%) reported the same about ovarian tissue cryopreservation. Fifteen (60%) of the participants revealed that they had never recommended sperm cryopreservation to any patients, 19 (76%) stated that they had not recommended ovarian tissue cryopreservation, 20 (80%) stated that there had been no instance where they had recommended oocyte cryopreservation, and, finally, all participants said that they had never recommended embryo cryopreservation. Eighteen (72%) of the participants indicated that they had never read any publication on fertility preservation; 20 (80%) stated that they had not read any publication about the subject in the last 6 months. Among the respondents, 20 (80%) expressed their approval of patients postponing their treatments for a short period  to accommodate the fertility preservation process. Fourteen (56%) of the participants said that the hospital in which their patients were treated had no clinic or assisted conception unit for fertility preservation and 17 (68%) stated that these should be established. Another 20 (80%) said that they had no knowledge about the costs of fertility preservation options. Of the participants, 22 (88%) said that they were not familiar with the recommendations on fertility preservation published by the American Society of Clinical Oncology (ASCO) and American Society of Reproductive Medicine (ASRM). Another 24 (96%) admitted that they did not know whether the Turkish Society of Hematology had published a guidebook on fertility preservation, and all participants asserted that the Turkish Society of Hematology should have a guidebook and that they would support its publication and sessions about fertility preservation at congresses of the Turkish Society of Hematology. It was found that most of the hematologists in the study were aware that the treatment modalities they used had an adverse effect on fertility. A majority of the hematologists stated that they have heard of the concept of fertility preservation. It was also found that most of the hematologists did not routinely inform their patients about fertility preservation options. Most of the hematologists that participated in the survey stated their lack of adequate knowledge about fertility preservation but, fortunately, most of them expressed their desire to learn more about the topic.As seen in the results obtained from the survey, a growing number of patients and their families are asking to be informed about the matter of fertility preservation and wish to discuss this with their doctors. It is our belief that it is very important that patients are informed about this subject. The same view has been expressed by ASCO and ASRM . Patients should be informed and thus made ready to make their own informed decisions. The active participation of patients’ families should also be ensured in sessions where patients are provided information about fertility preservation. This is important because the subject is not only a source of anxiety for the patient but also creates intense feelings in close relatives such as the patient’s spouse.Most of the hematologists in our study said that they did not routinely inform patients about fertility preservation before the start of treatment. In studies carried out abroad, half of male and female cancer patients were reported as not remembering being informed about fertility preservation . Those who did remember said that they were not happy with the quality and amount of information that they were given . Explaining the adverse effects on fertility of treatment modalities, informing patients about fertility preservation, or referring the patient to another physician for the purpose of obtaining this information is the responsibility of the patient’s attending physician [6].Because there is no test today to indicate which patients’ fertility will be affected by prescribed treatments and what the impact will be, it is our belief that all patients should be informed about this subject [4]. Fertility preservation alternatives should be explained in detail to all patients and patients should be given the chance to decide. The matter of fertility is an important topic that affects quality of life following treatment. In a study by Schover et al., it was found that 76% of young cancer survivors with no children wished to eventually be parents and, for this reason, were interested in learning about the positive and negative effects of treatment on fertility. In this context, it was also reported that patients were concerned about the negative impact of treatment on a possible pregnancy and on the fetus, should a pregnancy occur [10].As the majority of the hematologists participating in this study indicated in the questionnaire, there were no brochures or informative booklets available about fertility preservation that could be given to patients to read. Such booklets are generally made available to patients as standard procedure at clinics in Western countries. As the majority of hematologists indicated in the questionnaire in the present study, the availability of this kind of informative printed booklets will enhance the dialog of clinicians and patients on this subject and patients will find increased opportunity to bring up the matter for a more productive discussion. Organizations such as FertileHope [13] in the United States produce brochures of this kind and provide patients with support and guidance.The majority of the hematologists stated that no clinics dealing with fertility preservation existed in the hospital in which they worked. It was also observed that hematologists were not knowledgeable about the costs of possible fertility preservation options. It may be that the reason a large majority of the hematologists had not referred patients to a fertility preservation center was because they did not know where such centers dealing with fertility preservation were located or the probable cost. It is our belief that associations of professionals in the areas of fertility, hematology, and oncology should collaborate and share information, perhaps forming working committees among themselves if necessary. An oncofertility consortium has been established in the United States [14]. We feel it would be useful for the Turkish Society of Hematology to announce on its website the locations of centers involved in fertility preservation in all regions of the country so that hematologists can easily find the center closest to them.Another finding in our study was that some hematologists did not consider fertility preservation a priority topic at the time that a course of treatment was being planned for the patient. Other studies on this subject support this finding [15]. Clinicians are more liable to prioritize the discussion of the patient’s potential life-threatening complications. It is, however, true that as effective treatments cause 5-year survival and cure rates to rise, the topic of fertility will increasingly come to the fore as an issue of quality of life [10], and it is imperative that clinicians realize this. Patients and their families are often in a state of shock over life-threatening complications and in such a situation, the subject of fertility options may be completely overlooked or there may be a lack of interest in discussing the matter.It is very important that hematologists inform their patients in detail about fertility preservation before treatment begins [9]. The information presented to the patient should contain knowledge about the adverse effects of treatment on fertility. The hematologist should provide the patient and the patient’s family with the fertility preservation options and the success rates of fertility preservation modalities, also informing the patient that some options are still in their experimental stages. The knowledge that hematologists impart to their patients should also include information on the costs of fertility preservation options. It is important that the clinician give the patient the address and telephone number of a person or clinic that can be reached and from which information about fertility preservation can be acquired. We also feel that the hematologist’s own coordination with that person or clinic will be important in choosing a fertility preservation option that is suitable to the patient’s clinical status. It is also thought that obtaining written informed consent from the patient or their legal guardians will be important in terms of medicolegal matters.Based on this study with a limited number of hematologists, it is not possible to specify to what extent these results are generalizable to all hematologists. Thus, we assume that the results of this study can be generalized to a limited degree. Further research with larger numbers of participants should be conducted on the topic.It is our belief that publishing guidebooks to offer guidance to clinicians about fertility preservation options is important. The respondents to the questionnaire support the publication of such a guidebook by the Turkish Society of Hematology. ASCO and ASRM have made recommendations about fertility preservation in various publications . The British Fertility Society [16] has also made similar recommendations. Reviewing these recommendations allows clinicians to be more comfortable about this topic and act on it quickly and in coordination. We feel that it is important that a subcommittee that may be formed under the Turkish Society of Hematology prepare a guidebook that will provide hematologists with guidance on this issue.Most of the hematologists responding to the questionnaire believed that they did not know enough about fertility preservation, but fortunately they exhibited a desire to receive education on this topic. The respondents also supported the idea that the Turkish Society of Hematology should organize a session on fertility preservation at their regular congresses. An initiative of this kind by the Turkish Society of Hematology may be significant in terms of eliminating the lack of knowledge in this area. At the same time, we also think that making the topic of fertility preservation a standard part of the Society’s fellowship education program will be instrumental in enhancing the levels of knowledge. Meeting the needs of hematologists for more education and knowledge on the topic of fertility preservation and improving their knowledge, attitudes, and practices in this area is of great importance. The Turkish Society of Hematology may play a significant and key role in such efforts. The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/ or affiliations relevant to the subject matter or materials included.


