MicroRNAs (miRNAs) are a class of small noncoding RNA molecules that play important roles in carcinogenesis and tumor progression. In this study, we investigated the roles and mechanisms of miR-140 in human non-small cell lung cancer (NSCLC). We found that miR-140 is significantly downregulated in NSCLC tissues and cell lines. Both gain-of-function and loss-of-function studies demonstrated that miR-140 suppresses NSCLC cell proliferation, migration, and invasion in vitro. Importantly, overexpression of miR-140 effectively repressed tumor growth and metastasis in nude mouse models. Integrated analysis identified IGF1R as a direct and functional target of miR-140. Knockdown of IGF1R inhibited cell proliferation and invasion resembling that of miR-140 overexpression, while overexpression of IGF1R attenuated the function of miR-140 in NSCLC cells. Together, our results highlight the significance of miR-140 and IGF1R in the development and progression of NSCLC. Lung cancer is the number one cause of cancer-related death worldwide, and non-small cell lung cancer (NSCLC) accounts for at least 80% of all lung cancer cases ,2. DespiMicroRNAs (miRNAs) are a class of small noncoding RNA molecules that negatively regulate the expression of target genes by either mRNA degradation or translational inhibition . miRNAs miR-140 has attracted much attention because it is involved in the development and progression of various types of cancers, including breast cancer, osteosarcoma, colon cancer and hepatocellular carcinoma –20. Thes2 humid atmosphere at 37 °C.Human NSCLCs and their matched normal tissues (at least 5 cm away from primary tumor) were obtained from 30 patients at Zhongshan Hospital, Fudan University . The tissues were snap-frozen in liquid nitrogen and stored at -80 °C until RNA extraction. Written informed consent was obtained from each patient and this study was approved by the Medical Ethics and Human Clinical Trial Committee at Zhongshan Hospital. Five NSCLC cell lines  and a normal lung bronchus epithelial cell line BEAS-2B were purchased from American Type Culture Collection and cultured in DMEM  supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin . All cells were incubated in 5% COIGF1R and β-actin were performed using SYBR Premix Ex Taq . The primers used were as follows: IGF1R forward primer, 5’- GAGAAGGAGGAGGCTGAATACCG-3’; IGF1R reverse primer, 5’-GTGATGTTGTAGGTGTCTGCGGC-3’; β-actin forward primer, 5’- AGTGTGACGTGGACATCCGCAAAG-3’; and β-actin reverse primer, 5’-ATCCACATCTGCTGGAAGGTGGAC-3’. Real-time PCR was performed using the ABI 7900 real-time PCR machine. The relative expression of each gene was calculated and normalized using the 2−ΔΔCt method relative to U6 snRNA or β-actin.RNA was extracted from tissues and cell lines using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. The expression of mature miRNAs was assayed using TaqMan MicroRNA Assays . A two-step qRT-PCR was employed with specific primers for miR-140 designed by Applied Biosystems. U6 snRNA was amplified as an internal control. qRT-PCR analyses for 6) were infected with 1×107 lentivirus transducing units in the presence of 10 µg/mL polybrene . Empty lentiviral vector was used as a control. The miR-140 inhibitor and negative control were obtained from Genepharma . Oligonucleotide transfection was performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. Cells were collected 48 h after transfection.The miR-140 precursor and IGF1R siRNA were purchased from Origene . The pre-miR-140 sequence and IGF1R siRNA sequence were cloned into pCDH-CMV-MCS-EF1-coGFP constructs . The production and purification of lentivirus were performed previously described . Target 5’-ATACTCGAGTTTCCATGCAACCTCCTTCTGC-3’ (forward) and 5’-AGCAAGCTTTCCATCTTCCAAGGAGGAGGCT-3’ (reverse). Endonuclease restriction sites (XhoI/HindIII) were incorporated in primers to facilitate ligation into the pGL3 Basic Vector . Site-directed mutagenesis of the miR-140 seed sequence in the IGF1R 3’-UTR (Mut) was performed using the QuikChange™ Site-Directed Mutagenesis Kit . For luciferase assays, the reporter plasmid was cotransfected with a control Renilla luciferase vector into A549 and H157 cells in the presence of either miR-140 or miR-control. After 48 h, cells were harvested, and the luciferase activity was measured using the Dual-Luciferase Reporter Assay System .The coding sequence of IGF1R was purchased from Origene and cloned into pcDNA3.1 (+) to generate IGF1R expression vectors. The wild-type IGF1R 3’ UTR (WT) was amplified with the following primers: 3) were seeded in 96-well plates in 100 µl culture medium and cultured. The proliferation of the cells was assayed at the indicated time points using a CCK-8 kit  according to the manufacturer’s instructions. For cell cycle analysis, infected or transfected cells were fixed in 75% ethanol and stained with 50 µg/mL propidium iodide (PI). The cell cycle distribution was analyzed in a FACScan ﬂow cytometer . Cell apoptosis assays were performed using an Annexin V-FITC/PI Apoptosis Detection Kit (BD Biosciences). 1×104 cells were stained according to the manufacturer’s protocol and then analyzed with a flow cytometry (BD Biosciences) equipped with a CellQuest software (BD Biosciences).Cells (2×105 cells in serum-free media were added into the upper chamber of an insert precoated with Matrigel (BD Bioscience). The lower chamber was filled with DMEM with 10% fetal bovine serum. After 48 hours of incubation, the cells remaining on the upper surface of the membrane was removed, whereas the cells that had invaded through the membrane were stained with 20% methanol and 0.2% crystal violet, imaged, and counted under a microscope .Cell migration was assessed by wound healing assays. Cells were seeded in six-well plates and cultured to 100% confluence. Wounds were generated in the cell monolayer using a plastic pipette tip. The cells were then rinsed with PBS and cultured for another 48 hours. The spread of wound closure was observed and photographed under a microscope. For invasion assays, 1×10Western blotting analysis were performed as previously described . Briefly6 cells for each mouse). The mice were observed over 5 weeks for tumor formation. The tumor volume (V) was monitored weekly and calculated using the formula: V = 0.5 × length × width2. For in vivo metastasis assays, 1.5×106 A549-miR-140 or A549-miR-control cells were injected into the caudal vein of nude mice (5 per group). After 6 weeks, the mice were killed, and lung metastatic colonization was monitored and quantified.All animal experiments were approved by the Ethical Committee on Animal Experiments of the University of Fudan Animal Care Committee, Shanghai, China. For tumor growth assays, A549 cells infected with either the miR-140-overexpressing lentivirus or the control lentivirus were injected subcutaneously into the right scapulas of nude mice . The results showed that miR-140 expression was significantly decreased in NSCLC tissues compared with their matched normal tissues . In addiTo better understand the role of miR-140 in the development of NSCLC, we first constructed a lentiviral vector expressing miR-140 and established stable cell lines, denoted as A549-miR-140 and H157-miR-140 after lentivirus infection. Successful overexpression of mature miR-140 in these cells was confirmed by qRT-PCR . As showWe further investigated whether miR-140 could also inhibit cell migration and invasion in NSCLC. Using the wound healing assay, we found that overexpression of miR-140 dramatically suppressed tumor cell mobility in A549 and H157 cells compared with their corresponding controls . SimilarTo further determine the effect of miR-140 on NSCLC tumor growth and metastasis in vivo, A549-miR-140 cells and A549-miR-control cells were inoculated subcutaneously into the flank of nude mice and the animals were closely monitored for tumor growth for 5 weeks. The results illustrated that miR-140-overexpressing tumors were significantly smaller in size and tumor volume compared to the control tumors . FurtherTo elucidate the molecular mechanisms by which miR-140 executes its function, we searched for potential targets of miR-140 using different computational methods, such as TargetScan and miRanda. In particular, we focused on oncogenes. IGF1R was identified as one candidate target of miR-140, because the complementary sequence of miR-140 was identified in its 3’ UTR by TargetScan analysis . We founTo further examine whether miR-140 exerts its tumor suppressor function through downregulation of IGF1R, we performed gain-of-function and loss-of-function analyses. Firstly, A549 cells were infected with lentiviral constructs containing IGF1R siRNA or the negative control. Western blotting analysis confirmed that the expression of IGF1R was suppressed . As expemiRNAs have been reported to play important roles in carcinogenesis and tumor progression ,24. AlteIn this study, we showed that miR-140 expression was significantly decreased in NSCLC tissues and cell lines. Overexpression of miR-140 could effectively inhibit NSCLC cell proliferation, induce cell cycle arrest, enhance apoptosis, and suppress tumor growth in nude mice. Furthermore, miR-140 overexpression significantly repressed cell motility and invasion in vitro and tumor metastasis in vivo. Accordingly, knockdown of miR-140 promoted cell proliferation and invasion. These results suggest that miR-140 might be a novel tumor-suppressor miRNA in NSCLC.To elucidate the underlying mechanisms involved in the miR-140-induced inhibition on NSCLC growth and metastasis, we used different prediction algorithms to predict gene targets for miR-140. The insulin-like growth factor-1 receptor (IGF1R) oncogene, which is frequently overexpressed in many malignancies and functions as an important regulator of cell proliferation, survival, and metastasis –29, was In conclusion, the present study showed that miR-140 is significantly downregulated in NSCLC tissues and cell lines. Overexpression of miR-140 inhibits tumor growth and metastasis of NSCLC through directly targeting IGF1R. Our data suggest that the frequently downregulated miR-140 leads to the increased expression of IGF1R and in turn contributes to the development and progression of NSCLC.Figure S1Konckdown of miR-140 promotes cell proliferation, migration, and invasion of H520 cells.(A) H520 cells were transfected with anti-miR-140 or anti-control, and the expression of miR-140 was analyzed by qRT-PCR. (B) cell viability assay (CCK-8). (C) cell apoptosis assays. (D) cell cycle analysis. (E) Wound healing assays. (F) Transwell invasion assays. *P<0.05, **P<0.01.(TIF)Click here for additional data file.Figure S2IGF1R is upregulated in NSCLC tissues.The expression of IGF1R in NSCLC tissues and matched normal lung tissues was measured by qRT–PCR. **P<0.01.(TIF)Click here for additional data file.


