The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator

Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.

Transcriptional activation of glucose transporter 1 in orthodontic tooth movement-associated mechanical response

The interplay between mechanoresponses and a broad range of fundamental biological processes, such as cell cycle progression,growth and differentiation, has been extensively investigated. However, metabolic regulation in mechanobiology remains largely unexplored. Here, we identified glucose transporter 1(GLUT1)—the primary glucose transporter in various cells—as a novel mechanosensitive gene in orthodontic tooth movement(OTM). Using an in vivo rat OTM model, we demonstrated the specific induction of Glut1 proteins on the compressive side of a physically strained periodontal ligament. This transcriptional activation could be recapitulated in in vitro cultured human periodontal ligament cells(PDLCs), showing a time-and dose-dependent mechanoresponse. Importantly, application of GLUT1 specific inhibitor WZB117 greatly suppressed the efficiency of orthodontic tooth movement in a mouse OTM model, and this reduction was associated with a decline in osteoclastic activities. A mechanistic study suggested that GLUT1 inhibition affected the receptor activator for nuclear factor-κ B Ligand(RANKL)/osteoprotegerin(OPG)system by impairing compressive force-mediated RANKL upregulation. Consistently, pretreatment of PDLCs with WZB117 severely impeded the osteoclastic differentiation of co-cultured RAW264.7 cells. Further biochemical analysis indicated mutual regulation between GLUT1 and the MEK/ERK cascade to relay potential communication between glucose uptake and mechanical stress response. Together, these cross-species experiments revealed the transcriptional activation of GLUT1 as a novel and conserved linkage between metabolism and bone remodelling.

Transcriptional activation function of hepatitis B virus Pre S1 protein in yeast

OBJECTIVE: To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre Slprotein of HBV by two-hybrid system.METHODS: Yeast expression plasmids encoding fusion proteins of full length or portions of Pre Sl ofHBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeastreporter strain SFY526. Reporter gene product β-galactosidase activity was assayed as a measure oftranscriptional activation in yeast, Mammalian expression plasmid encoding fusion proteins of full lengthPre Sl and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell lineHuh-7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin-layerchromatography.RESULTS: The fusion proteins of full length Pre Sl protein and GAL4 DNA binding domain presentedtranscriptional activation function in yeast. The transcription activating sequence was localized to the 21 to47 amino acids of Pre Sl protein. Fusion proteins of full length Pre Sl and GAL4 DNA binding domaindid not show transcriptional activation function in mammalian cells.CONCLUSIONS: The transcription activating sequence of HBV Pre Sl protein in yeast overlaps thehepatocyte receptor binding site. The transcriptional activation function of HBV Pre Sl protein in yeastmay prevent researchers from using yeast two-hybrid system to clone HBV receptor interacting with Pre Slprotein. However, the Pre Sl protein does not show transcriptional activation function in mammaliancells. Mammalian two-hybrid system may be a practical method to clone the HBV hepatocyte receptorinteracting with Pre Sl protein.

Detection for Transcriptional Activity of Alternaria Tenuissim Protein Elicitor in Yeast Two-hybrid System

The peaT1 gene fragment was amplified from pGEM-6p-l-peaT1 by PCR, and recovered target gene was cloned into pLexA vector. After digestion and sequencing, the bait vector pLexA-peaT1 was transformed into yeast strain EGY48 [p8op-lacZ] by PEG/LiAC, and the transcriptional activity of bait vector was detected. The results showed that recombinant bait plasmid pLexA-PEMG1 was constructed, for the two bands of recombinant bait plasmid in agarose gel eleetrophoresis were expected after digesting by restriction endonuclease EcoR I and Xho I. Therefore, the recombinant bait plasmid could be used in yeast two-hybrid system to screen a cDNA library.