The production of antisense transcripts from the 3′ long terminal repeat (LTR) in human T-lymphotropic retroviruses has now been clearly demonstrated. After the identification of the antisense strand-encoded human T-lymphotropic virus type 1 (HTLV-1) bZIP (HBZ) factor, we reported that HBZ could interact with CRE-binding protein (CREB) transcription factors and consequently turn off the important activating potential of the viral Tax protein on HTLV-1 5′ LTR promoter activity. We have recently accumulated new results demonstrating that antisense transcripts also exist in HTLV-2, -3, and -4. Furthermore, our data have confirmed the existence of encoded proteins from these antisense transcripts (termed antisense proteins of HTLVs or APHs). APHs are also involved in the down-regulation of Tax-dependent viral transcription. In this review, we will focus on the different molecular mechanisms used by HBZ and APH-2 to control viral expression. While HBZ interacts with CREB through its basic zipper domain, APH-2 binds to this cellular factor through a five amino acid motif localized in its carboxyl terminus. Moreover, unlike APH-2, HBZ possesses an N-terminal activation domain that also contributes to the inhibition of the viral transcription by interacting with the KIX domain of p300/CBP. On the other hand, HBZ was found to induce T cell proliferation while APH-2 was unable to promote such proliferation. Interestingly, HTLV-2 has not been causally linked to human T cell leukemia, while HTLV-1 is responsible for the development of the adult T cell leukemia/lymphoma. We will further discuss the possible role played by antisense proteins in the establishment of pathologies induced by viral infection. One of the best studied associations between HTLV-1 and oncogenesis is the viral Tax protein, as demonstrated in primary T cells and various mouse models (in vitro (+ T cells at higher efficiency than HTLV-1 Tax (The human T-lymphotropic virus type 1 (HTLV-1) was the first pathogenic retrovirus to be isolated in humans . This vie models . It likee models . Howevere models . Selectie models . HTLV-2 e models . Howeverin vitro  and HTLVLV-1 Tax . Taken tLV-1 Tax . We haveLV-1 Tax . This regag, pol and env. In addition to these genes, they harbor genes coding for the Tax and Rex regulatory proteins . Tax acts in trans to activate transcription initiating from the viral promoter in the 5′ LTR and Rex regulates viral gene expression post-transcriptionally by facilitating cytoplasmic shuttling of incompletely spliced viral mRNAs. HTLV-1 encodes the accessory proteins p12, p13, and p30 whereas HTLV-2 encodes the p10, p11, and p28 accessory gene products . Studies have indicated that these proteins are dispensable for in vitro infection and transformation of T cells but are important for the ability of the virus to infect, spread, and persist in vivo . In 2009, we also characterized a spliced antisense mRNA in HTLV-2-infected cell lines, involved in the production of APH-2 . These similarities could suggest that the expression of antisense transcription in the HTLV (and STLV) retrovirus family has been conserved among the different viruses. Indeed, we have recently confirmed the synthesis of antisense proteins from HTLV-3 and -4 proviral DNA , leading to inhibition of their DNA-binding activity  involved in an interaction with the KIX domain of p300/CBP . Thus, HBZ not only inhibits expression of other viral proteins such as Tax but also stimulates its own expression. While the majority of HTLV-1-specific CD8+ T cells recognize the Tax protein, the frequency of HBZ-specific CD8+ T cells is significantly lower  and Fos  family members through their ZIP domains. AP-1 complexes are not limited to Jun and Fos dimers, since Jun and Fos proteins have been shown to dimerize with other bZIP proteins, including members of the ATF/CREB family and the Maf transcription factors. ATLL cells have been shown to contain constitutively activated AP-1 complexes, which are mainly constituted of JunD . HBZ, viFigure 2; in vivo . Furthermore, the modulation of AP-1 activation by Tax2B was greatly reduced when APH-2 was co-expressed. However, the above experiments were conducted in overexpression condition and it will therefore be mandatory to confirm these data with physiologically relevant expression levels. These results highlight a very complex interplay between Tax 2 and APH-2 in relation to other transcription factors and it will be important to determine how this type of interaction is affected in the context of ATF/CREB family members. Furthermore, we have not observed that HBZ interacts with Tax . However, cells highly expressing viral proteins are eliminated by the humoral response and CTL activity of the host . This dual action probably confers a survival advantage on HBZ-expressing cells and is consistent with the observation that HBZ favors the establishment of persistent infection in HTLV-1-inoculated rabbits . Further experiments are definitely needed to better understand the mechanism by which HTLV-2 infection could lead to lymphocytosis.Both HTLV-1 and HTLV-2 are able to establish a persistent infection in CD4phocytes . Moreovethe host . At this rabbits . On the  rabbits  and unab rabbits . HoweverThe discovery of HBZ has led to important new research avenues with potential association with HTLV-1-associated diseases. In fact, ATLL cells from infected patients consistently express HBZ and substantial evidence points toward its implication in viral persistence and ATLL cell survival. Recent evidences further suggest that HBZ lead toward a Treg phenotype. Future experiments will be needed to determine if similar attributes can be given to APH-2. As HBZ and APH-2 also differ mechanistically and functionally in their interaction with transcription factors such as Jun and CREB, it will be important to further expand on these differences and define the exact nature of these interactions. Several transcription factors known to interact with HBZ will also need to be analyzed in the context of APH-2 and in relation to the different cellular localizations of these proteins. In addition, the role played by HBZ on proliferation needs to be clarified as to how its transcript impacts IL-2-independent growth. These studies might also consider variation in the sequence of APH-2 transcripts, which could explain their different ability in inducing proliferation. Finally, these experiments should also be undertaken with equivalent antisense proteins of HTLV-3 and HTLV-4, i.e., APH-3 and APH-4 to determine how these proteins alter the various functions associated to HBZ. Finally, these comparative studies will provide a better understanding as to the mechanism by which HBZ is involved in ATLL development, which might also lead to new potential ATLL treatment.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.


Of the millions of HTLV-1 infected carriers worldwide, 3–5% will develop an aggressive T-cell neoplasm that is highly refractory to conventional therapy. The virus carries the Tax oncogene which constitutively activates the NFκB pathway. This co-option of signaling through NFκB provides for the HTLV-1 infected cell an escape from cell cycle arrest and apoptosis, a steady source of growth factors, and a mechanism by which the virus can activate its own target cell. Therapies that target the NFκB pathway sensitize adult T-cell leukemia/lymphoma (ATLL) cells to apoptosis. A focus on translational interrogation of NFκB inhibitors in animal models and ATLL patients is needed to advance NFκB-targeted ATLL therapies to the bedside. Despite intensive efforts to improve the overall survival, adult T-cell leukemia/lymphoma (ATLL) remains one of the hematologic malignancies with the poorest prognosis. ATLL is typically preceded by decades of clinical latency during which infected cells accumulate selectable traits leading to malignant transformation. Host pathways commandeered by the virus can be used as therapeutic targets and a constitutively activated NFκB pathway has emerged as an essential hit in the development of ATLL. Research into the mechanisms underlying HTLV-1 modulation of the NFκB pathway has revealed the extent to which HTLV-1 oncogenesis is dependent on the NFκB pathway [2.During decades of clinical latency HTLV-1 modulates host signaling pathways to promote proliferation of infected cells. Among the genes carried in the viral genome, the 40 kDa viral transcriptional transactivator (Tax) is sufficient to promote cellular transformation. The mechanism utilized by Tax to promote cellular transformation is multi-faceted, broadly involving activation of proliferation, dysregulation of cell cycle checkpoints, and promotion of genetic instability. While the affects of Tax expression in a cell are diverse, Tax depends on a central signaling pathway for its transforming activity. Mouse models in which Tax is expressed in the lymphocyte compartment have constitutively active NFκB and reproduce many aspects of HTLV-1 pathogenesis . Tax exp3.The NFκB family of transcription activators are involved in many biological processes including cell survival, stress responses, and development . NFκB is4.Lymphoma is commonly associated with constitutive NFκB activity and oncogenic human viruses, including Epstein-Barr virus (EBV), Human papillomavirus (HPV), Hepatitis B virus (HBV), and Hepatitis C virus (HCV) all carry viral factors that directly target and co-opt the NFκB pathway during oncogenesis. HTLV-1 uses Tax in multiple strategies to efficiently activate both the canonical and non-canonical NFκB pathways .Tax directly interacts with several NFκB members, including RelA, p50, p52, IKKγ, and the NFκB precursor protein p100 such that both arms of the NFκB pathway are involved in tumorigenesis . Rapid a5.Constitutive activation of NFκB family members and downstream effectors provides several selective advantages to malignant cells. One selective advantage offered by unregulated NFκB signaling is the ability to bypass checkpoint controls and p53-mediated apoptosis. In transformed cell lines as well as freshly isolated ATLL samples, NFκB activation supports the survival and proliferation of HTLV-1 infected cells. Moreover, lymphoma cell lines with constitutively activated NFκB are resistant to a variety of inducers of apoptosis including gamma-irradiation, etoposide, and combinations of cycloheximide and TNF or TRAIL, and resist the activation of both the intrinsic and extrinsic apoptotic pathways . Like AT6.in vitro and in vivo [HTLV-1 in humans is also associated with chronic inflammation which is mediated by NFκB. Chronic inflammation resulting from Tax-mediated canonical NFκB activity leads to HAM/TSP in some HTLV-1 carriers , but may in vivo ,28. More in vivo ,29. Prec in vivo .This inflammation-associated malignancy is Tax-dependent and promoter-dependent since SV40 large T under the same promoter does not reproduce the inflammation promoting nature of Tax tumors ,31. Inte7.+ T-cells. The NFκB pathway is essential for T-cell functions including T-cell development, activation, gene expression, cell cycle progression, survival, cytokine production, and apoptosis [HTLV-1 infects and transforms CD4poptosis . While Tpoptosis . This hapoptosis . Is ATLLpoptosis . In addipoptosis . Determi8.ex vivo and sensitizing cells to apoptosis [in vivo.NFκB regulates the expression of a wide variety of genes implicated in proliferation, angiogenesis, invasion, and metastasis and the dependence of HTLV-1 oncogenesis on the NFκB pathway makes it an ideal target for therapeutic attack. Repression of the NFκB pathway could make ATLL cells sensitive to apoptosis, slow their proliferation, or repress aspects of the immune response that promote malignancy. In tissue culture and mouse models, non-specific inhibitors of the NFκB pathway like sodium salicylate or cyclopentenone prostaglandins can increase the sensitivity of Tax-tumor cells to apoptosis and repress NFκB-inducible cytokines IL-6, IL-10, IL-15, and IFN-γ . Bortezopoptosis . Bortezopoptosis . Bay11-7poptosis . Over thpoptosis . Oridonipoptosis –44. Thes9.The majority of ATLL patients present with acute or lymphomatous ATLL, which results in a median survival of 0.5–2.0 years, despite intensive chemotherapy treatment ,46. To d10.Host pathways can be used as chemotherapeutic targets when they confer an essential trait to the cancer cell. A constitutively activated NFκB pathway represents such a target in the case of HTLV-1 mediated ATLL. The NFκB pathway provides an escape from cell cycle arrest and apoptosis, a steady source of growth factors, and a mechanism by which the virus can activate its own target cell. Accumulating evidence supports the concept that NFκB targeted therapies sensitize ATLL cells to apoptosis. Research in ATLL therapies should now focus on translational interrogation of NFκB inhibitors in animal models and ATLL patients.