Human T-cell lymphotropic virus type I (HTLV-I) was the first human retrovirus discovered , and it HAM/TSP results in demyelination of the spinal cord and clinical manifestations of this disease include progressive muscle weakness and hyperreflexia of the lower limbs, sensory disturbance, urinary incontinence, and impotence -11. ThesThe precise pathophysiology of HAM/TSP is not yet clear but, previous studies suggested that both HTLV-I subgroups, host genetic and immunological factors may be associated with the development of HAM/TSP, particularly cytokine gene polymorphisms , 15. ProIn patients with HAM/TSP, significantly higher proviral loads have been observed compared to asymptomatic carriers, suggesting that active HTLV-I viral replication plays a key role in the development of this disease. A recent study has shown that a high level of Tax expression and low CD8+ anti-viral efficiency are correlated with high proviral load (PVL) and HAM/TSP development .In a previous study the proviral load and host genetic risk factors for development of HAM/TSP between Iranian and Japanese HTLV-I infected individuals were compared, and it was found that the median HTLV-I proviral load of Iranian HAM/TSP patients was two-fold greater in HAM/TSP patients than in healthy carriers (HCs), whereas that of Japanese HAM/TSP patients was 13-fold greater than in HCs. In addition, The HTLV-I proviral load in Iranian HCs was significantly higher than Japanese HCs. These significant differences of proviral load between two populations reflect the role of genetic factors such as the human leukocyte antigen (HLA) genotype in populations. In this study the Iranian genome DNA samples were extracted from the whole blood but Japanese samples were from the peripheral blood mononuclear cells (PBMCs). The Iranian proviral load was probably underestimated in comparison with Japanese ones . The aimStudy populationIn this retrospective study, files of 102 HAM/TSP patients and 34 HCs from Iranian individuals whom were referred to ACECR Khorasan Central Medical Lab and Navid Medical Lab  were reviewed. All these subjects were resident of Khorasan province, Iran. Japanese study population consisted of 222 HAM/TSP patients and 184 HCs from Kogoshima, Japan and Brazilian population included 92 HAM/TSP patients and 242 HCs from Salvador, Brazil. HTLV-I infection was determined using a HTLV-I antibody serological test. The diagnosis of HAM/TSP was performed in accordance with the World Health Organization criteria. The demographic information , and neurological symptoms of HAM/TSP were collected from medical recorded files in Ghaem Hospital, Mashhad University of Medical Sciences . HTLV-I proviral load and clinical manifestation in Japanese and Brazilian populations obtained from published studies , 18-23.HTLV-I proviral load measurement4 and expressed as the number of HTLV-I proviruses per 104 PBMCs  were isolated from EDTA-treated blood samples by Ficoll density gradient . A real time PCR using a commercial absolute quantification kit  was performed to measure the proviral load of HTLV-I in PBMCs using specific primers, and a fluorogenic probe by a Rotorgen Q  Real-Time PCR machine. The HTLV-I copy number was reported as an actual amount of cellular DNA by means of quantification of the albumin gene as the reference gene. HTLV-I and albumin DNA concentrations were calculated from two 5-point standard curves. The normalized value of the HTLV- I proviral load was calculated as the ratio of ×1004 PBMCs .Statistical analysisData was analyzed by Nonparametric Mann-Whitney and Spearman’s correlation tests using SPSS/ver.19 software. Descriptive data were summarized as mean, standard error (SE), and percents. A p value < 0.05 was considered as statistically significant, and 95% confidence intervals (CI) were also estimated.HTLV-I proviral load4 PBMCs in an immune-compromised patient, and minimum HTLV-I proviral load was 10 copy number/104 PBMCs in a HCs subject. The mean age of HAM/TSP patients (43.07 ± 1.478 years) was higher than HCs group (39.68 ± 2.290 years). Characteristics and proviral load of HAM/TSP patients and HCs subjects are summarized in HTLV-I proviral load was determined in 136 Iranian individuals infected with HTLV-I. Of them, 102 subjects were HAM/TSP patients and 34 subjects were HCs. Eighty nine of them (65.4%) were females and forty seven (34.6%) were males. The mean age of them was 42.22 ± 1.250 years (range 11-79 years). Maximum HTLV-I proviral load was 2578 copy number/10Clinical manifestationsThe most common HAM/TSP neurological symptoms among Japanese were gait impairment (65%), urinary disturbance (33%), numbness of lower legs (13%), constipation (6%), and lumbago (9%) .The time+ T cells, age, gender, routes of transmission , and high antibody titers , Japanese  and Brazilian populations  were higher than Iranian , Japanese , and Brazilian  HCs , 18. The4 PBMCs), Japanese (798 ± 51copies/104PBMCs) and Brazilians (912.5 ± 778.6 copies/104 PBMCs) were greater than healthy carriers   -23. Mean04PBMCs) . In anot04PBMCs) -23. Anot04PBMCs) . Further04PBMCs) . These dHigh HTLV-I proviral load is associated with clinical progression in HAM/TSP ; howeverSeveral host genetic and viral factors have been identified for developing HAM/TSP. The results of this study demonstrated that different HTLV-I proviral load among Iranian and other endemic parts of the world might be also related to milieu and host genetic background. Although, high proviral load is associated with an increased risk of developing HAM/TSP in carriers, and consequently, clinical progression of disease, lower proviral load in Iranian population seems to have the same result for HAM/TSP occurrence. Therefore, other effective factors must be involved in HAM/TSP progression, and further studies are required to clarify other unknown risk factors involved in development of HTLV-I associated diseases. Such studies can help for opening a new insight toward understanding host, microbe, and environmental interaction in pathogenesis of the viral associated diseases for a better treatment.


The Tax protein is a key contributor to the oncogenic potential of the virus, and is also the key protein involved in viral replication. However, BLV infection is not sufficient for leukemogenesis, and additional events such as gene mutations must take place. In this review, we first summarize the similarities between the two viruses in terms of genomic organization, virology, and pathology. We then describe the current knowledge of the BLV model, which may also be relevant for the understanding of leukemogenesis caused by HTLV-1. In addition, we address our improved understanding of Tax functions through the newly identified BLV Tax mutants, which have a substitution between amino acids 240 and 265.Bovine leukemia virus (BLV) and human T-cell leukemia virus type 1 (HTLV-1) make up a unique retrovirus family. Both viruses induce chronic lymphoproliferative diseases with BLV affecting the B-cell lineage and HTLV-1 affecting the T-cell lineage. The pathologies of BLV- and HTLV-induced infections are notably similar, with an absence of chronic viraemia and a long latency period. These viruses encode at least two regulatory proteins, namely, Tax and Rex, in the pX region located between the  Retroviridae family and Deltaretrovirus genus, is the etiologic agent of EBL, although it remains unknown what causes SBL , which is the causative agent of adult T-cell leukemia (ATL) and a chronic neurological disorder known as tropical spastic paraparesis or HTLV-1-associated myelopathy HAM/TSP . Therefogag, pro, pol, and env essential genes, which are necessary for the production of infectious virions, and are flanked by two identical long terminal repeats . The gag, pro, pol, and env genes encode the internal structural proteins of the virion, the viral protease, the reverse transcriptase, and the envelope glycoproteins of the virion, respectively. The genome sequences of BLV and HTLV-1 are different, but have a unique sequence called the pX situated between the env gene and the 3′LTR and encoded by the regulatory gene . The pX sequence is not of host cell origin; that is, it is not an oncogene. It has been reported that both viruses have an ability to immortalize primary cells in vitro . Interestingly, the HTLV-1 genome codes for HBZ, a unique gene encoded by the minus strand chain . The major functions of the viral proteins encoded in the BLV and HTLV-1 pX regions are summarized in Table 1. The Tax protein has been extensively studied, and it is believed to play a critical role in leukemogenesis induced by BLV and HTLV-1  binding protein (CREB)/p300 /p300 . The HBZEB)/p300 . HoweverThe infection route of BLV and HTLV is by horizontal and vertical transmission. BLV is transmitted via direct contact , milk, aFigure 2, an infection with BLV is characterized by three progressive stages of disease, including an asymptomatic stage, PL, and lymphoma. Most BLV-infected cattle are asymptomatic, but approximately one-third of them suffer from PL characterized by a permanent and relatively stable increase in the number of B-lymphocytes in the peripheral blood. PL is considered to be a benign form of the disease resulting from the accumulation of untransformed B-lymphocytes. Finally, 1–5% of BLV-infected cattle develop B-lymphoma in various lymph nodes after a long latency period . These findings clearly demonstrated that the BLV proviral copy number increases with increasing severity of the disease. On the other hand, unlike BLV, HTLV-1 is associated with ATL and with the chronic neurological disorder, HAM/TSP, and induces not only a malignant tumor but also an inflammatory disease . By contrast, BLV lacks a known oncogene . Taken together, Tax may induce immortalization of only CD5+ IgM+ B-cells among BLV-infected B-cells, CD4+ T-cells, CD8+ T-cells, γ/δ T-cells, monocytes, and granulocytes in cattle, thereby conferring a selective transformation advantage to the infected CD5+ IgM+ B-cells by a second event.Most studies of BLV-induced leukemogenesis have focused on the Tax protein because it is believed to be a potent transcriptional activator of viral gene expression. In addition to its function as a transcriptional activator, Tax induces immortalization of primary REFs , 1998. F animals . Thus, BFigure 2A). The protein encoded by the p53 tumor suppressor gene plays a critical role in transducing a signal from the damaged DNA to genes that control cell cycle and apoptosis. Approximately half of the solid tumors induced by BLV in cattle , which plays a crucial role in determining immune responsiveness -DRB1 alleles encoding the Arg–Lys (RK) and the Ser–Arg (SR) motifs at positions 70–71 of the OLA-DRβ chain are associated with resistance (RK motif) and susceptibility (SR motif) to the development of lymphoma after experimental infection of sheep with BLV. The sheep with alleles encoding the RK motif produced neutralizing antibodies against BLV and interferon-γ, eliminated BLV completely, and did not develop lymphoma . A previous study found that, in sheep experimentally infected with BLV, the frequency of the TNF-α-824G allele, which has been associated with low transcription activity of the promoter/predicted enhancer region of the bovine TNF-α gene, was higher in animals with lymphoma than in asymptomatic carrier animals. In addition, a tendency was observed for increased BLV-provirus load in cattle homozygous for the TNF-α-824G/G allele compared to cattle homozygous for the TNF-α-824A/A or TNF-α-824A/G alleles. These data suggest that the observed polymorphism in the promoter region of the TNF-α gene could at least in part contribute to the progression of lymphoma in BLV infection (A polymorphism in the promoter region of the tumor necrosis factor (TNF)-α gene is one of several genetic changes involved in the development of lymphoma  signaling , since min vivo . In addiin vivo .Table 1 summarizes the functions of the Tax protein. The Tax open reading frame is mainly encoded in the pX region, and its translation occurs upstream of the pol stop codon. The Tax protein is modified by phosphorylation of two serine residues and is detected as a 34–38 kDa product . The microarray data also raised the possibility that BLV Tax regulates the innate immune response : the largest group of downregulated genes was related to the immune response, and the majority of these genes belonged to the interferon family of antiviral factors, such as interferon-induced protein with tetratricopeptide repeats 1 . Likewise, although the transient expression of TaxD247G induced apoptosis in transfected cells in vitro more effectively than TaxWT, higher level of protection against apoptosis was observed in PBMCs isolated from sheep infected with TaxD247G-encoded BLV compared to TaxWT-encoded BLV . These findings demonstrate that TaxD247G has an increased potential to induce apoptosis, which could be beneficial for BLV propagation like other viruses . Namely, the transactivation activity and target sequence specificity of BLV Tax might be limited or negatively regulated by this region. The most interesting point regarding the ability of TaxD247G to enhance BLV expression and apoptotic induction in vitro is that it might be suppressed in vivo or ex vivo. Thus, we hypothesize that there could be dominant mechanisms controlling the functions of TaxD247G ex vivo and in vivo, as shown in Figure 4. For HTLV-1, it has been reported that CD8+ cell-mediated cytotoxic T-lymphocytes (CTLs) target Tax-expressing cells, thereby reducing the number of infected cells (in vivo. Taken together, the findings discussed in this review suggest that there might be a dominant mechanism involved in the induction of apoptosis and expression of HTLV-1 in vivo. To address our hypothesis, it seems necessary to evaluate whether possible host responses against BLV infection, such as the induction of CTLs, genetic, and epigenetic alterations in apoptosis-regulatory genes, and DNA and chromatin modifications of BLV promoter for the suppression of viral expression, could be enhanced in animals infected with TaxD247G-encoded BLV. Thus, future investigations of the relationship between apoptosis and viral expression using BLV containing the mutant D247G Tax as a model will broaden our understanding of the replication and propagation of HTLV-1, and leukemia progression.BLV is the etiologic agent of EBL, which is the most common neoplastic disease in cattle. It infects cattle worldwide, thereby imposing a severe economic burden on the dairy cattle industry. In this review, we evaluated existing information on the mechanism of BLV-induced leukemogenesis. We propose that, since BLV Tax induces immortalization of only CD5ed cells . LikewisThe 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.