Isolation and Characterization of Transcriptionally Active Ty1-copia Retrotransposons in Fragaria × ananassa

One possible mechanism suggested for somaclonal variation is the activation of transposable elements. The activation of retrotransposons by stresses and external changes is commonly observed in plants. In previous study, we isolated the reverse transcriptase （RT） gene sequences of Ty 1-copia retrotransposons from tissue culture strawberry （Fragaria x ananassa） plant, but not the transcriptionally active sequence. For further understanding the relationship between retrotransposon and somaclonal varation, in this study, we isolated the transcriptionally active RT gene sequences from strawberry plants subjected to different abiotic stresses. These retrotransposons were activated by spraying strawberry leaves with 2 mmol L^-1 salicylic acid （SA）, 50 mmol L^-1 methyl jasmonate （MeJA）, 50 mmol L^-1 abscisic acid （ABA）, 50 mmol L^-1 2,4- dichlorophenoxyacetic acid （2,4-D） or by inducing callus growth in 2 types of MS media： first medium supplemented with 0.5 mg L^-1 6-benzylaminopurine （6-BA）, 0.5 mg L^-1 gibberellic acid （GA3）, 1.0 mg L^-1 thidiazuron （TDZ）, and 0.1 mg L^-1 2,4-D, and the second medium supplemented with 0.5 mg L^-1 6-BA, 0.5 mg L^-1 GA3, 2.0 mg L^-1 TDZ, and 0.02 mg L^-1 indole butyric acid （1BA）. Analysis of gene sequences of 17 RTs revealed that none of them contained stop codons and/or indels disrupting the reading frame. These different stress-origin transcriptionally active RTs were remarkably similar to each other- FATEXP2-8 and FATEYS9-7 showed 100% sequence identity. Analysis of pylogenetic of these transcriptionally active RTs and the RT sequences from genome showed that there were close phylogenetic relationships of most of the transcriptionally active RTs. The results of this study have contributed to the background information necessary for future studies for evaluating the relationship between retrotransposons and somaclonal variation.

SHP2 regulates skeletal cell fate by modifying SOX9 expression and transcriptional activity

Chondrocytes and osteoblasts differentiate from a common mesenchymal precursor, the osteochondroprogenitor（OCP）, and help build the vertebrate skeleton. The signaling pathways that control lineage commitment for OCPs are incompletely understood. We asked whether the ubiquitously expressed protein-tyrosine phosphatase SHP2（encoded by Ptpn11） affects skeletal lineage commitment by conditionally deleting Ptpn11 in mouse limb and head mesenchyme using ＂Cre-lox P＂-mediated gene excision.SHP2-deficient mice have increased cartilage mass and deficient ossification, suggesting that SHP2-deficient OCPs become chondrocytes and not osteoblasts. Consistent with these observations, the expression of the master chondrogenic transcription factor SOX9 and its target genes Acan, Col2a1, and Col10a1 were increased in SHP2-deficient chondrocytes, as revealed by gene expression arrays, q RT-PCR, in situ hybridization, and immunostaining. Mechanistic studies demonstrate that SHP2 regulates OCP fate determination via the phosphorylation and SUMOylation of SOX9, mediated at least in part via the PKA signaling pathway. Our data indicate that SHP2 is critical for skeletal cell lineage differentiation and could thus be a pharmacologic target for bone and cartilage regeneration.

Diffusion-weighted magnetic resonance imaging reflects activation of signal transducer and activator of transcription 3 during focal cerebral ischemia/reperfusion

Signal transducer and activator of transcription（STAT）is a unique protein family that binds to DNA,coupled with tyrosine phosphorylation signaling pathways,acting as a transcriptional regulator to mediate a variety of biological effects.Cerebral ischemia and reperfusion can activate STATs signaling pathway,but no studies have confirmed whether STAT activation can be verified by diffusion-weighted magnetic resonance imaging（DWI）in rats after cerebral ischemia/reperfusion.Here,we established a rat model of focal cerebral ischemia injury using the modified Longa method.DWI revealed hyperintensity in parts of the left hemisphere before reperfusion and a low apparent diffusion coefficient.STAT3 protein expression showed no significant change after reperfusion,but phosphorylated STAT3 expression began to increase after 30 minutes of reperfusion and peaked at 24 hours.Pearson correlation analysis showed that STAT3 activation was correlated positively with the relative apparent diffusion coefficient and negatively with the DWI abnormal signal area.These results indicate that DWI is a reliable representation of the infarct area and reflects STAT phosphorylation in rat brain following focal cerebral ischemia/reperfusion.