There are few clinical trials indicating type-l IFNs efficacy in HTLV-I infected patients,[2b (PDferon®-B) on released particles of HTLV-I by HTLV-I transformed MT2 cell line.HTLV-I is causative agent of Acute T-cell Leukemia (ATL) and HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP). To date there isn't an established treatment available for HTLV-I infection. Type-I IFNs are well known for their antiviral, anti proliferative and immunomodulatory effects. Antiviral mechanism of these IFNs varies depending on the virus and host type and could fluctuate between virus entry to its replication and release. IFN-α patients, neverthepatients, Here we 2b ( PDferon®-B). 12 and 36 hours post treatment, MT2 cells were collected and cell lysates were analyzed for expression of HTLV-I Gag, Tax and HBZ genes by gene specific primers utilizing real time RT-PCR method .As a representative of HTLV-I free particles released in supernate by MT2 cells, virus Gag gene copy number in supernate was evaluated at aforementioned hours post treatment.MT2 cells were treated with 1000 U/ml of IFN-α 2b.There was no significant difference in expression of HTLV-I Gag, Tax and HBZ genes between cell lysates of treated and untreated MT2 cells at both 12 and 36 hours post treatment. At 12 hours post treatment, released Virus titer was not changed in supernate of treated cells while a significant decrease in viral particles was observed in collected supernate of treated cells at 36 hours after treatment with IFN-α2b (PDferon®-B) treatment does not have any effect on expression of both structural and non structural genes of HTLV-I at 12 and 36 hours of treatment thus Reduction of HTLV-I titer at the 36th hour post treatment suggests that IFN-α2b ( PDferon®-B) does not exert its antiviral effects at viral transcription level. The significant decrease in viral titer in supernate by IFN-α 2b ( PDferon®-B) could be due to its interfering effect at any posttranscriptional level. Our results are in accordance with previous studies that have shown the similar effects of other members of type-l IFN family such as IFNα2a. [2b in treatment of HTLV-I infection.Our result shows that IFN-α


The neuromuscular junction has been extensively employed in order to identify crucial determinants of synaptogenesis. At the vertebrate neuromuscular synapse, extracellular matrix and signaling proteins play stimulatory and inhibitory roles on the assembly of functional synapses. Studies in invertebrate species have revealed crucial functions of early morphogens during the assembly and maturation of the neuromuscular junction. Here, we discuss growing evidence addressing the function of Wnt and Bone morphogenetic protein (BMP) signaling pathways at the vertebrate neuromuscular synapse. We focus on the emerging role of Wnt proteins as positive and negative regulators of postsynaptic differentiation. We also address the possible involvement of BMP pathways on motor neuron behavior for the assembly and/or regeneration of the neuromuscular junction. The vertebrate neuromuscular junction (NMJ) has been extensively used as a model of synaptogenesis essentially due to its larger size and simplicity, as compared to central synapses. As a consequence, several proteins that have been demonstrated to play crucial roles in the behavior of central synapses, were first described at the NMJ. In addition, the NMJ is the final target of therapeutic strategies aimed to regenerate peripheral synaptic connections after neurodegenerative or traumatic injury of the spinal cord. In this regard, it is fair to assume that the more we increase our knowledge on the strategies employed by development in the original wiring of the nervous system, the more we will be able to precisely imitate them for successful regeneration. Therefore, research directed to unveil the mechanisms accounting for NMJ assembly, maturation and maintenance contributes to the basic knowledge of synaptic behavior as well as to potential strategies for regeneration.At the molecular level, it has been demonstrated that both nerves and muscles secrete molecules that regulate key aspects leading to the assembly of functional neuromuscular synapses ,2. For iCumulative evidence obtained from different model organisms, either invertebrate or vertebrate, has demonstrated that signaling molecules that act as morphogens during early development, such as Wnts and BMPs, regulate crucial events later that lead to the formation of proper neuronal connections. Members of both signaling pathways have been involved in several features of neuronal behavior, such as differentiation, axonal guidance and synaptogenesis.in vivo role of well described molecules playing crucial roles on the establishment of pre and postsynaptic terminals at the vertebrate NMJ. We will additionally focus on current data, mainly obtained in invertebrate systems, indicating that Wnts and BMPs potentially activate different signaling pathways to modulate, positively and negatively, the formation of the vertebrate NMJ.In this review, we will first summarize current views on the end-plate band. Presynaptic differentiation is accompanied by morphological changes in motor terminals that contact the muscle fiber and begin to accumulate synaptic vesicles containing acetylcholine and other presynaptic components. In turn, postsynaptic differentiation is characterized by increased expression of several genes coding for postsynaptic proteins, including the acetylcholine receptors (AChRs), in the few myonuclei localized beneath the junction and the aggregation of these proteins in a small invaginated and folded fraction of the muscle membrane to shape the mature NMJ [in vivo studies that have underscored the potential physiological role of different signaling molecules acting as pro and anti-synaptogenic signals at both sides of the vertebrate NMJ.During vertebrate embryonic development, motor nerve trunks penetrate peripheral regions where myotubes have been recently differentiated. Later on, motor neuron axons branch to innervate a variable number of skeletal muscle fibers in a discrete central region of the muscle named the ture NMJ . The prein vivo presynaptic differentiation, there are good examples of signaling molecules affecting motor neuron behavior at the NMJ. One example of such proteins are members of the ephrin-A family of bidirectional signaling molecules, which are differentially expressed by developing skeletal muscles along the anteroposterior axis [in vitro experiments showing that rostral and caudal motor neurons bear different sensitivity to ephrin-A5 on neurite outgrowth [Even though little is known about the identity and function of muscle-derived molecules regulating ior axis . Consistutgrowth , transgeutgrowth .in vivo showed that they control different sequential features of the vertebrate NMJ formation. Thus, whereas signaling through the FGF receptor 2b is required for the onset of presynaptic terminals, the presence of laminin-β2 is crucial for their maturation [in vivo [in vivo approaches reveal that multiple signaling pathways are likely required to refine the correct differentiation and positioning of functional presynaptic terminals at the vertebrate neuromuscular synapse.A comprehensive genetic approach conducted by Fox and colleagues analyzed the potential role of members of the fibroblast growth factor (FGF) family and extracellular matrix proteins, such as laminins and collagens, in presynaptic differentiation at the NMJ . Interesturation . Interesturation , which hturation . On the turation . Remarka[in vivo ,9. TakenThe idea that neural inputs induce postsynaptic differentiation is supported by genetic studies showing that ablation of specific genes expressed by motor neurons results in severe defects in the morphology of the NMJ –12. In aAgrin is a motor neuron-secreted heparan sulfate proteoglycan extensively characterized by its ability to aggregate AChRs and other postsynaptic proteins in cultured muscle cells –17. In sThe crucial role of neural-derived molecules on postsynaptic differentiation has, however, been challenged by the findings showing that AChR clusters do form even before motor axons contact muscle fibers ,26,27. FA relevant emerging concept in the field is the existence of negative signals for postsynaptic differentiation that act at the most abundant non-innervated (“extrasynaptic”) domains of the muscle membrane. Several lines of evidence reveal that the neurotransmitter acetylcholine acts as such a signal. Indeed, mutant mice for choline acetyltransferase (ChAT), the sole synthetic enzyme for acetylcholine, display wider endplate bands containing more innervated AChR clusters than wild-type littermates ,34. In aWnt ligands comprise a family of 19 secreted signaling proteins with a wide range of developmental functions including cell fate decisions during early embryogenesis and specific cellular responses such as cell proliferation, differentiation and survival . Interes2+ levels that activate specific protein kinases [N-terminal c-Jun kinase and functions mainly in polarity and morphogenesis [Wnt ligands signal through their cognate seven-pass transmembrane G-protein coupled Frizzled (Fz) receptors from which ten members have been described in vertebrates . Three d kinases . In turnogenesis .Along with the three pathways described, Wnt proteins activate several other signaling cascades . This heDrosophila NMJ, the Wnt orthologue Wingless (Wg) is secreted by motor terminals whereas its DFz2 receptor is expressed both at pre- and post-synaptic cells [C-terminal polypeptide is cleaved and transported into the nucleus to possibly activate transcription of target genes [At the ic cells . In agreic cells . Ultrastic cells . A novelet genes ,53. Thiset genes ,53.Drosophila NMJ. In motor neurons, Wg signaling is transduced through GSK-3β to regulate the formation of synaptic boutons and the recruitment of synaptic components. Indeed, GSK-3β mutants display abnormal synaptic boutons and NMJ growth by affecting presynaptic differentiation through a mechanism involving local β-catenin-independent changes in the dynamics of the microtubule cytoskeleton [Wnt signaling also affects presynaptic differentiation at the skeleton ,55.Drosophila NMJs, studies in C. elegans have demonstrated that Wnt signaling inhibits neuromuscular synaptogenesis. In this model system, the most proximal segment of the DA9 motor neuron is normally asynaptic whereas the remaining fraction of the axon form functional synapses with muscles along the dorso-ventral axis [In contrast to the positive effects of nerve-derived Wg on the development of ral axis . Mutant ral axis . Wnt/linral axis . These fral axis . Taken tin vivo evidence supports a role for Wnt signaling during early steps of postsynaptic differentiation at the vertebrate NMJ. For instance, Dvl1 mutant mice diaphragms display postsynaptic domains organized in a wider end-plate band than control littermates [in vitro data obtained in cultured muscle cells that positioned Dvl as a key organizer of postsynaptic differentiation by regulating the function of the agrin receptor MuSK [Important termates , a phenotermates ,12. In ator MuSK . Considein vitro studies revealed that Wnt3 activates Rac1 in a more efficient way than agrin does, whereas agrin preferentially increases Rho activity, suggesting that Wnt and agrin signaling pathways cross-talk at the vertebrate NMJ . Consisiew, see ,64, Wnt3iew, see . Future in vivo support of these findings, electroporation of Wnt3a in post-natal muscles dispersed stabilized AChR clusters of mature NMJs [In sharp contrast with the effect of Wnt3, the highly identical ligand Wnt3a plays negative roles on AChR clustering . In suppure NMJs . In culture NMJs . Consisture NMJs , thus suMore recent evidence demonstrates that Wnt ligands also affect early aneural pre-patterning of AChR clusters at the vertebrate NMJ . In zebrDrosophila NMJ [In summary, Wnt ligands affect different features of the vertebrate NMJ formation. Regarding postsynaptic differentiation, they have the ability to modulate the early assembly of aneural AChR clusters, possibly to distribute nascent neuromuscular synapses. In areas of synaptic contact, motor neuron-derived Wnts could act through local pathways as positive inputs for postsynaptic differentiation; in turn, Wnt/β-catenin signaling, possibly activated by non-neuronal Wnt ligands, could negatively regulate postsynaptic differentiation in non-innervated muscle regions  Figure . Even thhila NMJ ,55, a poDrosophila and C. elegans [Bone morphogenetic proteins (BMPs) belong to the transforming growth factor-β (TGF-β) superfamily and were originally named for their ability to induce ectopic bone formation ,73. Over elegans .Xenopus, Drosophila, zebrafish and mouse have shown the central role of BMPs during the development of dorsal-ventral patterning, patterning of the body axes, early patterning of the central nervous system, skeletogenesis, and development of heart, kidney, eyes and limb buds, among others [Besides their role in bone formation, different studies have shown that BMPs are multifunctional proteins with effects on diverse biological processes ranging from patterning and specification of several tissues and organs during embryonic development to adult tissues homeostasis. Genetic studies in g others –77.Drosophila NMJ. Additionally, we will focus on some in vivo evidence obtained in vertebrate models, showing changes in the expression of BMP signaling effectors upon spinal cord injury and regeneration. We discuss these findings on the light of the potential role that BMP pathways may have during vertebrate NMJ assembly and regeneration.In neuronal adult tissue, BMPs regulate several features of cell behavior. For instance, dendritogenesis, number of neurites, length of neurites and branch points have been shown to be stimulated or inhibited by different BMPs in diverse neuronal types, including cultured sympathetic, cortical, hippocampal, cerebellar and peripheral neurons –84. The Early events in BMP signaling involve the formation of heteromeric complexes of two types of transmembrane receptors with serine/threonine kinases activity, named type I and type II. BMPs binding to a preformed heteromeric complex of BMPRII and BMPRI initiate the classical Smad signaling pathway ,86. UponC-terminal tail following its kinase domain that is not required to induce Smad dependent or independent signaling [C-terminal tail of BMPRII is also present in its corresponding homologues in C. elegans, Drosophila and Xenopus [N-terminal kinase (JNK) [An interesting feature of BMP-mediated signaling is the fact that BMPRII bears a long ignaling . Remarka Xenopus –93. Some Xenopus , the c-Jse (JNK) , and these (JNK) . In a nese (JNK) ,95. ThusDrosophila have demonstrated that a key component of BMP signaling affecting the NMJ is wishful thinking (Wit), the BMPRII homolog, as Wit mutant larvae show a significant reduction in size and function of the NMJ [Wit mutants showed specific loss of the phosphorylated form of the Smad homolog Mad in embryonic motor neurons and decreased neurotransmitter release. In addition, ultrastructural examination of the neuromuscular synapse showed complete presynaptic membrane detaching from the postsynaptic membrane in certain areas [Wit mutant phenotype could be largely rescued by the expression of Wit in motor neurons, revealing a major function of this receptor in presynaptic cells at the invertebrate neuromuscular synapse [Genetic and biochemical evidence obtained at the invertebrate NMJ shows that retrograde BMP signaling plays crucial roles in presynaptic motor neurons to regulate synaptic growth, assembly and maintenance ,97–101.  the NMJ ,99. Wit in areas ,99. Impo synapse ,99.trio in motor neurons, which is required for normal synaptic growth at the NMJ. Gbb expression in muscle and overexpression of activated Tkv and Sax in motor neurons enhance the transcription of trio [Since Wit function is required in the presynaptic cell, it could transduce signals from the muscle to the motor neuron cell to coordinate synaptic growth with muscle growth. Impaired motor neuron synaptic growth and function has also been demonstrated in loss of function mutants either for the BMP homolog Glass bottom boat (Gbb), the BMPRI Saxophone (Sax) and/or Thickveins (Tkv) or the Smad homologs Mad and Medea ,102–104. of trio . Even th of trio . OverallC-terminal domain of Wit to interact with the actin cytoskeleton modulator DLIMK1 in motor neurons to stabilize the NMJ. Remarkably, this interaction is not required for Smad-mediated synaptic growth [In addition, mutants of the canonical BMP signaling cascade, including Gbb, Wit, Tkv, Mad and Medea, showed an increased number of sites of “synaptic footprints”, organized postsynaptic muscle membranes that lack opposing presynaptic neuronal markers; therefore, synaptic footprints identify regions of the NMJ where the terminal nerve once resided and has retracted . These fc growth . TherefoDrosophila models of motor diseases have also been connected to BMP signaling. Hereditary Spastic Paraplegia (HSP) is a group of genetic disorders characterized by retrograde axonal degeneration of the corticospinal tract and the posterior columns in the spinal cord that result in progressive spasticity and weakness of the lower limbs [Drosophila ortholog of the human HSP gene NIPA1 (Nonimprinted in Prader-Willi/Angelman), display a 2-fold increase in synaptic boutons. Importantly, presynaptic overgrowth at the NMJ in mutants is recovered by the specific expression of Spict in neurons, as well as by mutations in Tkv, Sax, Wit, Gbb and Medea, suggesting that Spict inhibits the BMP pathway in motor neurons [Drosophila models of motor diseases, such as amyotrophic lateral scelerosis and spinal muscular atrophy, correlate with down-regulation of BMP signaling . AtA physiological role for BMPs in adult motor neurons comes from experimental models of injury. For instance, the expression of BMP-2 mRNA is induced in motor neurons after crush injury of the facial nerve in rabbits . SimilarSpinal cord regeneration is strongly impaired by the formation of a glial scar that inhibits axonal regrowth at injured sites . Since BDifferent mutated genes have been identified in HSPs . Atlastiatl1) in developing zebrafish induced a prominent loss of larvae motility, associated with abnormal axon pathfinding and multiple aberrant branching of spinal motor neurons [atl1 morphants showed a significant increase in the amount of nuclear pSmad1/5/8, suggesting that atlastin inhibits BMP signaling. Consistently, atl1 knockdown defects were rescued by genetically or pharmacologically inhibiting the BMP pathway [For instance, morpholino-dependent knockdown of the ortholog of human atlastin ( neurons . Remarka pathway . As for  pathway . TogetheDrosophila mutants of Spict and zebrafish knockdown of atl1 display motor neuron presynaptic overgrowth and abnormal morphological motor neurons, respectively [Even though little is known regarding the molecular mechanisms by which HSP proteins modulate BMP signaling, recent evidence reveals that they could modulate the bioavailability of BMP receptors at the cell membrane. For instance, atlastin1 localizes to punctate vesicular structures in neurites and the cell soma of zebrafish spinal neurons, where it displays partial colocalization with BMPRI in late endosomes, which are distributed all along spinal axons . Therefoectively ,120. Thein vivo evidence points to an anterograde effect of Wnt signals on postsynaptic differentiation, whereas BMP ligands act more likely through retrogradely-induced mechanisms to induce presynaptic effects. It is still to be determined if, as in Drosophila, Wnt signaling affects motor neuron behavior in the context of the vertebrate NMJ.In the present review we have focused on recent evidence revealing that the well described roles of Wnt and BMP signaling at the invertebrate neuromuscular synapse could also take place at the vertebrate NMJ. As in invertebrate species, Remarkably, animal models of injured spinal cord show increased expression of BMP signaling molecules. In addition, BMP pathways are modulated by proteins involved in vertebrate motor neuron dysfunction. Therefore, even though the role of BMP signaling at the vertebrate NMJ has not been elucidated, this evidence points to a key role for BMPR-mediated signaling during vertebrate motor axon extension towards muscle cells for the assembly, maintenance and/or regeneration of the neuromuscular synapse. In this regard, the reported cross-talk between Wnt and BMP pathways ,122 coul