The Interleukin 28B (IL28B) rs12979860 polymorphisms was recently reported to be associated with the human T-cell leukemia virus type 1 (HTLV-1) proviral load (PvL) and the development of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP).In an attempt to examine this hypothesis, we assessed the association of the rs12979860 genotypes with HTLV-1 PvL levels and clinical status in 112 unrelated Brazilian subjects ).All 112 samples were successfully genotyped and their PvLs compared. Neither the homozygote TT nor the heterozygote CT mutations nor the combination genotypes (TT/CT) were associated with a greater PvL. We also observed no significant difference in allele distribution between asymptomatic carriers and patients with HTLV-1 associated HAM/TSP.Our study failed to support the previously reported positive association between the IL28B rs12979860 polymorphisms and an increased risk of developing HAM/TSP in the Brazilian population. Infecti of ATLL -11.Cytokines play an indispensable role in the defense against viral infection, both indirectly through determination of the predominant pattern of the host response and directly through significant inhibition of viral replication . In atte.,[n =112), in correlation to PvL.Recently, three independent genome-wide association studies identified several polymorphisms, such as rs8099917, rs12979860, and 12980275, in or near the IL28B gene in patients infected with hepatitis C virus genotype 1 who had previously been treated with a combination of pegylated IFN-α and ribavirin -23. The ., was desc.,-27. InteAll patients were Brazilian and were recruited from the HTLV-1 outpatient clinic at the University of Sao Paulo, Brazil. We were unable to recruit more subjects, which would have enhanced the power of the study, and the total number of subjects was thus limited to 112. Thus, our study has an 80% power to detect a minimum odds ratio of 1.5 for the SNP, assuming α = 0.05 and the two tailed test.Peripheral blood samples (5 ml) were collected from the 112 HTLV-1 positive individuals. Infected individuals were identified by the HTLV enzyme immunoassays Murex HTLV I + II  and Vironostika HTLVI/II , and infection was confirmed by HTLV BLOT 2.4 . Of these, 81 (72.3%) were asymptomatic carriers (ACs), 24 (21.4%) had HAM/TSP and 7 (6.3%) had ATLL. All ACs were diagnosed as HTLV-1 carriers at the time of blood donation. The clinical status of HAM/TSP was determined based on WHO criteria for HTLV-1 associated diseases . Diagnos3 PBMC cells, and HTLV-1 genotypes [Laboratory measurements included peripheral blood lymphocyte counts, quantitative measurement of HTLV-1 PvLs with the use of sensitive SYBR Green Real-Time PCR with a detection limit of 1 copy per 10enotypes . The resTaq polymerase on an automated sequencer , according to the protocols recommended by the manufacturer.Based on the published human chromosome 19q13 sequence (NCBI Reference Sequence: NT_011109.16), a 293 base pair segment upstream of the IL-28 gene, flanking the rs12979860 polymorphism between nucleotides 12006835 and 120071103, relative to the chromosome 19 translational start codon, was analyzed. This region was PCR-amplified from genomic DNA using the forward primer 5'-GCTCAGCGCCTCTTCCTCCTGCG-3' and the reverse primer 5'-GGCAGGGCTCCCTTCTGTGATTGACC-3'. The PCR conditions consisted of an initial denaturation step at 95°C for 3 minutes, followed by 35 cycles of 95°C for 30 seconds, 58°C for 30 seconds and 68°C for 1 minute, and a final extension at 68°C for 5 minutes. Negative controls without the DNA template were included in each experiment. After termination of the PCR cycle, the products were purified using a QIA quick PCR purification kit (Qiagen). Complementary DNA strands from each amplicon were directly sequenced by cycle sequencing using the same primers used for the PCR, BigDye terminator chemistry and χ2 test with 1 degree of freedom. Differences between PvL variables were assessed by means of Student’s t test with the non-parametric U-Mann–Whitney test, while those between allelic frequencies variables were evaluated using the Pearson χ2 test with Yate's correction or Fisher’s exact test, when appropriate. A p value < 0.05 was considered significant. The data were analyzed with Stata statistical software .Continuous variables, including PvLs, were presented as the median (range) or mean ± standard deviation, while allelic frequencies were estimated by direct allele counting and were expressed as frequencies (%). Deviation from Hardy-Weinberg equilibrium was tested by a standard n = 70) of the study population. The median measurements of CD4, CD8 and CD25 lymphocyte percentage by flow cytometry  were 45%, 26%, and 22%, respectively, in 65% of subjects. The main characteristics of the study population are given in Table In total, 112 blood samples from HTLV-1 infected individuals were included in the study. The participants' ages ranged between 24 and 73 years, and the median age was 55 years. Females constituted 62.5% , compared to ACs.The median PvL and the frequency of clinical status among subjects are shown in Figure n = 7), and because PvL values in individuals with this genotype and in individuals that have the heterozygote CT genotype were not significantly different (p = 0.4), subjects that were homo- or heterozygotes for the T mutated allele (CT/TT) were grouped together for further analysis. Next, we analyzed the relationship between the PvL levels and the rs12979860 alleles. As shown in Figure p = 0.7).In the entire group, the frequencies of the CC, CT and TT genotypes of rs12979860 were 45%, 48% and 7%, respectively, reflecting a C allele frequency of 93%. The calculated distribution of the rs12979860 alleles according to the Hardy-Weinberg equilibrium was 69% for the C allele and 31% for the T allele. Because of the small number of subjects who were homozygous for the TT allele , ATLL patients were excluded from analysis. The frequency of the rs12979860 CC genotype was 43.2% among the 81 AC subjects and 54.2% among the 24 patients with HAM/TSP (p > 0.5). Similarly, the frequency of the CT/TT genotypes was not appreciably different between the two groups , and a significantly higher median HTLV-1 PvL was found in individuals with the CT/TT variant than in those with the CC variant (p = 0.01). In contrast to this report, our data do not support the IL28B gene polymorphism as a novel genetic susceptibility marker, either for HTLV-1-associated HAM/TSP nor ATLL, despite involving more than twice as many cases of HAM/TSP as the recent study [In this study, we sought to replicate specific findings of association between the polymorphism rs12979860 in the vicinity of the IL28 gene and the HTLV-1 PvL, which is itself a predictive factor for development of HAM/TSP or ATLL ,32-36. Hnt study .et al.[et al. [et al.[Several recent studies have also assessed the association of the IL28B rs12979860 SNP in other viral infections in which IFN plays a critical role, and the results of those studies were similar to ours. For instance, the study by Rallon et al. found a .[et al.  also fou.[et al.  reported. [et al. reported. [et al., had no We observed no compelling evidence of an association between the IL28B allelic variants and HTLV-1 PvL. Additionally, we were unable to confirm the hypothesis that the IL28B CT/TT polymorphisms might be a genetic marker for HTLV-1-associated HAM/TSP risk. These findings do not support the clinical utility of either testing for the IL28B in all asymptomatic HTLV-1 individuals, or closer follow-up of IL28B CT/TT carriers, as these steps could put us at risk of diverting financial resources from research areas that might make more of a difference in our understanding of the etiological mechanisms of HTLV-1 associated disease.The authors declare that they have no competing interests.SSS wrote the manuscript and directed the study. YN, JP, FEL, ACS and EGK were responsible of the clinical management of patients and acquisition of data, ACC, ACSO RP participated in the design of study, performed the experiments and the statistical analysis. FEL, EGK and ECS contributed to drafting the manuscript. All authors read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/374/prepub


Induction of MMPs and TGFß1 by Hh signaling expands on the current understanding of how Hh signaling affects fibrosis and tumorigenesis. These transgenic models will be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases which are induced by Hh signaling activation.Hedgehog (Hh) signaling is frequently up-regulated in fibrogenic pancreatic diseases including chronic pancreatitis and pancreatic cancer. Although recent series suggest exclusive paracrine activation of stromal cells by Hh ligands from epithelial components, debates still exist on how Hh signaling works in pathologic conditions. To explore how Hh signaling affects the pancreas, we investigated transgenic phenotypes in zebrafish that over-express either Indian Hh or Sonic Hh along with green fluorescence protein (GFP) to enable real-time observation, or GFP alone as control, at the ptf1a domain. Transgenic embryos and zebrafish were serially followed for transgenic phenotypes, and investigated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry. Over-expression of Ihh or Shh reveals virtually identical phenotypes. Hh induces morphologic changes in a developing pancreas without derangement in acinar differentiation. In older zebrafish, Hh induces progressive pancreatic fibrosis intermingled with proliferating ductular structures, which is accompanied by the destruction of the acinar structures. Both myofibroblasts and ductular are activated and proliferated by paracrine Hh signaling, showing restricted expression of Hh downstream components including Patched1 (Ptc1), Smoothened (Smo), and Gli1/2 in those Hh-responsive cells. Hh ligands induce matrix metalloproteinases (MMPs), especially MMP9 in all Hh-responsive cells, and transform growth factor-ß1 (TGFß1) only in ductular cells. Aberrant Hh over-expression, however, does not induce pancreatic tumors. On treatment with inhibitors, embryonic phenotypes are reversed by either cyclopamine or Hedgehog Primary Inhibitor-4 (HPI-4). Pancreatic fibrosis is only prevented by HPI-4. Our study provides strong evidence of Hh signaling which induces pancreatic fibrosis through paracrine activation of Hh-responsive cells  Hh ligands are secreted glycoproteins and they initiate hedgehog signaling upon binding to Patched (Ptc) receptors. The signaling is transmitted through Smoothened (Smo)'s activation, resulting in the Gli-mediated transcriptional up-regulation of Hh target genes. This signaling plays a critical role in both physiologic and pathologic conditions by participating in cell differentiation and tissue patterning during early embryonic development and in tissue homeostasis as well as tumorigenesis in adult organs in vitro study revealed enhanced migration of pancreatic stellate cells by exogenous Ihh The pancreas is one of the organs where Hh signaling is strictly controlled. Although inactivation of Hh signaling is a crucial event for proper pancreatic development and differentiation, this signaling is frequently reactivated in fibrogenic pancreatic diseases. For instance, chronic pancreatitis and pancreatic ductal adenocarcinoma, with several components of Hh pathway are frequently and often markedly up-regulated in early stages of those conditions It has been well-documented that Hh signaling relies on paracrine action for proper patterning of the gastrointestinal tract during murine development in vitro studies in vivo evidence that Hh ligands cause pancreatic fibrosis by paracrine activation of myofibroblasts, as well as ductular cells.Since the early 1980s, the zebrafish has been widely used for the study of genetics and developmental biology, and is often exploited as a disease model In order to express transgenes from a zebrafish pancreas, we took advantage of Tg zebrafish When transgene expression was evaluated by GFP expression or by ISH, it was found to be spatiotemporally restricted to the Ptf1a domain . In contThe endocrine or exocrine differentiation was not compromised by Hh over-expression . To visuAll Hh-expressing zebrafish from independent lines revealed a varying degree of pancreatic fibrosis and the desmoplasia was accumulated as the zebrafish aged . Among tThe fibrotic changes were typically observed in the pancreas between the liver and gut, where the exocrine pancreas surrounded the principal islet. Interestingly, this corresponded to the area where the transgene expression was most robust. A prominent fibrotic area revealed a discernable whitish plaque in the entire dissected viscera and corresponded with the spot showing strong GFP expression . On ISH Proliferating myofibroblasts were invariably positive for α-SMA . The majInterestingly, proliferation of ductular structures was also noted at the age of three months, showing dense fibrotic bands intermingled with ductules . Along wThe Hh signaling has been considered as a mediator of gastrointestinal tumorigenesis for many years, and Pdx1-Shh mice have shown metaplastic change and PanIn-like lesions In order to identify differentially expressed genes, GFP-expressing pancreases were dissected under a fluorescence microscope and pancreas samples were extracted from 4–5 of each transgenic zebrafish, which were processed for RT-PCR. Among the Hh components, real-time RT-PCR revealed up-regulation