β-Arrestin-2 enhances endoplasmic reticulum stress-induced glomerular endothelial cell injury by activating transcription factor 6 in diabetic nephropathy

BACKGROUND Glomerular endothelial cell(GENC)injury is a characteristic of early-stage diabetic nephropathy(DN),and the investigation of potential therapeutic targets for preventing GENC injury is of clinical importance.AIM To investigate the role ofβ-arrestin-2 in GENCs under DN conditions.METHODS Eight-week-old C57BL/6J mice were intraperitoneally injected with streptozotocin to induce DN.GENCs were transfected with plasmids containing siRNA-β-arrestin-2,shRNA-activating transcription factor 6(ATF6),pCDNA-β-arrestin-2,or pCDNA-ATF6.Additionally,adeno-associated virus(AAV)containing shRNA-β-arrestin-2 was administered via a tail vein injection in DN mice.RESULTS The upregulation ofβ-arrestin-2 was observed in patients with DN as well as in GENCs from DN mice.Knockdown ofβ-arrestin-2 reduced apoptosis in high glucose-treated GENCs,which was reversed by the overexpression of ATF6.Moreover,overexpression ofβ-arrestin-2 Led to the activation of endoplasmic reticulum(ER)stress and the apoptosis of GENCs which could be mitigated by silencing of ATF6.Furthermore,knockdown ofβ-arrestin-2 by the administration of AAV-shRNA-β-arrestin-2 alleviated renal injury in DN mice.CONCLUSION Knockdown ofβ-arrestin-2 prevents GENC apoptosis by inhibiting ATF6-mediated ER stress in vivo and in vitro.Consequently,β-arrestin-2 may represent a promising therapeutic target for the clinical management of patients with DN.

Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway

BACKGROUND Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved.Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process.AIM To assess the influence of interleukin-10(IL-10)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)following their interaction with macrophages in an inflammatory environment.METHODS IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment.In this study,we investigated its impact on the proliferation,migration,and osteogenesis of BMSCs.The expression levels of signal transducer and activator of transcription 3(STAT3)and its activated form,phos-phorylated-STAT3,were examined in IL-10-stimulated macrophages.Subsequently,a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling.RESULTS IL-10-stimulated macrophages underwent polarization to the M2 type through substitution,and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs.Mechanistically,STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages.Specifically,IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response,as evidenced by its diminished impact on the osteogenic differentiation of BMSCs.CONCLUSION Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs.The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs’osteogenic differentiation.

Central role of Yes-associated protein and WW-domain-containing transcriptional co-activator with PDZ-binding motif in pancreatic cancer development

Pancreatic ductal adenocarcinoma(PDAC) remains a deadly disease with no efficacious treatment options. PDAC incidence is projected to increase, which may be caused at least partially by the obesity epidemic. Significantly enhanced efforts to prevent or intercept this cancer are clearly warranted. Oncogenic KRAS mutations are recognized initiating events in PDAC development, however, they are not entirely sufficient for the development of fully invasive PDAC.Additional genetic alterations and/or environmental, nutritional, and metabolic signals, as present in obesity, type-2 diabetes mellitus, and inflammation, are required for full PDAC formation. We hypothesize that oncogenic KRAS increases the intensity and duration of the growth-promoting signaling network.Recent exciting studies from different laboratories indicate that the activity of the transcriptional co-activators Yes-associated protein(YAP) and WW-domaincontaining transcriptional co-activator with PDZ-binding motif(TAZ) play a critical role in the promotion and maintenance of PDAC operating as key downstream target of KRAS signaling. While initially thought to be primarily an effector of the tumor-suppressive Hippo pathway, more recent studies revealed that YAP/TAZ subcellular localization and co-transcriptional activity is regulated by multiple upstream signals. Overall, YAP has emerged as a central node of transcriptional convergence in growth-promoting signaling in PDAC cells. Indeed, YAP expression is an independent unfavorable prognostic marker for overall survival of PDAC. In what follows, we will review studies implicating YAP/TAZ in pancreatic cancer development and consider different approaches to target these transcriptional regulators.