S-glutathionylation is the process of mixed disulfide formation between glutathione (GSH) and protein thiols. Until recently, protein-S-glutathionylation was associated with increased cellular oxidative stress, but S-glutathionylation of key protein targets has now emerged as a physiologically important redox signaling mechanism, which when dysregulated contributes to a variety of disease processes. In this review, we will explore the role of thiol oxidative stress and protein-S-glutathionylation in monocyte and macrophage dysfunction as a mechanistic link between oxidative stress associated with metabolic disorders and chronic inflammatory diseases, including atherosclerosis.Atherosclerosis is a chronic inflammatory disease involving the accumulation of monocytes and macrophages in the vascular wall. Monocytes and macrophages play a central role in the initiation and progression of atherosclerotic lesion development. Oxidative stress, which occurs when reactive oxygen species (ROS) overwhelm cellular antioxidant systems, contributes to the pathophysiology of many chronic inflammatory diseases, including atherosclerosis. Major targets of ROS are reactive thiols on cysteine residues in proteins, which when oxidized can alter cellular processes, including signaling pathways, metabolic pathways, transcription, and translation. Protein- Monocyte recruitment and migration are functional characteristics of the innate and adaptive immune system ,2. Recrui.e., smooth muscle cell migration, extracellular matrix deposition, and platelet activation -cysteine) in order to label intracellular glutathione, then separated the proteins by two-dimensional electrophoresis, and analyzed by mass spectrometry [l-[35S]-cysteine into cellular proteins [S-glutathionylated proteins that contain radioactive GSH can also be useful to identify S-glutathionylated proteins by Western blot, utilizing radiography, or by mass spectrometry [S-glutathionylated proteins couple enrichment techniques with mass spectrometry [N-ethyl maleimide (NEM) or iodoacetamide (IAM) to prevent further modification [S-glutathionylated proteins [S-glutathionylated proteins in cell lysates or tissues are matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) [One of the first proteomic studies used for the identification of trometry . Althougproteins . S-glutatrometry . Global trometry ,55,66. Afication ,55. Oxidproteins ,66. For proteins ,58,67–69LDI-TOF) ,55,65 anLDI-TOF) . Of thesS-glutathionylated proteins have been established as bona fide redox signaling proteins. Ras and PTP1B are two representative examples that have best fulfilled the signaling criteria outlined in Few S-glutathionylated in vitro using various reagents, including S-nitrosoglutathione (GSNO) [2O2, GSSG, and diamide [S-glutathionylated on residue C118 which increases ras activity and upregulates downstream signaling pathways, fulfilling the requirement for criteria #1 and #3 [S-glutathionylation on C118 was detected in vascular smooth muscle cells in response to angiotensin II (AngII)-induced hypertropic signaling [−) or oxLDL treatment in bovine aortic endothelial cells (BAECs) resulted in p21ras S-glutathionylation, increasing ras activity and subsequent downstream activation of Mek/Erk and phosphatidylinositide-3 kinase (PI3K) and protein kinase B (Akt) signaling [S-glutathionylation, thereby reducing insulin-induced Akt phosphorylation, which was restored with the C118S mutant [S-glutathionylation on C118 and thereby activation of the rapidly accelerated fibrosacroma (Raf)/Mek/Erk pathway [Ras proteins are a family of small GTPases that modulate downstream signaling pathways important in proliferation, differentiation, and migration, and are known oncogenes . Ras wase (GSNO) , GSH and diamide –74. Ras 1 and #3 ,75–77. Fignaling . Overexpignaling . Also inS mutant . MechaniS-glutathionylation studies used Grx-1 overexpression to demonstrate the reversibility of the modification, meeting criteria #5. Using catalase overexpression or an unspecific Nox inhibitor , H2O2 production was found to be necessary for ras-S-glutathionylation, although the specific Nox isoform involved was not identified [S-glutathionylation for activation of downstream signaling pathways in response to various sources of oxidative stress and mechanical strain.Many of the ras-entified . These s2O2-induces sulfenic acid formation and S-glutathionylation of its catalytic cysteine [S-glutathionylated protein that meets most of the criteria listed in Protein tyrosine phosphatase 1B (PTP1B) belongs to the larger family of cysteine-based phosphatases (CBP) that contains a nucleophilic and catalytic cysteine that is susceptible to oxidation. . PTP1B icysteine . PTP1B i−/−) mice [−/−) mice [−/− mice increased lesion area by 60% [−/− mice fed a diet supplement with L-2-oxo-4-thiazolidin carboylate (OTC), a cysteine precursor, show a significant increase in GSH levels and a reduction in both their lipid peroxide content and their ability to oxidize LDL [−/− mice fed a diet supplemented with buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, showed depleted GSH levels, resulting in increased lipid peroxide content and increased LDL oxidation [The contributions of redox-sensitive mechanisms that regulate monocyte and macrophage function and their importance in the development of atherosclerosis is not well established. Recent evidence suggests that alterations in the intracellular redox environment of monocytes and macrophages not only change their phenotype and functionalities, but also directly affect the development and progression of atherosclerotic lesions. For example, overexpression in mouse macrophages of glutathione reductase (GR), the enzyme responsible for reducing GSSG to GSH, decreases atherosclerosis lesion formation in low-density lipoprotein receptor deficient mice (LDLR/−) mice . Alterna/−) mice . Conversa by 60% . Pharmacdize LDL . OTC-fedxidation . Atheros−/− mice correlates with macrophage chemotactic activity in vivo, macrophage recruitment into atherosclerotic lesions and the severity of atherosclerosis in these mice [S-glutathionylation occurs within aortic lesions in particular macrophages in these atherosclerotic lesions, indicating that hyperglycemia and hyperlipidemia induce thiol oxidative stress in macrophages within atherosclerotic lesions [S-glutathionylation.The thiol redox status of peritoneal macrophages in diabetic LDLRese mice . The sam lesions . CollectS-glutathionylation may represent a key mechanistic link between oxidative stress and inflammation. Protein S-glutathionylation has been reported in alveolar macrophages (AMs), primary human blood monocytes, human monocyte-derived macrophages, THP-1 human monocytic cell-line, mouse peritoneal macrophages (PMs), and mouse macrophage cell-lines, but only few S-glutathionylated proteins have been validated as actual redox signaling proteins. Many of the studies addressing protein-S-glutathionylation in monocytes and macrophages measured changes in global S-glutathionylation. For example, lipopolysaccharide (LPS)-induced-stress increases protein-S-glutathionylation in AMs and promotes an inflammatory response by AMs, which was exacerbated with Grx-1 deficiency [S-glutathionylation, which correlates with macrophage viability [S-glutathionylation, which correlates with increased ROS formation and decreased macrophage recruitment [S-glutathionylation in oxLDL-treated mouse PMs were associated with increased oxidative stress, as measured by a decrease in the GSH/GSSG ratio, and increased cell death [S-glutathionylation for monocyte and macrophage function, but only few studies have actually identified specific S-glutathionylated proteins in monocytes and macrophages , two key proteins involved in monocyte motility, are diseases ,49.S-glutathionylation at cysteine 374 [S-glutathionylated even under resting cell conditions in both fibroblasts [The actin cytoskeleton, through a series of coordinated polymerization and depolymerization events, mediates chemokine-directed cell adhesion and migration necessary for cellular motility ,91. Overeine 374 –96. Interoblasts ,95 and Troblasts .S-glutathionylation. The ratio of filamentous (F) to globular (G) actin is used as an indicator of actin polymerization and actin turnover [S-glutathionylation accelerates actin turnover. These data suggest that by increaseing actin dynamics, metabolic stress enables monocyte to more effectively respond to cues from inflamed issues. However, the physiologic significance of this shift to a more proinflammatory monocyte phenotype is not yet clear. Nox4 colocalizes with actin in human monocyte-derived macrophages, providing a mechanism for the specific oxidation of actin thiols by Nox4 [S-glutathionylation and accelerated migration, indicating the functional relevance and reversibility of actin-S-glutathionylation. Our study identified Nox4-derived H2O2 as the oxidant mediating actin-S-glutathionylation, providing a molecular mechanism for this important oxidative modification that regulates actin dynamics and cell motility , also known as dual-specific phosphatases, have the same catalytic cysteine residue as PTP1B, a well-known site of tivation . MKPs cotivation ,104. In ormation .S-glutathionylation of 14-3-3zeta, targeting 14-3-3zeta for caspase-dependent degradation [S-glutathionylation did not affect binding of 14-3-3zeta to SSH1L, suggesting that degradation and overall loss of total 14-3-3zeta increases the pool of free, unbound SSH1L phosphatase, thereby preventing the phosphorylation and inactivation of cofilin in response to chemokine activation. Increases in free, active SSH1L phosphatase in monocytes resulted in cofilin phosphorylation and activation, and accelerated monocyte chemotaxis in response to MCP-1 [Cofilin, an actin binding protein that promotes actin depolymerization, undergoes phosphorylation and dephosphorylation in response to various extracellular stimuli that also trigger changes in the actin cytoskeleton ,106. Theradation . 14-3-3zto MCP-1 .bona fide redox signaling proteins. While some of these proteins meet a several of the criteria set out by Mieyal and coworkers that would allow them to be classified as redox-regulated molecules, most S-glutathionylated proteins do not meet all criteria, primarily because no intracellular mechanism has yet been identified that would account for the rapid and effective formation of this oxidative modification. Future studies should focus on verifying all the criteria required before classifying individual S-glutathionylated proteins as true redox signaling proteins. Research on protein-S-glutathionylation, particularly in monocytes and macrophages, is still in its infancy and future studies on redox sensitive mechanisms are likely to provide important new insight into the (dys)regulation of monocyte and macrophage function, particularly the molecular details that link oxidative stress associated with metabolic disorders to chronic inflammatory diseases. In summary, protein-S-glutathionylation is a thiol oxidative modification important in redox signaling and provides an important new mechanistic link between oxidative stress and inflammation.Redox signaling is evolving as an important signaling paradigm contributing to the regulation of cellular functions. Evidence is emerging that many cysteine-containing proteins are redox regulated, but to date very few have been validated as