in most of the downstream components including Ptc1, Smo, Gli1, and Gli2a as well as transgenes compared to the control, which suggested the presence of cells with active Hh signaling . The sigHistologic expression of the Hh signaling components was assessed by either IHC or ISH, depending on the availability of an antibody that was cross-reactive to zebrafish antigen. Though Ptc1 theoretically counteracts the activation of Smo, the Hh ligand needs Ptc1 receptor to bind and initiate Hh signaling MMPs function in the regulation of the extracellular matrix (ECM) organization by degrading ECM gives way to cellular migration. Thus, induction of MMPs is necessary for the progression of fibrosis. RT-PCR showed that induction of MMP9 was the most striking among the MMP genes evaluated in this study. While MMP2 was modest, MT1MMPs were mildly elevated. An immunostaining analysis revealed that both Hh-responsive myofibroblasts and ductular cells strongly expressed MMP9 with nil expression in acinar cells, which suggests that activated Hh signaling was responsible for induction of MMP9 .TGFß family members also play important roles in fibrosis as well as tumorigenesis. Crosstalk between Hh and TGFß signaling has been found, and both genes are often co-expressed in epithelial compartments in vivo screening of candidate drugs due to a lower cost and a higher efficiency than with mouse models. To investigate the feasibility of phenotypic reversal by Hh inhibitors, Tg and control embryos were treated with the maximal tolerable dose (MTD) of either cyclopamine  or HPI-4 The zebrafish model has been spotlighted for its feasibility in Next, groups of 12-day old Tg larvae were treated with Hh inhibitors for an extended period of up to 6 weeks. On histologic observation, the maximal tolerable dose (5 uM) of HPI-4effectively prevented pancreatic fibrosis but induced prominent fatty infiltration of the pancreas , which min vivo evidence that Hh signaling operates in a paracrine mode in the pancreas.For the first time, current study presents a zebrafish model to study pancreatic fibrosis in which molecular events relevant to Hh-induced fibrosis can be explored. Zebrafish have recently been seen to simulate human disease in both molecular and histopathologic levels Among the three members of Hh ligands, Ihh and Shh expression is broader and strictly controlled in various organs, including the gastrointestinal system In vitro studies have provided evidence of the autocrine activation of Hh signaling in keratinocytes, medulloblastoma, and renal cell carcinoma cells in vivo evidence that secreted Hh ligands cause pancreatic fibrosis by paracrine activation of responsive cells. The embryonic phenotypes in current models are not dramatic, they simply show morphologic changes of the exocrine pancreas. The development of the endocrine pancreas as well as other endodermal organs including the liver and the intestine were not affected by Hh over-expression. The exocrine markers such as trypsin, elastase, and CPA are properly and timely expressed in acinar cells. These findings strongly support the fact that Hh-expressing acinar cells are not influenced by this signaling but undergo proper differentiation. Although it is not clear whether the mode of action is dependent on cell type, the result suggests that a paracrine mechanism is highly involved in the pancreas.The canonical Hh pathway involves ligands, receptors, intracellular mediators, and transcription factors. In the present study, aberrant expression of Ihha and Shha molecules in the exocrine pancreas caused progressive fibrosis by paracrine action. This leads to the destruction of acinar structures which mimics desmoplasia occurring in human chronic pancreatitis and pancreatic cancer. Although the paracrine action of Hh signaling during embryonic development has been well-documented Chronic pancreatitis and pancreatic cancer represent human diseases that are accompanied by progressive pancreatic fibrosis. In both conditions, Hh ligands are considerably over-expressed in metaplastic ductal and cancer cells and play central roles in desmoplasia  [7], [8]We also demonstrated that Hh-responsive myofibroblasts and ductular cells invariably express downstream components of Hh signaling. However, none of the acinar cells expressed these genes at either the mRNA or protein level. It is unclear as to how the Hh signaling exerts paracrine action in the pancreas, so it is crucial to determine responsiveness to secreted Hh molecules. In the current study, Hh-responsive myofibroblasts invariable expressed Ptc1 and Smo even in control, suggesting that expression of Ptc1 or Smo, or both determines Hh-responsiveness. Considering that Hh signaling is initiated by ligand-binding to the Ptc receptor, expression of the Ptc gene is mandatory for the initiation of Hh signaling. A recent observation has implicated that over-expression of Smo in pancreatic cancer-associated fibroblasts is a potential determinant for Hh-responsiveness Our study suggests that the aberrant expression of Hh molecules does not induce tumors. We followed the transgenic zebrafish for more than one year without observing any evidence of tumor foci or PanIn-like lesions; as opposite to Pdx1-Shh mice that developed metaplastic duct and PanIn-like lesions with over-expressed Ptc1 and Smo in vivo during embryonic development In vitro studies have shown that TGFß cooperates with canonical Hh signaling to activate Gli proteins and Hh target gene expression We identified TGFß1 and MMPs as important mediators of Hh signaling. Recent observations have demonstrated that Hh signaling accelerates pancreatic tumorigenesis through tumor-stromal interaction by providing favorable conditions for tumor cells in vitro observations that have demonstrated either exogenous Hh molecules or ectopic expression of Gli1 or Hh molecules induced MT1-MMP and MMP9 in cultured cells in vivo environment enables exploration of epiphenomena manifested by the complex interaction of different types of cells. Thus, reflection of what really happens in the context of the physiologic and pathologic conditions is more than an in vitro study can provide.Similarly, MMPs play roles by remodeling the extracellular environment, which is an important step in the progression of fibrosis as well as tumorigenesis. In this study, Hh-responsive cells demonstrated the striking up-regulation of MMP9 with modest elevation of MMP2 and MT1-MMPs.This factor is consistent with in vivo screening for small molecules, in which the effect of given drugs as well as toxicity can be simultaneously monitored under physiologic conditions. A visible short-term phenotype can facilitate high-throughput screening of candidate drugs. Though in this model, we could not thoroughly explain how the morphologies of a developing pancreas were changed by Hh expression, we could observe phenotypic reversal by treatment with Hh inhibitors. We also demonstrated that pancreatic fibrosis and destruction were effectively prevented by the treatment of HPI-4, a ciliogenesis inhibitor, but not with cyclopamine. Although failure by cyclopamine may be attributed to the dose limitation such as the high toxicity in the larval stage, this finding implies that targeting downstream of Smo may be beneficial for obtaining a therapeutic effect. Though the mechanism for differential sensitivity between embryos and larvae was unclear at the time, the acquisition of a toxicity profile in a physiologic context is an additional benefit of using the zebrafish as a model for drug screening. This study provides in vivo evidence that inhibition of Hh signaling is a viable option for the prevention of pancreatic fibrosis which has a detrimental effect on chronic pancreatitis and pancreatic cancer.While chronic pancreatitis accompanies the infiltration of inflammatory cells, the pancreatic pathology in these Hh-expressing zebrafish lacks an inflammatory reaction. Considering that the eventual pancreatic dysfunction in human chronic pancreatitis results from long-standing fibrotic change, acinar destruction and prevention of fibrosis is one of the main therapeutic targets. As a model, the zebrafish uniquely allows In conclusion, aberrant expression of either Ihha or Shha causes progressive pancreatic fibrosis through paracrine activation of Hh-responsive cells. We identified TGFß and MMPs as important genes induced by Hh signaling in responsive cells. These transgenic models will be a valuable platform in exploring the mechanism of fibrogenic pancreatic diseases caused by Hh signaling activation.It was not necessary to obtain approval by the Laboratory Animal Committee at Yonsei University College of Medicine. The current committee does not request approval when non-mammalian models are used for experiments. This study, however, was strictly carried out to minimize suffering. All live images of embryos were taken under anesthesia using E3 media with 0.3 mg/mL tricaine. All adult zebrafish to be processed for experiments were euthanized by immersion in an ice-water bath.Transgenic constructs were generated by modifying JD21-UAS:GFP-Kras, a kind gift from Steven D. Leach, which allows Tol2- to mediate transgenesis and is designed to co-express the transgene along with green fluorescence protein (GFP) which enabled real-time observation , Fig. S1Each injection mixture was made by reconstituting Tol2-transposase mRNA (20 ng/ul) and a transgene construct (20 ng/ul) in Danieu's buffer mixed with 0.03% phenol red. Single-cell stage Tg embryos were transferred to a molded agarose dish and 4 pL of injection mixture was introduced by yolk injection using a MMPI-2 micro injector. Approximately 50% of injected embryos survived. On day two, embryos showing GFP at the Ptf1a domain were selected using a fluorescence microscope, raised until adulthood, and out-crossed to generate F1 transgenic zebrafish. The utilization of Tol2-mediated transgenesis greatly enhanced the transgenic efficiency that 25–50% of F0 zebrafish from each construct gave rise to F1 offspring expressing transgenes. In each clutch of F1 embryos, approximately 10% showed transgene expression. Among the F1 progenies, embryos showing faithful expression were selected and raised to produce F2 progenies. All transgenes were transmitted into normal Mendelian ratios. Transgenic zebrafish were raised in a standardized aquaria system  according to standard protocols. Embryos to be processed for whole mount examination of GFP expression or ISH analyses were placed in 0.003% phenylthiourea at 24 hours post-fertilization (hpf) to inhibit pigmentation.Histologic evaluation was performed in a subset of F2 transgenic zebrafish at 1,3,6,9, and 12-month(s). Hematoxylin and eosin (H&E) staining and IHC were performed according to the standard protocols. Primary antibodies used for immunohistochemistry were rabbit anti-α-smooth muscle actin (α-SMA) , rabbit anti-Smoothened (Smo) , rabbit anti-Gli1 , rabbit anti-Gli2 , mouse anti-Transforming growth factor ß1 (TGFß1) , rabbit anti-matrix metalloproteinase 9 (MMP9) , mouse anti-cytokeratin (CK) AE1/AE3 , mouse anti-proliferating cell nuclear antigen (PCNA) , and rabbit anti-phosphohistone H3 (pHH3) . Horse radish peroxidase (HRP)-conjugated secondary antibodies were utilized and colored using DAB solution. Slides were counterstained with hematoxylin, dehydrated, and mounted with Histomount (Zymed Co.).A western blot hybridization was performed as previously described ISH was performed either using 4% paraformaldehyde-fixed whole embryos or on 4-um sections of 4% paraformaldehyde-fixed, paraffin-embedded tissues as described previously Whole mount immunofluorescence was ISH was performed using 4% paraformaldehyde-fixed whole embryos essentially as described previously Photographs were obtained using an Olympus BX51 for slide sections and an Olympus MVX10 for whole mount embryos. If not indicated, all section images were taken with a 10× eye lens and a 40× objective lens. If needed, zoom functions were used to obtain further magnified images.RT-PCR was performed using the exocrine pancreas dissected under a fluorescence microscope from three-month old zebrafish. For each group, samples were collected from five to six zebrafish and processed for RNA extraction. Real-time, quantitative RT-PCR was performed as previously described To antagonize Hh signaling, either cyclopamine  or Hh Primary Inhibitor-4 (HPI-4)  was used Figure S1Schematic illustration for the generation of transgene constructs.(TIF)Click here for additional data file.Figure S2IHC for Hh ligands in human pancreas.  Immunostaining for Ihh and Shh in a normal pancreas showing nil expression. (C) IHC for Ihh in chronic pancreatitis. Metaplastic ducts are strong positive for Ihh expression (arrows). (D) IHC for Shh in pancreatic cancer. Ductal cancer cells (arrow) and neighboring metaplastic ducts (arrowhead) are positive for Shh expression. Microscopic images are 400×.Bars, 50 µm.(TIF)Click here for additional data file.Table S1Primers used for the generation of transgene constructs. F-UAS-Seq was used for sequence verification of constructs. Underlined GCCACC sequence was inserted to satisfy Kozak sequence for proper transcription. Underlines, restriction enzyme sequences.(DOCX)Click here for additional data file.Table S2Primers used for TA cloning to generate riboprobes.(DOCX)Click here for additional data file.Table S3Primers used for RT-PCR.(DOCX)Click here for additional data file.