Regulation and function of signal transducer and activator of transcription 3

Signal transducer and activator of transcription 3(STAT3), a member of the STAT family, is a key regulator of many physiological and pathological processes. Significant progress has been made in understanding the transcriptional control, posttranslational modification, cellular localization and functional regulation of STAT3. STAT3 can translocate into the nucleus and bind to specific promoter sequences, thereby exerting transcriptional regulation. Recent studies have shown that STAT3 can also translocate into mitochondria, participating in aerobic respiration and apoptosis. In addition, STAT3 plays an important role in inflammation and tumorigenesis by regulating cell proliferation, differentiation and metabolism. Conditional knockout mouse models make it possible to study the physiological function of STAT3 in specific tissues and organs. This review summarizes the latest advances in the understanding of the expression, regulation and function of STAT3 in physiological and tumorigenic processes.

Inhibition of signal transducer and activator of transcription 3 expression by RNA interference suppresses invasion through inducing anoikis in human colon cancer cells

AIM:To investigate the roles and mechanism of signal transducer and activator of transcription 3 (STAT3) in invasion of human colon cancer cells by RNA interference. METHODS: Small interfering RNA (siRNA) targeting Signal transducer and activator of transcription 3 (STAT3) was transfected into HT29 colon cancer cells. STAT3 protein level and DNA-binding activity of STAT3 was evaluated by western blotting and electrophoretic mobility shift assay (EMSA), respectively. We studied the anchorage-independent growth using colony formation in soft agar, and invasion using the boyden chamber model, anoikis using DNA fragmentation assay and terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL), respectively. Western blot assay was used to observe the protein expression of Bcl-xL and survivin in colon cancer HT29 cells. RESULTS: RNA interference (RNAi) mediated by siRNA leads to suppression of STAT3 expression in colon cancer cell lines. Suppression of STAT3 expression by siRNA could inhibit anchorage-independent growth, and invasion ability, and induces anoikis in the colon cancer cell line HT29. It has been shown that knockdown of STAT3 expression by siRNA results in a reduction in expression of Bcl-xL and survivin in HT29 cells. CONCLUSION: These results suggest that STAT3 siRNA can inhibit the invasion ability of colon cancer cells through inducing anoikis, which antiapoptotic genes survivin and Bcl-xL contribute to regulation of anoikis.These studies indicate STAT3 siRNA could be a useful therapeutic tool for the treatment of colon cancer.

Signal transducer and activator of transcription 3 promotes the Warburg effect possibly by inducing pyruvate kinase M2 phosphorylation in liver precancerous lesions

BACKGROUND Study shows that signal transducer and activator of transcription 3(STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2(PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.AIM To investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.METHODS A model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with Nmethyl-N'-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen(PCNA), STAT3,and PKM2 were examined by Western blot and immunofluorescence.RESULTS We found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liverprecancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression,PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells.Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.CONCLUSION The Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

Silencing of signal transducer and activator of transcription 3 expression by RNA interference suppresses growth of human hepatocellular carcinoma in tumor-bearing nude mice

AIM： To explore the effect of silencing of signal transducer and activator of transcription 3 （STAT3） expression by RNA interference （RNAi） on growth of human hepatocellular carcinoma （HCC） in tumorbearing nude mice in vivo.METHODS： To construct the recombinant plasmid of pSilencer 3.0-H1-STAT3-siRNA-GFP （pSHI-siRNA- STAT3） and establish the tumor-bearing nude mouse model of the HCC cell line SMMC7721, we used intratumoral injection together with electroblotting to transfect the recombinant plasmid pSHI-siRNA- STAT3 into the transplanted tumor. The weight of the nude mice and tumor volumes were recorded. STAT3 gene transcription was detected by semi-quantitative reverse transcription polymerase chain reaction （RT- PCR）. Level of protein expression and location of STAT3 were determined by Western blotting and immunohistochemical staining. STAT3-related genes such as survivin, c-myc, VEGF, p53 and caspase3 mRNA and protein expression were detected in tumor tissues at the same time. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay was used to detect apoptosis of tumor cells.RESULTS： The weight of the treated nude mice increased, and the tumor volume decreased markedly compared with those of the mock-treated and negative control groups （P 〈 0.01）. The results of RT-PCR and Western blotting showed that mRNA and protein levels of STAT3 declined markedly in the treated group. The change in STAT3-related gene expression in tumor tissues at the mRNA and protein level also varied, the expression of survivin, VEGF and c-myc were obviously reduced, and expression of p53 and caspase3 increased （P 〈 0.01）. Most of the tumor tissue ceils in the treated group developed apoptosis that was detected by TUNEL assay.CONCLUSION： Silencing of STAT3 expression by RNAi significantly inhibits expression of STAT3 mRNA and protein, and suppresses growth of human HCC in tumor-bearing nude mice. The mechanism may be related to down-regulation of survivin, VEGF and c-myc and up-regulation of p53 and caspase3 expression. Accordingly, the STAT3 gene may act as an important and effective target in gene therapy of HCC.