Most patients with HTLV-1 infection are asymptomatic, however 3% of individuals develop a progressive neurological disorder, HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Factors leading to this complication are not well defined, but are associated with high levels of pro-virus and cytolytic T cells. During chronic viral infections, virus-specific CD8+ T cells undergo altered patterns of differentiation and can become restrained from effector activity. We hypothesized that suppression of immune receptors T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) and PD-1 would result in partial reversal of a T cell restraint profile. In turn this increased cytolytic activity may mediate neuro-immunopathology of HAM/TSP.We investigated the expression of Tim-3 and PD-1 on T cells in 22 serially recruited HTLV-1 asymptomatic and HAM/TSP patients and 7 HTLV-1-seronegative matched controls, their distribution on HTLV-1-specific T cells and the relationship with T cell function.Using flow cytometry, we found that patients with HAM/TSP had significantly lower levels of Tim-3+ PD-1- expressing CD8+ (p=0.002) and CD4+ (p=0.004) T cells compared to healthy uninfected controls. HTLV-1 Tax11-19, HLA-A*02 restricted CD8+ T cells among HAM/TSP individuals expressed markedly lower levels of Tim-3. Furthermore, we found that the frequency of Tim-3 expressing Tax11-19 specific CD8+ T cells inversely correlated with the number of IFN-γ secreting cells in response to the Tax Tax11-19 peptide.We propose that this low expression of Tim-3 on HTLV-1 Tax-specific T cells may lead to persistent and deleterious effector T cell pool, resulting in more inflammation and disease progression.


Here we demonstrate that Cx40.8 exhibits novel differential subcellular localization in vivo, depending on the growth status of the fin. During fin ontogeny, Cx40.8 is found at the plasma membrane, but Cx40.8 is retained in the Golgi apparatus during regeneration. We next identified a 30 amino acid domain of Cx40.8 responsible for its dynamic localization. One possible explanation for the differential localization is that Cx40.8 contributes to the regulation of Cx43 in vivo, perhaps modifying channel activity during ontogenetic growth. However, we find that the voltage-gating properties of Cx40.8 are similar to Cx43. Together our findings reveal that Cx40.8 exhibits differential subcellular localization in vivo, dependent on a discrete domain in its carboxy terminus. We suggest that the dynamic localization of Cx40.8 differentially influences Cx43-dependent cell proliferation during ontogeny and regeneration.Connexins (Cx) are the subunits of gap junctions, membraneous protein channels that permit the exchange of small molecules between adjacent cells. Cx43 is required for cell proliferation in the zebrafish caudal fin. Previously, we found that a Cx43-like connexin,  Connexins, the subunits of gap junction channels, are part of a large multigene family that includes about 20 genes in mammals CX43 cause the craniofacial and limb skeletal malformations associated with oculodentodigital dysplasia (ODDD) cx43 cause short bony fin ray segments associated with the short fin (sof) phenotype b123sof  allele that has reduced mRNA and protein levels j7e1sof  codes Cx43-F30V, j7e2sof  codes Cx43-P191S, and j7e3sof  codes Cx43-F209I). Each missense allele can form gap junction plaques, but channels exhibit aberrant ionic coupling properties Gap junctional intercellular communication (GJIC) is necessary for normal tissue development, evidenced by the identification of mutations in connexins that cause human disease cx43 is up-regulated in the proliferating cells of the blastema sof alleles exhibit reduced levels of cell proliferation during regeneration cx43-gene knockdown also causes reduced levels of cell proliferation The zebrafish caudal fin consists of 16–18 segmented bony rays, where fin length depends on the number and size of bony segments cx40.8, a cx43-like gene in the zebrafish genome cx40.8 gene shares 80% nucleotide identity with cx43, and is co-expressed with cx43 in the population of dividing cells during fin regeneration. Therefore Cx40.8 is a reasonable candidate for influencing Cx43 function. However, Cx40.8-EGFP localized to intracellular vesicles and not to gap junction plaques when expressed alone in HeLa cells cx43 and cx40.8 are both expressed in the proliferating cells of the regenerating fin, and since Cx43 and Cx40.8 can co-localize to common gap junction plaques in HeLa cells, one possibility is that Cx40.8 influences Cx43 function in dividing cells. Here, we test this hypothesis during zebrafish fin regeneration. Strikingly, we find that Cx40.8 exhibits differential subcellular localization either to the plasma membrane (during ontogeny) or to the Golgi apparatus (during regeneration). This dynamic localization is dependent on a 30 amino acid sequence immediately following the fourth transmembrane-spanning domain (TM4) of Cx40.8. We also show that that the channel properties of Cx40.8 are similar to Cx43, suggesting that Cx40.8 does not directly influence Cx43-based GJIC. Together, these findings reveal that Cx40.8-dependent subcellular localization is correlated with the growth rate of fins, perhaps by regulating a non-channel function of Cx43.The zebrafish genome includes a large connexin gene family (n = 37) in vivo, an antibody was generated against an internal peptide located in the carboxy terminus ). In the transgenic line, GalT-GFP is expressed under the control of the zebrafish β-actin promoter , resulting in GFP-labeled Golgi apparatus in most cells of the fin. We found that Cx40.8 immunostaining co-localizes with GalT-GFP  with GFP . Results show that Cx40.8-BCD-EGFP forms gap junction plaques at the plasma membrane, resembling the behavior of Cx43, whereas Cx43-bcd-EGFP remains intracellular, resembling the behavior of Cx40.8 . Thus, tTo establish conclusively that the Cx40.8 “b” domain is responsible for the intracellular localization of Cx40.8 in HeLa cells, two additional constructs were tested. The first construct deletes the “b” region from Cx40.8 (i.e. Cx40.8-cd-EGFP) and results in localization of Cx40.8-cd-EGFP into gap junction plaques at the plasma membrane . Thus, “Although EGFP fusions to connexins are widely utilized for following subcellular localization, it remained possible that such fusions could lead to aberrant localization. For example, Cx43-EGFP fusions fail to bind to the recycling factor ZO-1, leading to reduced turnover and expanded gap junctions We previously found that Cx40.8 is capable of co-localizing to gap junction plaques in HeLa cells when co-transfected with Cx43 j) was measured while transjunctional voltage (Vj) was varied up to ±180 mV in 30 mV steps. Voltage was stepped in one cell of the pair while the other cell remained clamped at 0 mV. Step duration was either 400 ms or 4 s. Representative current families from the 4 s protocol for cell pairs expressing Cx43-EGFP and Cx40.8-cd-EGFP constructs are shown (j) declining to 50.4±21.5% at Vj of +180 mV (n = 4), confirming results obtained previously from paired oocyte recordings j from the 4 s protocol was plotted as a function of Vj, and normalized to the conductance at ±30 mV . We performed dual whole cell voltage clamp in N2a cells expressing either Cx43-EGFP or Cx40.8-cd-EGFP. Transjunctional current . Also, the juxta-TM4 domain of mammalian Cx43 has been shown to bind microtubules, which may influence its localization to the plasma membrane can cause Cx43 to be retained intracellularly (i.e. Cx43-bBCD). However, recall that the proximity of the juxta-TM4 domain to TM4 is also significant, since adding the “b” domain to Cx43 following its native “B” domain (i.e.Cx43-BbCD) does not appear to alter Cx43 trafficking. Perhaps this domain is uniquely situated to influence the trafficking or retention of connexins.Previous studies have found that the juxta-TM4 domain is required for connexin trafficking to the plasma membrane. For example, the mammalian Cx32-TM4 sequence  has been shown to be required for localization to the plasma membrane in vivo. This and other possibilities will be explored in future experiments.At least two other connexins have been found in intracellular compartments and not at the plasma membrane. For example, mouse Cx46 is retained in a compartment of the trans-Golgi network in ROS osteosarcoma cells, although the functional significance of this finding has not been revealed Cx40.8 is a unique example of a connexin whose subcellular localization depends on the growth status of a tissue. Differential localization appears to be regulated by its juxta-TM4 domain. It is of interest to define the mechanism of this “switch.” For example, the Cx40.8 “b” sequence may interact with a hypothetical retention factor in the Golgi apparatus, preventing it from trafficking to the