The ability for a neuron to distinguish important input signals from background noise is essential for proper circuit function. Several mechanisms exist to enhance signal detection. For example, short-term facilitation enhances burst transmission, while information from single spikes is transmitted more effectively through synapses that exhibit depression . HoweverWe used a combination of modeling and experimental methods to examine the role of the Cav3-KCa3.1 complex in simultaneously enhancing detection of parallel fiber bursts and suppressing background inputs. The Cav3-KCa3.1 complex was simulated using a calcium-diffusion model with 10 hemispherical compartments around the Cav3 channel and the KCa3.1 channel placed within 50 nm of the calcium source. The Cav3-KCa3.1 complex was included in a single-compartment membrane model with HCN and synaptic conductances to determine its effect on excitatory postsynaptic potential (EPSP) summation. Whole-cell current-clamp recordings of Purkinje cells were also made from acute slices of rat cerebellum (P18-P25). Parallel fibers were stimulated using a monopolar stimulating electrode in the cerebellar molecular layer.2+ (100 μM) to block Cav3 current removed this supralinear increase in rate of decay, confirming that the Cav3-KCa3.1 complex dynamically changes temporal summation during repetitive input trains.We simulated 5 pulse 50 Hz trains of facilitating and non-facilitating EPSPs in the model. The Cav3-KCa3.1 complex reduced the summation of both sets of inputs. However, the facilitating inputs achieved a higher degree of summation than non-facilitating inputs, even though charge transfer for both trains was identical. Examination of the KCa3.1 current showed that synaptic facilitation compensated for the increase in KCa3.1 activation during the input train, allowing EPSPs to summate to higher voltages. When the Cav3-KCa3.1 complex was not included in the model, facilitating and non-facilitating inputs reached similar degrees of summation. Our computational results were confirmed by our current clamp recordings. Facilitating inputs evoked by direct PF stimulation generated more depolarization and spikes in Purkinje cells than non-facilitating simulated EPSPs (simEPSPs). During trains of simEPSPs, a supralinear increase in the rate of EPSP decay was observed for consecutive inputs, indicating an increased recruitment of hyperpolarizing current during the train. Application of NiOur results demonstrate that the Cav3-KCa3.1 complex significantly affects the temporal summation of synaptic inputs. Specifically, the Cav3-KCa3.1 complex allows facilitating inputs from single PF sources to summate and generate spike output in Purkinje cells, while background inputs are quickly suppressed, improving the ability to respond to important sensory information.


HIV-associated nephropathy (HIVAN) is the leading cause of end-stage renal disease in patients with HIV-1 infection. While HIVAN involves all portions of the kidney, marked changes in podocyte proliferation and dedifferentiation are central to the pathogenesis of this disease. This review focuses on identifying the genes and signaling pathways that trigger terminally differentiated quiescent podocytes to lose their normal markers of maturity and reenter the cell cycle. The parallels between these alterations to malignant transformation are quite striking.HIV-associated nephropathy (HIVAN) is the leading cause of chronic kidney failure in patients infected with HIV . The majThe HIV virus directly infects tubular cells and podocytes leading to the development of HIVAN. Not surprisingly, the HIV-1 transgenic mouse (Tg26 mice) model in which renal cells express viral proteins has greatly advanced our knowledge of the pathogenesis of HIVAN. The Tg26 mice develop proteinuria, progressive renal failure, and renal pathologic findings identical to HIVAN patients . Reciprogag, pol, and env leads to the development of massive proteinuria, severe collapsing FSGS, and microcystic tubular dilatation , synaptopodin, podocalyxin, common acute lymphoblastic leukemia antigen (CALLA), and ezrin . The losin vitro . This isgag, pol, env), two regulatory genes (tat and rev), and four accessory genes . In HIV-1 infected hosts, the 27-kDa protein Nef is at an early phase in the viral replication cycle and performs several functions  which normally prevent mature podocytes from reentering the cell cycle  . PhosphoActivation and signaling of the MAPK family plays a role in mitogenesis and cell differentiation. Nef can activate this downstream signaling pathway via Raf and Src activation . MAPK sinef and downstream signaling pathways in vitro  are binding sites for the SH3 domain of the Src kinase family and PAK2. Using immortalized murine podocytes, mutations of Nef in the PxxP region have been shown to have the following effects in vitro: inhibition of Stat3 and MAPK1, 2 phosphorylation, reduction of cyclin E expression, restoration of synaptopodin expression, and reversal of Nef-induced cell proliferation (105R106 domain did increase synaptopodin expression, mutation at this site failed to change phosphorylation of Stat3 or MAPK1, 2 and only minimally decreased cell proliferation (105R106 motif may exert some of Nef’s effects leading to the HIVAN phenotype in podocytes, it is likely through a signaling cascade independent of Stat3 and MAPK1, 2 activation. A summary of known pathways of nef signaling and its downstream effectors is presented in Two Nef domains (PxxP and Rferation . While mferation . The datin vitro. Immunostaining for phospho-Stat3 is markedly elevated in the glomeruli of transgenic mice with podocyte-specific nef expression compared to control littermates (nef generated mice that no longer exhibited the HIVAN phenotype (in vivo evidence that nef induces the myriad of renal cortical abnormalities that characterize the HIVAN phenotype via activation of the Src family of tyro-sine kinases.We confirmed these signaling pathways are critical to mediating Nef’s effect on the HIVAN phenotype termates . Also, itermates . Additiohenotype . This stnef interaction and activation of PAK2 in the murine model contributes to the HIVAN phenotype (nef alleles but lacking the Nef—PAK2 association do not exhibit kidney disease. This identifies Nef binding and activating PAK2 as critical to the development of HIVAN. Activation of PAK2 alone, however, is not sufficient to develop kidney disease. Transgenic mice expressing nef mutants that were able to bind and activate PAK2 failed to exhibit kidney disease (Vincent et al. have proposed that henotype . Activathenotype . HIV tra disease .In vitro and in vivo evidence indicates that Nef induces these changes in the podocyte via Src kinase-dependent activation of Stat3 and MAPK1,2 signaling pathways. Additionally, Nef interaction with PAK2 may contribute to some of the features of the HIVAN phenotype. There are still several downstream targets that have yet to be identified which potentiate the development of podocyte proliferation and dedifferentiation. Evidence also indicates that Nef may activate other pathways, in addition to Stat3 and MAPK1, 2, as part of the pathogenesis of HIVAN. Continued studies are needed to identify downstream effectors of Nef and therapeutic interventions that may abrogate the activity of these signaling pathways.HIVAN is characterized by marked proteinuria, renal insufficiency and characteristic pathologic changes on kidney biopsy. Marked podocyte proliferation and dedifferentiation occurs within the collapsing glomerulopathy found in HIVAN. These podocyte changes are not seen in other glomerular diseases. HIV-1 Nef is the critical protein mediating reentry of podocytes into the cell cycle and down-regulation of mature podocyte differentiation markers.