New insights into p53 activation

The tumor suppressor p53 is a multifunctional, highly regulated, and promoter-specific transcriptional factor that is uniquely sensitive to DNA damage and cellular stress signaling. The mechanisms by which p53 directs a damaged cell down either a cell growth arrest or an apoptotic pathway remain poorly understood. Evidence suggests that the in vivo functions of p53 seem to balance the cell-fate choice with the type and severity of damage that occurs. The concept of antirepression, or inhibition of factors that normally keep p53 at bay, may help explain the physiological mechanisms for p53 activation. These factors also provide novel chemotherapeutic targets for the reactivation of p53 in tumors harboring a wild-type copy of the gene.

Activation of JAK-STAT pathway is required for platelet-derived growth factor-induced proliferation of pancreatic stellate cells

AM: To clarify the role of Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway in platelet-derived growth factor (PDGF) induced proliferation in activated pancreatic stellate cells (PSCs). METHODS: PSCs were isolated from rat pancreas tissue, and used in their culture-activated, myofibroblast-like phenotype. STAT-specific binding activity was assessed by electrophoretic mobility shift assay. Activation of Src, JAK2, STAT1, STAT3, and ERK was determined by Western blotting using anti-phosphospecific antibodies. Cell proliferation was assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine. RESULTS: PDGF-BB induced STAT-specific binding activity, and activation of Src, JAK2, STAT1, STAB, and ERK. Ethanol and acetaldehyde at clinically relevant concentrations decreased basal activation of JAK2 and STAT3. PDGF-induced activation of STAT1 and STAT3 was inhibited by a Src inhibitor PP1 and a JAK2 inhibitor AG490, whereas PDGF-induced activation of ERK was inhibited by PP1, and not by AG490. PDGF-induced proliferation was inhibited by PP1 and AG490 as well as by STAT3 antisense oligonucleotide. CONCLUSION: PDGF-BB activated JAK2-STAT pathway via Src-dependent mechanism. Activation of 3AK2-STAT3 pathway, in addition to ERK, may play a role in PDGF-induced proliferation of PSCs.

Downregulation of signal transducer and activator of transcription 3 by sorafenib:A novel mechanism for hepatocellular carcinoma therapy

Hepatocellular carcinoma is one of the most common cancers worldwide,and a leading cause of cancer-related death.Owing to unsatisfactory clinical outcomes under the current standard of care,there is a need to search for and identify novel and potent therapeutic targets to improve patient outcomes.Sorafenib is the first and only approved targeted therapy for the treatment of hepatocellular carcinoma.Besides functioning as a multiple tyrosine kinase,sorafenib also acts via a kinase-independent mechanism to target signal transducer and activator of transcription 3(STAT3) signaling in hepatocellular carcinoma cells.STAT3 is a key regulator of inflammation,cell survival,and tumorigenesis of liver cells,and the high percentage of hepatocellular carcinoma cells with constitutively active STAT3 justifies targeting it for the development of novel therapeutics.Sorafenib inactivates STAT3 and STAT3-related signaling by inducing a conformational change in and releasing the autoinhibition of Src homology region 2 domaincontaining phosphatase-1.This phosphatase negatively regulates STAT3 activity,which leads to the subsequent apoptosis of cancer cells.The novel anti-cancer property of sorafenib will be discussed in this review,not only adding information regarding its mechanism of action but also providing an innovative approach for the development of cancer therapeutics in the future.