plasma membrane. Association between the retention factor and Cx40.8 may be reduced at the completion of regeneration, allowing the synthesized Cx40.8 to move to the plasma membrane. Once located at the plasma membrane, we suggest that Cx40.8 negatively influences Cx43 activity, contributing to reduced levels of cell proliferation associated with ontogenetic growth. It remains unclear if Cx40.8 forms homomeric heterotypic gap junction channels with Cx43 in vivo, and/or if Cx40.8 and Cx43 also hetero-oligomerize in common connexons that are initially retained in the Golgi. Both newly translated Cx43 and Cx40.8 must travel through the Golgi apparatus during fin regeneration. We have not identified a large pool of Golgi-retained Cx43 by immunofluorescence, however this does not preclude the possibility that a small portion of Cx43 is retained with newly assembled Cx40.8 connexons. Biochemical analyses will be required to determine the composition of the connexons and gap junction channels in vivo.In conclusion, we find that Cx40.8 exhibits an unusual change in subcellular localization depending on the growth state of the zebrafish fin. Localization appears to depend on its juxta-TM4 domain, which may represent a common domain for the regulation of connexin trafficking . Continued analyses will define the novel mechanisms of regulating both Cx40.8 protein location and Cx40.8-dependent regulation of Cx43 activity.This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols used for this manuscript were approved by Lehigh's Institutional Animal Care and Use Committee (IACUC) . Lehigh University's Animal Welfare Assurance Number is A-3877-01.Tg zebrafish were generated using Invitrogen Gateway technology to fuse amino acids 1–6 from human B4GALT1 with GFP under the control of the beta-actin promoter and inserted into the zebrafish genome using sites derived from the tol2 transposon. Lines were maintained on the TAB14 background.The fish used in this study were derived from the C32 strain and were raised in a 14 light:10 dark photoperiod at 25°C www.qcb.com). QCB completed peptide synthesis, immunization, rabbit maintenance, and affinity purifications of bleeds. Escherichia coli lysates expressing GST-Cx40.8CT fusion protein were grown to confluency before adding 0.3 mM IPTG to induce protein expression. Lysates were prepared using 50 µg/ml lysozyme in lysis buffer . In addition, 2.2 N NaOH and 8% BME were added to the mixture. Total protein was precipitated using TCA and pellets were resuspended in SDS buffer. Samples were diluted in SDS buffer and decreasing volumes of sample were loaded in a total volume of 20 µl. For the antibody competition, identical gels were prepared. The anti-Cx40.8 antibody was either used directly (1∶2000) or following pre-incubation with the peptide made as the Cx40.8 antigen. Wildtype fins (5 dpa regenerates) were homogenized in homogenization buffer  using a 5 mm generator . Protein samples were separated using 12% SDS-PAGE before transfer to nitrocellulose membranes. Following transfer, blots were rinsed in 40% isopropanol, rinsed in distilled water, and then blocked in 5% milk in SuperTBST for 30 min at room temperature. Blots were then incubated with anti-Cx40.8 (1∶2000) for 1 hr at room temperature, rinsed for 40 min in TBST, and then incubated with peroxidase-conjugated goat anti-rabbit IgG  for 1 hr at room temperature. Following incubation, blots were rinsed in TBST for 40 min at room temperature. Using the Amersham ECL Plus Western Blotting Detection System , blots were developed and exposed to X-ray film .Amino acids 269–285 (CSAPVPNLGYNLDTVDK) of zebrafish Cx40.8 were chosen as the antigen in conjunction with Quality Controlled Biochemicals , 5 dpa regenerates were harvested and processed identically except for the antibodies. For primary antibodies, the rabbit Cx40.8 antibody (1∶200) and the mouse EGFP  antibodies were used together. The secondary antibodies were the goat anti-rabbit Alexa 546 and the goat anti-mouse Alexa 488. In the experiments with Brefeldin A , 70 nl of a 10 µg/ml solution was injected into the dorsal fin rays of the regenerating caudal fin. After 2 hours, fins were harvested and processed as above. TO-PRO3 (Molecular probes) was added at 1∶1000 with the secondary antibodies. Laser scanning confocal imaging was performed on whole fins. Images were acquired using a 40X N.A. 1.4 PlanApo DIC objective on an inverted microscope  equipped with an LSM510META scan head. Argon ion (488), 543 HeNe (546), and 633 HeNe (far red) lasers were used to generate the excitation lines and multitrack sequential excitation was utilized to avoid bleed-through between fluorophores. Files were exported as TIFF files.Wildtype fins  were harvested and fixed in 2% PFA in 0.1 mM phosphate buffer (PB) for 30 min at room temperature. Fins were washed (3×10 min) in 25 mM PB and incubated in Trypsin/EDTA (Gibco) for 10 min on ice. Fins were next blocked  for 30 min at room temperature and incubated with Cx40.8 (1∶200) antibody overnight at 4°C. After incubation, fins were washed in 25 mM phosphate buffer (3×5 min) and incubated in goat anti-rabbit Alexa 488  antibody and propidium iodide for 2 hr at room temperature followed by 1× PBS washes (3×10 min). For http://www.genewiz.com/). Cx43-bcd, Cx40.8-BCD, Cx40.8-cd, Cx43-bBCD, and Cx43BbCD were all generated by a common strategy using two sequential rounds of PCR. For example, Cx43-bcd was generated as follows. Two chimeric internal oligos were generated. The first internal oligo shared 20 bases of 3′ complementarity to the Cx43-TM4 followed by 20 bases of complementarity to the start of the Cx40.8 carboxy tail. When paired with a 5′ oligo specific to the first 20 bases of the Cx43 coding sequence (and using Cx43-EGFP plasmid as template), the first half of Cx43 was amplified through the end of TM4, with the first 20 bp of Cx40.8 as an overhang. The second internal oligo had a 5′ overhang of the final 20 bases of Cx43-TM4 followed by the first 20 bases of the Cx40.8 carboxy tail. When paired with a 3′ oligo specific to the final 20 bases of Cx40.8 (but not the stop codon), and using Cx40.8-EGFP plasmid as template, the carboxy tail of Cx40.8 with the last 20 bp of Cx43-TM4 as an overhang is amplified. Together, these two products (sharing 40 bases of overlap across TM4 and the start of the carboxy tail) served as template for the next PCR reaction. Using the ‘outside’ oligos for amplification (i.e. the 5′ cx43 oligo and the 3′ cx40.8 oligo), a chimeric connexin of Cx43 through TM4 and the carboxy tail of Cx40.8 was generated. This final product was subcloned into pN1-EGFP and sequenced. The same strategy was used to generate Cx40.8-BCD, Cx40.8-cd, Cx43-bBCD, and Cx43BbCD. All internal oligos were HPLC-purified to insure that they were the correct length. The untagged constructs were generated by isolating the EcoRI fragments from their pN1-EGFP backbones. The EcoRI fragments were subcloned into the pIRES2 vector. Orientation was determined using a BamHI digest.The Cx43-EGFP, Cx43-mApple and Cx40.8-EGFP were previously described 2 and 37°C and grown in tissue culture dishes with minimal essential media supplemented with 10% FBS, antibiotics, and antimycotics (Invitrogen). The cells were seeded onto poly-L-lysine coverglasses, incubated overnight, then transfected with Superfect (Qiagen) for 3 hours using 2 µg of plasmid DNA and imaged 21–24 hours later. Transfections were evaluated by standard immunofluorescence on a Nikon Eclipse E80 microscope. For double transfections, 1 µg of each plasmid was transfected. Analyses were completed on a Nikon Eclipse TE2000-U using the 100× lens in combination with a 1.5× optivar to increase the primary magnification.HeLa and N2a cells (ATCC) were stored at 5% COFor dual whole cell voltage clamp recording, the poly-L-lysine cover glass was placed into a chamber at room temperature perfused with oxygenated extracellular solution . N2a cells were imaged on a Nikon (EF-4) Physiostation equipped with epifluorescence and infra red differential interference contrast optics. GFP positive cell pairs expressing connexin constructs were located by fluorescence and imaged using a Hammamatsu C5700 video camera . Cells contacting multiple GFP positive neighbors were excluded.Patch pipettes were pulled from thick walled borosilicate glass capillary tubes (WPI 1B120F-4) using a two-stage puller  to a resistance of 4–6 MΩ when back-filled with internal solution . Patch pipettes were then brought into contact with cells using motorized micromanipulators  and gigaohm seals were established. Whole cell configuration was achieved and cells were initially voltage clamped to 0 mV using a Multiclamp 700B amplifier . Series resistance was compensated at 60–80%. The signal was filtered at 1–2 kHz and digitized at 10 kHz with a Digidata 1440 data acquisition board. Recording protocols were generated using Clampex 10.2 software .j) was varied up to ±180 mV in steps of 30 mV. Step duration was either 400 ms or 4 s. Recordings were analyzed using Clampex 10.2 software. Transjunctional conductance values were calculated from mean transjuctional current (Ij) amplitudes from a 15 ms window measured at 5 ms after pulse onset. Steady state Gj was computed from Ij measured at 5 ms before the termination of the pulse. Gj was maximal and generally linear at voltages <60 mV so steady state Gj was normalized to the conductance value calculated at ±30 mV. Data in the results text are expressed as percent of maximum Gj ± SD. Error bars in figures represent SE.Voltage clamp methods followed Figure S1Cx43-mApple and Cx40.8-EGFP co-assemble in common gap junction channels. High resolution fluorescence microscopy was used to provide evidence for co-association of Cx43-mApple and Cx40.8-EGFP in common gap junction channels. Plane views are shown. Top: In HeLa cells co-transfected with Hs-Cx43-EGFP and Hs-Cx43-mApple, both green and red plaques completely overlap. Middle: In HeLa cells co-transfected with Hs-Cx26-GFP and Hs-Cx43-mApple, discrete domains of green and red plaques are observed. Bottom: In HeLa cells co-transfected with Dr-Cx40.8-EGFP and Dr-Cx43-mApple, both green and red plaques completely overlap.(TIF)Click here for additional data file.