Ursolic acid (UA) is a triterpenoid compound with multiple biological functions. This compound has recently been reported to possess an anti-obesity effect; however, the mechanisms are less understood.As adipogenesis plays a critical role in obesity, the present study was conducted to investigate the effect of UA on adipogenesis and mechanisms of action in 3T3-L1 preadipocytes.β), peroxisome proliferator-activated receptor γ (PPARγ), CCAAT element binding protein α (C/EBPα) and sterol regulatory element binding protein 1c (SREBP-1c), respectively. Ursolic acid increased the phosphorylation of acetyl-CoA carboxylase (ACC) and protein expression of carnitine palmitoyltransferase 1 (CPT1), but decreased protein expression of fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Ursolic acid increased the phosphorylation of AMP-activated protein kinase (AMPK) and protein expression of  1 (Sirt1). Further studies demonstrated that the anti-adipogenic effect of UA was reversed by the AMPK siRNA, but not by the Sirt1 inhibitor nicotinamide. Liver kinase B1 (LKB1), the upstream kinase of AMPK, was upregulated by UA. When LKB1 was silenced with siRNA or the inhibitor radicicol, the effect of UA on AMPK activation was diminished.The 3T3-L1 preadipocytes were induced to differentiate in the presence or absence of UA for 6 days. The cells were determined for proliferation, differentiation, fat accumulation as well as the protein expressions of molecular targets that regulate or are involved in fatty acid synthesis and oxidation. The results demonstrated that ursolic acid at concentrations ranging from 2.5 µM to 10 µM dose-dependently attenuated adipogenesis, accompanied by reduced protein expression of CCAAT element binding protein β (C/EBPUrsolic acid inhibited 3T3-L1 preadipocyte differentiation and adipogenesis through the LKB1/AMPK pathway. There is potential to develop UA into a therapeutic agent for the prevention or treatment of obesity. Obesity has become an epidemic in developed countries and also many developing countries. The rates of obesity and overweight have been continuing to grow in adults, and unfortunately that the situation has been worsening by penetrating into the child and adolescent population. In addition to morbidity, obesity is associated with many metabolic complications, including type-II diabetes, insulin resistance, hyperlipidemia, hypertension and coronary heart disease in vitro and in vivo studies suggests that UA possesses many nutritional and pharmacological functions, including anti-inflammatory Although a number of drugs have been developed and used to treat obese patients through regulating appetite, fat absorption and fat oxidation γ) and inhibits differentiation of preadipocytes into adipocytes + concentration, and modulates cellular energy metabolism γAdipogenesis is determined by multi-processes, which include preadipocyte proliferation, differentiation, and fatty acid oxidation and synthesis, and controlled by a number of molecular factors. Emerging evidence suggests that AMP-activated protein kinase (AMPK) functions as a sensor of cellular energy status. Once activated, it switches on the catabolic pathways and simultaneously switches off the ATP-consuming anabolic pathways α (Thr 172), AMPKα, fatty acid-binding protein 4 (FABP4), β-actin, carnitine palmitoyltransferase 1 (CPT1), acetyl-CoA carboxylase (ACC) and pACC were purchased from Santa Cruz Biotechnology, Inc. . Antibodies against liver kinase B1 (LKB1), pLKB1, PPARγ, CCAAT element binding protein α (C/EBPα) and C/EBPβ were from Cell Signaling Technology, Inc. . Fatty acid synthase (FAS) antibody was from Novus Biologicals . BM chemiluminescence blotting substrate kit was from Roche Diagnosis . LKB1 and AMPK small interfering RNAs (siRNAs), Opti-MEM, reduced serum medium and lipofectamine RNAiMAX were obtained from Life Technologies .Ursolic acid, nicotinamide, radicicol, insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone, propidium iodide, ribonuclease (Rnase) and protease inhibitor were purchased from Sigma . High glucose Dulbecco's modified Eagle's medium (DMEM) was from Mediatech, Inc. . Fetal bovine serum (FBS) was from PAA Laboratories . Bovine calf serum (BCS) and adipogenesis assay kits were purchased from Cayman Chemical Company . Lipolysis assay kits were purchased from Zen-Bio, Inc. . The BCA protein assay kit was from Thermo Scientific . RIPA lysis buffer was from Millpore . Protein loading buffer was from Bio-Rad . Antibodies against sterol regulatory element binding protein 1c (SREBP-1c), pAMPK3T3-L1 mouse embryo fibroblasts were obtained from American Type Culture Collection  and cultured in DMEM containing 10% BCS until confluent, and were then maintained in the same medium for an additional 2 days. Differentiation was induced 2 days post-confluence (day 0 of differentiation) by adding 0.5 mM IBMX, 1 µM dexamethasone, and 5 µg/mL insulin in DMEM with 10% FBS. After 2 days of incubation, culture medium was changed to fresh DMEM containing 10% FBS and 5 µg/mL insulin. Two days later, the medium was replaced with DMEM supplemented with 10% FBS and incubated for another two days. UA was added two days after confluence (day 0) and maintained during cell differentiation until the time when the cells were harvested for the following described tests.4 cells/cm2 and differentiated with induction medium in the presence of 2.5 to 20 µM UA for 6 days, as described above. Cells were treated with MTT assay reagents (1 mg/mL) for 4 hours and the resulting formazan was solubilized in 150 µL dimethyl sulfoxide (DMSO) and further diluted 10 times with DMSO. The absorbance was measured at 570 nm on a Varioskan Flash spectral scanning multimode plate reader .3T3-L1 preadipocytes were seeded onto 96-well plates at a density of 103 cells/cm2 in 96-well plates. The cells were grown in DMEM containing 10% BCS supplemented with increasing doses of UA for 24, 48, and 72 hours. MTT solution (1 mg/mL) was added to the medium and incubated for 4 hours. The purple formazan crystals were dissolved in 150 µL DMSO and the absorbance was read at 570 nm on the Varioskan Flash spectral scanning multimode plate reader as described above.3T3-L1 preadipocytes were seeded at a low density of 3×103 cells/cm2. The cells were treated with different concentrations of UA for 24, 48, and 72 hours, respectively. The number of adherent cells was determined by direct counting using a hemocytometer and a mini-automated cell counter from Orflo (http://www.orflo.com) after being trypsinized. Three independent experiments were performed with triplicates in each experiment.3T3-L1 preadipocytes were seeded onto 24-well plates at a low density of 3×10Three days post-confluence, 3T3-L1 preadipocytes were differentiated in the presence of 0, 2.5, 5, 10 µM ursolic acid for 24 hours. The cells were collected and fixed overnight with 70% ethanol at 4°C. Following wash with PBS, the cells were stained with propidium iodide solution containing 20 µg/ml of RNase for 30 minutes. Fluorescence-activated cell sorting (FACS) analysis was performed on a Becton–Dickinson FACScan system and data were analyzed using the Flowjo software .3T3-L1 preadipocytes were seeded onto 96-well plates and were induced to differentiate in the presence or absence of ursolic acid as described above. Lipid accumulation was measured 6 days after the induction using a commercial adipogeneis assay kit. Briefly, the cells were fixed with lipid fixative solution, stained with Oil Red O and washed with distilled water. Intracellular lipids were extracted with the extraction solution included in the kit. The absorbance was read at 520 nm on the same plate reader as described above. Images were captured under a Nikon inverted microscope (ECLIPSE TE200) after washing with distilled water.3T3-L1 preadipocytes were seeded onto 96-well plates and induced to differentiate 2 days after confluence. The differentiated Cells were treated with different concentrations of ursolic acid for 24 and 48 hours to detect cell viability as described above. Lipolysis was measured using a commercial 3T3-L1 lipolysis assay kit. Briefly, cells were washed twice with 200 µL wash buffer and then treated for 3 hours with 150 µL of 10 µM ursolic acid suspended in the assay buffer in a humidified incubator at 37°C. One hour prior to the assay, glycerol standards were prepared following the kit's instructions . At the end of the incubation, 100 µL of medium was collected from each well and transferred to a new plate with addition of 100 µL of the glycerol reagent. After 15 minutes at room temperature, the absorbance was measured at the wavelength of 540 nm on the Varioskan Flash spectral scanning multimode plate readerβ expression was determined 2 days after the induction of cell differentiation in the presence or absence of the indicated concentrations of UA. The expression of each protein was present as fold of the loading control, β-actin.After 6-day differentiation in the presence of UA, 3T3-L1 adipocytes were collected and lysed in ice-cold RIPA lysis buffer for 30 minutes. Protein content was determined using a BCA protein assay kit. Equal amount of protein for each sample was loaded and separated on a 10% SDS-PAGE. After electrophoretic separation, the proteins were transferred to a nitrocellulose filter using a semi-dry transfer cell (Bio-Rad) at 13 V for 1 hour, blocked with 5% skim milk for 1 hour at room temperature, and incubated with primary antibodies at 4°C overnight. The nitrocellulose filters were then incubated with horseradish-peroxidase conjugated secondary antibody at room temperature for 3 hours. Immunoreactive proteins were detected using the chemiluminescent ECL assay and quantified using the Molecular Imager software (Bio-Rad). C/EBP3T3-L1 preadipocytes were transfected with LKB1 or AMPK siRNA oligonucleotide duplexe 1 day post the confluence with lipofectamine RNAiMax. Generally, 75 nM siRNA was transfected with 4 µl/well of lipofectamine in a 6-well plate. Lipofectamine RNAiMax and siRNA were individually diluted in 50 µL Opti-MEM medium, mixed, incubated for 20 minutes at room temperature, and then added to each well. The medium was removed and replaced with the induction medium in the presence or absence of UA after 24 hours of transfection. The effectiveness of siRNA knockdown was determined after 24 hours of transfection and on day 6 of cell differentiation, respectively, by measuring the expression of LKB1 or AMPK using the Western blotting.The statistical analyses were performed using SPSS 13.0 statistical program . The treatment effect was determined using one-way ANOVA and followed by a post-hoc Dunnett's or Bonferroni's multiple comparisons test, where a P value less than 0.05 was considered significant. Data are presented as means ± SD.).3T3-L1 preadipocytes were induced to differentiate in the presence of 0, 2.5, 5, 10, 15, or 20 µM of UA for 6 days. The MTT assay revealed that UA at concentrations of 2.5 to 10 µM did not affect cell viability while 15 µM was toxic Fig. 1A.To investigate the effect of UA on preadipocyte proliferation, 3T3-L1 preadipocytes were grown in basal DMEM supplemented with different concentrations of UA for 24, 48 and 72 hours, respectively. As shown in Fig. S1C), indicating that UA stimulated lipolysis in mature 3T3-L1 adipocytes.3T3-L1 preadipocytes underwent morphological changes from the spindle-like features to round shape and accumulated intracellular lipids after adding the induction reagents. Ursolic acid at the doses of 2.5, 5, or 10 µM decreased intracellular fat content compared to the control as revealed by microscopic examination following Oil Red O staining in differentiated 3T3-L1 adipocytes Fig. 2A.β, C/EBPα, PPARγ and SREBP-1c. Normally, C/EBPβ functions quickly following the induction of preadipocyte differentiation, followed by the expression of C/EBPα and PPARγ. As shown in β on day 2 and subsequently inhibited the expressions of PPARγ and C/EBPα on day 6 after the induction of differentiation. SREBP-1c expression was also significantly down-regulated by UA at 10 µM after 6 days  and reduced the expression of FAS and FABP4 at the concentrations of 5 and 10 µM, respectively Fig. 4D.AMPK and Sirt1 are two important regulators of preadipocyte differentiation and adipogenesis. In this study, UA at concentrations of 5 µM or 10 µM increased AMPK phosphorylation (pAMPK) and thus activation, while showing no effect on its total protein expression Fig. 4E.Fig. S2A, the AMPK siRNA significantly reduced AMPK protein expression, indicative of the effectiveness of AMPK siRNA transfection. The transfection of AMPK siRNA significantly increased lipid storage in differentiated 3T3-L1 cells as compared with the control . To further explore the role of LKB1 in the upregulation of UA on adipogenesis, LKB1 siRNA and its destabilizer radicicol were used, respectively. As shown in Fig. S2B and 2D, total LKB1 expression was inhibited by either radicicol or LKB1 siRNA. Consequently, the increased phosphorylation of AMPK by UA was diminished when LKB1 siRNA or inhibitor was applied. , suggesting that UA activated AMPK via LKB1. To further evaluate the effect of LKB1 knockdown on adipogenesis in the presence of UA, we detected two key adipogenic proteins, FAS and FABP4. As shown in To understand whether UA modulates AMPK activity in 3T3-L1 preadipocytes through its upstream regulator LKB1, we further investigated the effect of UA on the expression and activation of LKB1. It was observed that UA at 10 µM increased the LKB1 activity by increasing its phosphorylation Fig. 7AUrsolic acid is present in many fruits and plants and the major component of several traditional medicine herbs. It is well known to that UA possess a wide range of biological functions γ, C/EBP members and SREBP-1c Adipose tissue is determined by the number and size of adipocytes. The increase in the number of adipocytes involves the proliferation of preadipocytes. Therefore, the growth and proliferation of preadipocytes has a profound implication in the development of obesity therapeutics or health products. Our results suggest UA did not affect preadipocyte number, cell cycle or apoptosis. However, UA significantly inhibited the differentiation of preadipocytes. After treatment with 10 µM of UA for 6 days, adipogenesis in differentiated 3T3-L1 cells was reduced by approximately 30%. Further studies demonstrated that several transcription factors were involved in the regulation of UA on adipogenic differentiation, including PPARγ is sufficient to initiate and control adipogenesis in growing fibroblast cells by binding to PPAR response elements located in the promoter of target genes β is responsible for the initial transcriptional activation of C/EBPα gene β expression in 3T3-L1 preadipocytes during the first 48 hours after the induction of differentiation, and subsequently PPARγ, C/EBPα and SREBP-1c expressions on day 6, suggesting that UA inhibited preadipocyte differentiation through altering the expression of transcriptional factors involved at different differentiation stages. C/EBPα, PPARγ and SREBP-1c coordinately drive the expression of adipocyte-specific genes, such as FAS, ACC and FABP4, which determine the later stages of adipocyte differentiation and associated biosynthesis of fatty acids and triacylglycerols PPARγ functions through coordinating the expression of genes responsible for the establishment of mature adipocyte phenotype. The expression of PPARγ expression in cultured adipocytes α, PPARγ and their downstream protein FAS and phosphorylation of ACC, were interestingly reversed by blocking AMPK expression using AMPK siRNA.Accumulating evidence suggests that AMPK and Sirt1 act as intracellular energy sensors and regulate energy metabolism through a concert process, and thus are of great interest in recent years as molecular targets in obesity research. AMPK directly modulates fatty acid synthesis and oxidation by altering the expression and activation of enzymes and proteins involved in fat metabolism. AMPK also regulates preadipocyte differentiation and adipogenesis LKB1 and calmodulin kinase kinase β are two main upstream kinases of AMPK β and the activity of PPARγ affect adipogenesis through a different way than their expressions. In addition, we used only 3T3-L1 adipocyte cell line to study the effect of UA on differentiation and adipogenesis. Our results and the above-mentioned items could be confirmed in other origins of adipocytes, such as human preadipocytes and pluripotent mesenchymal stem cells.Although we identified AMPK as the regulator through which UA exerted its anti-adipogenic effect, it is not possible to rule out other regulators involved in adipocyte differentiation and adipogenesis, such as Wnt signaling, GATA factors, and KLFs in vivo study to evaluate the effects of UA on anti-obesity and improving the fatty liver in rats with high fat diet–induced obesity. The results revealed that UA at doses of 0.25% and 0.50% significantly reduced body weight and fat content, increased the fatty acid oxidation in skeletal muscle but without affecting the food intake (unpublished data). Thus, both in vitro and in vivo studies suggest the potential of UA to be an anti-obesity agent. Of course, the side effect of UA should be evaluated prior to the clinical application, especially in obese patients who are also diabetic as described in our studies Encouragingly, we have conducted an Taken together, our results have demonstrated that UA inhibits 3T3-L1 preadipocyte differentiation and lipid accumulation by regulating the transcriptional factors and their downstream lipogenic targets via the activation of LKB1/AMPK pathway. UA is a promising naturally-occurring therapeutic agent for the prevention and treatment of obesity.Figure S1Effect of UA on the viability and lipolysis in mature 3T3-L1. (A–B) Differentiated 3T3-L1 adipocytes were incubated in different concentrations of UA for 24, 48 hours, respectively. MTT reagent was added to the medium. After 4 hours of incubation, the medium was aspirated and 150 µL DMSO was added to each well. The absorbance was read at 570 nm. (C) Mature adipocytes were treated with 10 µM ursolic acid for 3 hours. Lipolysis was quantified by measuring absorbance at 520 nm. Data are expressed as means ± SD (n = 3). * P<0.05 and ** P<0.001 vs. the control.(TIF)Click here for additional data file.Figure S2Effectiveness of AMPK and LKB1 siRNA, and the effect of radicicol on the expression of LKB1, pAMPK and AMPK. (A) and (D) 3T3-L1 preadipocytes were transfected with AMPK or LKB1 siRNA oligonucleotide duplexes 1 day post the confluence with lipofectamine RNAiMax. The effectiveness of siRNA knockdown after 24 hours of transfection and on day 6 of cell differentiation was determined by measuring the expression of AMPK and LKB1 using the Western blotting as described in the (TIF)Click here for additional data file.


W69 and W106, two putative GPX genes, were obtained by de novo transcriptome sequencing of salt-treated wheat (Triticum aestivum) seedlings. The purified His-tag fusion proteins of W69 and W106 reduced H2O2 and t-butyl hydroperoxide (t-BHP) using glutathione (GSH) or thioredoxin (Trx) as an electron donor in vitro, showing their peroxidase activity toward H2O2 and toxic organic hydroperoxide. GFP fluorescence assays revealed that W69 and W106 are localized in chloroplasts. Quantitative real-time PCR (Q-RT-PCR) analysis showed that two GPXs were differentially responsive to salt, drought, H2O2, or ABA. Isolation of the W69 and W106 promoters revealed some cis-acting elements responding to abiotic stresses. Overexpression of W69 and W106 conferred strong tolerance to salt, H2O2, and ABA treatment in Arabidopsis. Moreover, the expression levels of key regulator genes  involved in salt, H2O2 and ABA signaling were altered in the transgenic plants. These findings suggest that W69 and W106 not only act as scavengers of H2O2 in controlling abiotic stress responses, but also play important roles in salt and ABA signaling.Oxidative stress caused by accumulation of reactive oxygen species (ROS) is capable of damaging effects on numerous cellular components. Glutathione peroxidases  are key enzymes of the antioxidant network in plants. In this study,  It is known that certain plant glutathione-S-transferase (GST) displayed also strong GPX activity (GST/GPX) toward organic hydroperoxides catalysing their reduction to the less toxic alcohols GPX gene was strongly expressed in normal tissues of high photosynthetic efficiency as well as in tissues stimulated by oxidative stresses GST/GPX gene confers salt and chilling tolerance due to enhanced ROS scavenging and reduces membrane damage in transgenic tobacco plants Arabidopsis GPX3 plays dual roles in H2O2 scavenging and ABA signal transduction Arabidopsis play a role in cross talk between photooxidative stress and immune responses Plant GPXs are involved in responses to many abiotic stresses and hormones Triticum aestivum L.) is one of the most important cereal crops in the world; however, ROS accumulation in cells induced by environmental stresses can cause severe oxidative damage to wheat growth and grain yield de novo transcriptome assembly technology. The isoenzymatic characteristics, subcellular localization, and transcript accumulation of the recombinant protein when transgenic plants were subjected to different stresses were investigated.Wheat (de novo assembly (unpublished data). Two up-regulated putative GPX cDNA fragments, designated as W69 (GenBank Accession No. KF031945) and W106 (GenBank Accession No. KF031946), were selected from the total transcript-derived assembled unigenes. Full-length cDNA of W69 and W106 were cloned successfully from wheat cDNA by reverse transcription PCR (RT-PCR) using special primers sets. The predicted W69 protein consists of 214 amino acids with a calculated molecular mass of 21.3 kD, and W106 protein consists of 211 amino acids and calculated molecular mass of 19.2 kD. Multiple sequence alignment of W69 and W106 with other reported GPXs from animals and plants showed that some motifs of GPXs were conserved among different species   . A numberomoters .W69 and W106 promoter regions have multiple abiotic stress-responsive elements . As shown in The elements . To asseW69 and W106 were rapidly activated to peak within 0.5 h after salt treatment, then declined to normal levels after 1 h, and were barely detectable within 24 h. Both were also rapidly induced by drought and ABA, reached a maximum level at 4 h and 1 h, respectively, and then declined. W69 was strongly induced by drought, but only weakly by ABA. In contrast, W106 was highly activated by ABA, but weakly by drought. In addition, W106 was immediately activated by H2O2 and absent after 6 h; however, the transcript of W69 was barely detectable over a 24 h treatment with H2O2. These results implied that the GPX subfamily may function mainly in oxidative responses induced by salt, H2O2 and ABA stresses, but with some divergence in roles between W69 and W106.Both E. coli BL21 cells was observed after IPTG induction. Samples of total proteins and purified soluble protein fractions were separated on SDS-PAGE. W69 (21 kDa) and W106 (19 kDa) were successfully expressed  program for predicting subcellular localization, indicated that both W69 and W106 with a chloroplastic N-terminal transit peptide would most probably localize in chloroplasts. To investigate the biological activity of the W69 and W106 proteins, the full-length cDNAs of W69 and W106 were fused in frame with the green fluorescent protein (hGFP) gene under the control of the CaMV 35S promoter, and transferred into wheat mesophyll protoplasts. Fluorescence of both W69-GFP and W106-GFP was specifically detected in chloroplasts, whereas the control expressing hGFP alone showed fluorescence distributed throughout the protoplasts .W69 and W106 seedlings had similar phenotypes to wild-type seedlings under normal conditions. As expected, salt stress reduced the growth of both transgenic seedlings and wild-type seedlings to some extent . Therefore, overexpression of W69 and W106 resulted in enhanced early tolerance to high salt stress.Transgenic e extent . At 150 e extent . Furthertrations . In addiWe also examined tolerance to drought stress in the transgenic plants, but observed no detectable increases in tolerance (data not shown).Arabidopsis were transferred to MS medium plates supplemented with 1 mM H2O2. At 15 d, the seedlings of W69 and W106 grew vigorously and were larger than wild-type seedlings. At 1 mM H2O2, the transgenics had longer primary roots than wild-type plants. There were also significant increases in the number and