The innate ability of the human cell to silence endogenous retroviruses through RNA sequences encoding microRNAs, suggests that the cellular RNAi machinery is a major mean by which the host mounts a response against contemporary retroviruses, such as HIV-1 and HTLV-1. Several recent publications have identified cellular miRNAs that target and hybridize to specific sequences of both the HIV-1 and HTLV-1 transcripts. However, much like the variety of host immune responses to retroviral infection, the virus itself contains mechanisms that assist in the evasion of viral inhibition through manipulation of the cellular RNAi pathway. Retroviruses can hijack both the enzymatic and catalytic components of the RNAi pathway, in some cases to produce novel viral miRNAs that can either assist in active viral infection or promote a latent state of infection. Here, we propose that HTLV-1 viral proteins contribute to the dysregulation of the RNAi pathway by altering expression of key components of the pathway. A survey of uninfected and infected cell lines revealed that Drosha was present at lower levels in all HTLV infected lines. Additionally, transfection of HeLa cells with Tax shows colocalization of Tax and Drosha in the nucleus (speckles), suggesting that the HTLV-1 viral transactivator physically interacts with Drosha and targets it to specific areas of the cell. This data suggests the direct interaction of HTLV-1 viral components with RNAi machinery proteins which may lead to their dysregulation in infected cells.


The growth hormone secretagogue receptor (GHSR), also known as the ghrelin receptor, is involved in mediating a wide variety of biological effects of ghrelin, including: stimulation of growth hormone release, increase of food intake and body weight, modulation of glucose and lipid metabolism, regulation of gastrointestinal motility and secretion, protection of neuronal and cardiovascular cells, and regulation of immune function. Dependent on the tissues and cells, activation of GHSR may trigger a diversity of signaling mechanisms and subsequent distinct physiological responses. Distinct regulation of GHSR occurs at levels of transcription, receptor interaction and internalization. Here we review the current understanding on the intracellular signaling pathways of GHSR and its modulation. An overview of the molecular structure of GHSR is presented first, followed by the discussion on its signaling mechanisms. Finally, potential mechanisms regulating GHSR are reviewed. Two mechanisms have been reported to mediate the GHSR1a-induced [Ca2+]i signaling: the dominant phospholipase C (PLC)/inositol  triphosphate (IP3) signaling pathway and the debated protein kinase A (PKA)/cAMP pathway. Ligand binding activates the GHSR1a, induces the dissociation of the Gαq/11-subunit which subsequently stimulates the production of PLC. PLC cleaves the membrane lipid phosphoinositol 4,5 diphosphate  P2) into IP3 and diacylglycerol (DAG). IP3 binds with IP3 receptor to trigger the release of calcium from stores inside the endoplasmic reticulum, which contributes to the initial rise in [Ca2+]i. DAG activates the protein kinase C (PKC) which inhibits potassium channels leading to membrane depolarization, subsequent opening of voltage-gated calcium channels and extracellular calcium influx i independent of the concentration of IP3. Pretreatment with the Gαs subunit activator cholera toxin (CTX), the adenylate cyclase inhibitor MDL-12,330 A, or the PKA inhibitor H-89 blocks the effect of adenosine on GHSR1a-induced [Ca2+]i signaling i signaling relates to the stimulation of growth hormone release. In pituitary cells, both non-endogenous and endogenous GHSR1a agonists stimulate growth hormone release in a [Ca2+]i dependent manner. GHSR1a-induced increase in [Ca2+]i may trigger the release of neurotransmitters/hormones and gene expression. In the arcuate nucleus, GHSR1a induces [Ca2+]i signaling in NPY neurons [2+]i signaling employed by GHSR1a has been reported to either stimulate or attenuate insulin release in the isolated pancreatic islet cells i signaling is only observed in cells transfected with GHSR1a, but not in native cells. These results suggest a potential interaction between GHSR1a and the adenosine receptor. Such an interaction appears to occur at the intracellular signaling levels. Further experiments reveal that adenosine signals through adenosine receptor 2b to potentiate the GHSR1a-mediated [Ca2+]i signaling by a mechanism involving Gαs/cAMP/PKA mediated phosphorylation and its subsequent activation of IP3 receptors i mobilization, which is completely restored after blockade of the 5-HT2C receptor i and calcium/calmodulin kinase IV (CaMK IV)/cAMP responsive element-binding protein (CREB) signaling pathways. PLC/IP3 signaling is the first specifically associated with the GHSR1a constitutive activity [3 production and [Ca2+]i mobilization are mediated via Gαq/11 protein. Constitutive activity of GHSR1a is also detected by CRE reporter gene assay and serum response element (SRE) luciferase assay -substance P, an inverse agonist for GHSR1a, significantly decreases food intake and body weight likely by reducing gene expressions of neuropeptide Y (NPY) and uncoupling protein 2 (UCP2) in the hypothalamus [ghrelin or GHSR1a gene knockout mice. GHSR1a constitutive activity increases limbic seizures in rodents. GHSR1a knockout mice demonstrate a higher seizure threshold than their wild-type littermates when treated with pilocarpine. Inverse agonism and desensitization/internalization of the GHSR1a attenuate limbic seizures in rats and epileptiform activity in hippocampal slices [Emerging evidences supports the physiological relevance of GHSR1a constitutive activity in growth hormone release, food intake and neural activation. The association of GHSR1a constitutive activity with PLC/IPtion Phe2Leu affec stature . This cothalamus . All thel slices . Other sl slices , hippocal slices , and arol slices . Further5.Studies on the GHSR1a have revealed many fundamentals on its molecular structure, intracellular signaling, constitutive activity, and interaction with other GPCRs. Many of these findings are relevant to the physiological functions of GHSR1a and of interest for translational research. Future opportunities for GHSR1a research are four-fold: (1) to define the mechanism underlying the tissue specific response in GHSR1a-mediated intracellular signaling; (2) to examine the tissue specificity of GHSR1a constitutive activity and ligand-induced response and their physiological relevance; (3) to explore the integrative function of GHSR1a in the intricate regulatory network governing energy metabolism and metabolic homeostasis; (4) to evaluate the potential applications of GHSR1a agonists, antagonists or inverse agonist in the treatment of obesity and its related metabolic diseases.


Understanding the regulation of radial growth that underlies wood development is of great importance for future use of tree products as a renewable resource. For this purpose, we have performed a detailed analysis of cytokinin function in the regulation of cambial development in a tree stem. Furthermore, we have identified potential cytokinin signaling response genes active during the secondary development in Arabidopsis and currently studying the best candidate genes also in Populus. In the cambium, expression of cytokinin receptor genes peaks in the actively dividing cells. This indicates that cytokinin signaling pathway is a major regulator of cambial cell proliferation. We have previously shown in transgenic Populus trees that we can slow down the cambial activity by reducing the level of cytokinin signaling in it . To studWe have shown that the cambial cell division activity, and through it the production of stem biomass, can be enhanced or decreased through elevated or decreased cytokinin signaling. Together, our results show that cytokinins are major rate-limiting hormonal regulators of cambial development. Our study provides potential for applied research towards future breeding of faster growing tree cultivars.