total lengths of the lateral roots compared to wild-type  found in the promoters of W69 and W106 might be responsible for the responses of genes to ABA. The CRT/DRE and GT1GMSCAM4 elements present in the promoters of the two isoenzymes might be responsible for genetic responses to NaCl. More importantly, ARE, an antioxidant-responsive element in rat is also present in the promoters of the two isoenzymes, and might be related to genetic response to oxidative stress  seedlings grown hydroponically at 25°C for 10 d were subjected to various abiotic stresses. Seedlings were exposed to air on filter paper for rapid induction of water stress. To mimic salinity and ABA treatments, roots were submerged in 100 mM NaCl or 100 µM ABA, respectively. For hydrogen peroxide treatment, seedlings were transferred to solutions containing 0.1 mM H2O2. Materials were collected at 0, 1, 2, 4, 16, 12, and 24 h after various treatments. Harvested plants were dropped immediately into liquid nitrogen and stored at –80°C for RNA extraction.Wheat  method.GPX genes were measured by Quantitative real-time PCR (Q-RT-PCR), using the ABI Prism 7300 system . Total RNA was extracted from young leaves of 2-week-old wheat plants using Trizol reagent according to the manufacturer's protocol  and DNase I digestions were applied . First-strand cDNA was synthesized with AMV Reverse Transcriptase . Each PCR was repeated three times in total volumes of 20 μl containing primer (4 μM), cDNA (40 ng) and 9 μl RealMasterMix . Quantification of the target gene expression under each stress was carried out by the relative 2−△△CT method actin gene was used as an internal control for normalization of template cDNA. The gene-specific primers were as follows: W69, 5'- ACAGAGGATTCGGGAGGACT-3' and 5'- AGGAGGACCTTCCCCTTGTA-3'; W106, 5'-ACAAGGGGAAAGTCCTGCTT-3' and W106-RTR 5'-CTGGTTCCTGTCCAGCAAAT-3'; wheat actin, 5'- CTCCCTCACAACAACCGC-3' and 5'- TACCAGGAACTTCCATACCAAC-3'. Q-RT-PCR was also performed to analysis the expression of stress- and ABA-responsive genes, using 2-week-old transgenic and wild-type Arabidopsis seedlings. The actin gene was used as an internal control. The specific primers were as follows: RbohD (At5g47910), 5'-CCTCAACAACACCACCTCCT-3' and 5'-GTAAGAGGCCGTTGGAATCA-3'; ABI1 (At4g26080), 5'-TGAAGAAGCGTGTGAGATGG-3' and 5'-CTGTATCGCCAGCTTTGACA-3'ABI2 (At5g57050), 5’-TGCGGCGAGTAAAAGAAGAT-3' and 5'-TTCCTTTTTGCAAAGCCATC-3'; SOS1 (At2g01980): 5'-ACCGGCAGATCTAATGAACG-3'and 5'-CTCCGCTACTGTCGATGTCA-3'; Actin2 in Arabidopsis, 5'-GGTAACATTGTGCTCAGTGGTG-3' and 5'-C GACCTTAATCTTCATGCTGC-3'.Expression patterns for the two 5'-TACGGATCCATGGGGGCGTCCGAATCT-3' and 5'-TAGCTCGAGCTTCTGCGAGTCGGAAGATTCC-3') and W106-PET (5'-TACGGATCCAACATGGGTGCGGCAGAGT-3' and 5'-TAGCTCGAGAACCTCCAACAGCTTCTTGATG-3'). The PCR product was cut by BamHI and XhoI and then subcloned into corresponding sites of pET-28a (Novagen) with a Histidine-tag at the N-terminus. The fusion plasmids and empty vector (PET-28a) were transformed into E. coli BL21 (DE3) strain. E. coli cells carrying pET-W69, pET-W106, and PET-28a were grown in 40 ml LB medium containing 50 mg/ml kanamycin at 37°C to D600  = 0.5 and induced with 0.8 mM isopropyl-1-thio-bD-galactopyranoside (IPTG) at 22°C. Cells were extracted at 4 h and were collected by centrifugation at 3000 g for 10 min, resuspended in pre-chilled sodium phosphate buffer , and disrupted by sonication. His-tag fusion pET-W69/pET-W106/and/PET-28a proteins were purified using Ni-NTA Agarose  according to the manufacturer's instructions. Protein concentrations were quantified using the Bradford method. Purified proteins from each sample were loaded for sodium dodecyl sulfate polyacrylamide gel electrophoresis.The two GPX cDNAs were amplified by reverse transcription PCR (RT-PCR) using the primer sets W69-PET . The reaction was started by addition of H2O2 or t-butyl hydroperoxide. The absorbance was immediately read at OD340 nm and again at 4 min using a Perkin-Elmer Lambda 35 UV/VIS spectrometer . The unit activity (U/L) is the amount of GPX that produces 1 μM of oxidized glutathione (GSSG) per min at pH 7.6 and room temperature.Enzyme activity was measured spectrophotometrically using a Glutathione Peroxidase Assay Kit  with minor modifications. W69 and W106 genes were respectively cloned into 16318hGFP vectors, fused with the GFP reporter gene under the control of the 35S promoter W69-hGFP and W106-hGFP fusion plasmids were transformed to common wheat mesophyll protoplasts by the PEG-mediated method Green fluorescent protein (GFP)-fused GPXs, coexpression plasmids was constructed for the expression studies. The full open reading frames of the W69 and W106 were separately ligated into the modified pBI121 vector under control of the CaMV 35S promoter Arabidopsis transformants carrying W69 or W106 were generated using the vacuum infiltration method. Transformed plants were cultured on Murashige and Skoog (MS) medium containing 0.8% agar and 50 μM Kanamycin in a day/night regime of 16/8 h under white light (50 photons m−1s−1) at 22°C for 2 weeks and were then transferred to soil. The T3 generation seeds obtained were used for further analysis.Fragments of Arabidopsis plants were grown on 10×10 cm MS agar plates. They were routinely kept for 3 days in the dark at 4°C to break dormancy and transferred to a tissue culture room under a day/night of 16/8 h under white light (with 50 photons m−1s−1) at 22°C for 3 d.Seeds of transgenic (T3 generation) and wild-type 2O2 treatment, the plants were placed on MS agar plates supplemented with H2O2 (1–5 mM) for 7 days. For ABA treatment, the seeds of transgenic and wild-type Arabidopsis were grown on MS agar plates supplemented with 0.5–1 μM ABA for 7–14 days. Seeds were considered germinated when radicles had completely emerged from the seed coat.For salt treatment, 7-day-old seedlings were transferred to MS agar plates containing 150 mM NaCl for 7 days. For drought treatment, the seedlings were placed on MS agar plates with mannitol (100–200 mM) for 7 days. For HArabidopsis were measured with a ruler, and lateral roots were counted and measured with a dissection microscope. The fresh weight was measured on an analytical balance immediately after plant harvest. The samples used for dry weight determinations were measured after 48 h at 70°C.The root lengths of


The antisense transcript-encoded HBZ protein acts upon viral and cellular gene expression through its modulation of the activation of transcription factors such as Jun family members. A possible link between HBZ and ATL development has also been highlighted. HTLV-3 and HTLV-4 are two newly discovered human retroviruses closely related to HTLV-1; no known diseases have yet been associated to these viruses. Based on previous in silico analyses, our aim was to test for the presence of antisense transcripts in these new viruses and to functionally assess these viral proteins.By RT-PCR analyses, HTLV-3 and HTLV-4 were shown to produce spliced antisense transcripts termed APH (Antisense Protein of HTLV)-3 and APH-4. Confocal microscopy analyses of cells expressing Myc- or GFP-tagged APH-3 and APH-4 showed distinct localization but partial colocalization with HBZ to the nucleus and the nucleoli. Using LTR-luciferase constructs, we demonstrated that APH-3 and APH-4 inhibited Tax1 and Tax3 transactivation of respective LTRs. In transfection experiments with a collagenase promoter-driven luciferase reporter construct, we showed that APH-3 and APH-4 modulated JunB- and c-Jun-dependent transcription differently from HBZ. Deletion mutants indicated that this upregulation of the transactivation potential of Jun factors was mediated through the atypical bZIP domain of APH-3 and APH-4.We show that HTLV-3 and HTLV-4 express new viral proteins, APH-3 and APH-4, which modulate Jun-dependent transcription differently from HBZ. These data underscore the importance of our study on APH-3 and APH-4 to help in better understanding the role of HBZ in the development of ATL.


Because of the low prevalence of Human T Lymphotropic Virus type I (HTLV-I) in comparison with Khorasan Province, considering HTLV-I as an etiology of spastic paraparesia, it may be neglected in evaluation of spastic paraparesis in the other regions of Iran. Some reports of spastic paraparetic patients due to HTLV-I infection in West Azarbaijan, caused us to reconsider the importance of HTLV-I epidemiology in the other areas of the country.All spastic paraparetic patients who referred to Motahari and Imam Khomeini educational hospitals of Urmia from September 2004 to September 2007 were assessed for HTLV-I infection antibodies.In our 3 years study, 11 cases were diagnosed as Human T Lymphotropic Virus type I Associated Myelopathy/Tropical Spastic Paraparesis .The mean age of patients at the time of diagnosis was 45.8 years. Dorsal and cervical MRI of all patients was normal. Serum Enzyme-Linked Immuno-Sorbent Assay (ELISA) and Western blot (WB) for anti HTLV-I antibody in all patients was positive. Four patients underwent for lumbar puncture in which were normal in respect of cells and biochemistry, but positive for anti-HLTLV-I antibodies.HAM/TSP detection in West Azarbaijan in spite of its long distance from Khorasan Province shows the importance of anti-HTLV-I Ab assay in the blood and CSF of every spastic paraparetic patient all over the country. During the past 25 years, Human T Lymphotropic Virus type I (HTLV-I) epidemiology has been studied many times getting more advanced day by day. Geographical distribution of virus in the world was previously demonstrated. Although several studies showed the high prevalence of HTLV-I in Japan southwest, however the prevalence of virus in adjacent countries like Korea, China, Eastern Russia and Khorasan in Iran is low. HTLV-I cAll patients presented with spastic paraparesia and referred to Motrahari and Imam Khomeini hospitals in Urmia, from September 2004 to September 2007 were assessed for anti HTLV-I Ab.All patients after systemic and neurologic examinations underwent dorsal and cervical Magnetic Resonance Imaging (MRI). After ruling out of compressive lesions all patients’ blood were tested for anti HTLV-I Ab with ELISA and WB methods. Only four patients allowed us to do lumbar puncture for Cerebro- spinal Fluid (CSF) analysis . The results are presented in Eleven patients  were diagnosed as HAM/TSP. The mean age for diagnosis was 45.8 years and for beginning of symptoms based on history was 36.3 years old. Three patients had a history of blood transfusion . Three patients were under treatment for primary progressive multiple sclerosis. All patients underwent dorsal and cervical MRI which were normal.Using ELISA and WB methods showed anti-HTLVI Ab in all patients. Only four patients underwent lumbar puncture in which all were normal for cell and biochemistry but positive for anti-HTLV-I antibodies.Epidemiologic studies have been done over the past 25 year's demonestrating worldwide distribution of HTLV-I with different prevalence rates. Because of wide distribution of HTLV-I infection all over the world, doing epidemiological studies in every region (even non-endemic areas) is essential. In some nonendemic areas such as rural West African and SoutA positive blood result for HTLV-I by ELISA in all patients indicates the high sensitivity of this test and repeatedly, proving the fact that ELISA is the best screening test in HTLV-I assay. Normal dorsal and cervical MRI in patients who referred to us with spastic paraparesia, reinforces the suspicion for viral and infectious etiologies such as HTLV-I. The presence of upper motor neuron signs with less severity in upper limbs is an interesting finding in which should be considered in HAM/TSP.Reporting 11 HAM/TSP patients in West Azarbaijan during a three years interval indicates the distribution of HTLV-I in Iran that remind us of the necessity for evaluation of any patient who refer with spastic paraparesia or spastic tetraparesia for HTLV-I.Regarding the possibility of HTLV-I transmission by blood transfusion, assessing the patients with the history of blood transfusion in past decades should be considered. Finally we are emphasizing that HAM/TSP should be considered in patients with subjective complains such as urinary incontinence or frequency, distal paresthesia, dysesthesia, muscle stiffness and gait disturbances.