Characterization of ribulose-1,5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica(Laminariales,Phaeophyta)

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments, and consequently may have highly efficient ribulose-1, 5-bisphosphate carboxylase/ oxygenase （Rubisco） activity for carbon assimilation. In our study, we cloned the full-length Rubisco gene from S.japonica （SJ-rbc）. It contained an open reading frame for a large subunit gene （SJ-rbcL） of 1 467 bp, a small subunit gene （SJ-rbcS） of 420 bp, and a SJ-rbcL/S intergenie spacer of 269 bp. The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa, 5.81 and 15,84 kDa, 4.71, respectively. After induction with 1 mmol/L isopropyl-β-D- thiogalactopyranoside for 5 h and purification by Ni2＋ affinity chromatography, electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein. Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations： increased expression during the day but suppressed expression at night. This observation implied that Rubisco played a role in normal gametophytic growth and development. In juvenile sporophytes, mRNA levels of SJ-rbcL, carbonic anhydrase, Calvin-Benson- Bassham cycle-related enzyme, and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance. Similarly, expression of these genes was up-regulated under blue light irradiance at 350 umol/（m2.s）. Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.

Repression of interferon-γ expression in T cells by Prosperorelated Homeobox protein

Interferon-gamma （IFN-γ） is a major proinflammatory effector and regulatory cytokine produced by activated T cells and NK cells. IFN-γ has been shown to play pivotal roles in fundamental immunological processes such as inflammatory reactions, cell-mediated immunity and autoimmunity. A variety of human disorders have now been linked to irregular IFN-γ expression. In order to achieve proper IFN-γ-mediated immunological effects, IFN-γ expression in T cells is subject to both positive and negative regulation. In this study, we report for the first time the negative regulation of IFN-γ expression by Prospero-related Homeobox （Proxl）. In Jurkat T cells and primary human CD4＋ T cells, Proxl expression decreases quickly upon T cell activation, concurrent with a dramatic increase in IFN-γ expression. Reporter analysis and chromatin immunoprecipitation （CHIP） revealed that Proxl associates with and inhibits the transcription activity of IFN-γ promoter in activated Jurkat T cells. Co-immunoprecipitation and GST pull-down assay demonstrated a direct binding between Proxl and the nuclear receptor peroxisome proliferator-activated receptor gamma （PPARγ）, which is also an IFN-γ repressor in T cells. By introducing deletions and mutations into Proxl, we show that the repression of IFN-γ promoter by Proxl is largely dependent upon the physical interaction between Proxl and PPARγ. Furthermore, PPARγ antagonist treatment removes Proxl from IFN-γ promoter and attenuates repression of IFN-γ expression by Proxl. These findings establish Proxl as a new negative regulator of IFN-γ expression in T cells and will aid in the understanding of IFN-γ transcription regulation mechanisms.

Effects of the Ratio of Lysine to Digestible Energy Level in the Diet on the Expression and Activity of Transcription Factors Involved in Lipogenesis in Rongchang Pigs

This study was conducted to determine the effects of varying the ratio of lysine to digestible energy level On the activity and gene expression of the transcription factors peroxisome proliferator-activated receptor-γ （PPAR-γ） and CCAAT/enhancer-binding protein-or and -β （C/EBP-α and C/EBP-β） to better understand the regulatory mechanisms controlling adipogenesis in fat and muscle tissue of the Rongchang pig. A total of 144 castrated Rongchang pigs weighing approximately 20 kg were used in a 2 ×2 factorial design experiment. Diets were formulated to contain a high （14.22 MJ/kg） or low （13.11 MJ/kg） digesti- ble energy （DE） level. Within each energy level, pigs were fed diets containing a high lysine： DE ratio （0.67,0. 53, or 0. 42） or a low lysine ： DE ratio （0.49,0.38 ,or 0.30） during the periods from 20 to 50 kg, 50 to 80 kg, and 80 kg to slaughter, respectively. Each diet was fed to six replicate pens, each containing nine pigs. When the pigs reached average live weights of 20,35,60, and 90 kg ,one pig from each of the replicates was chosen at random and slaughtered.Samples of back fat and longissimus dorsi muscle were collected for the assessment of transcriptional factor. The results showed that feeding a high DE level significantly increased （ P 〈 0.05 ） the expression of PPAR-T at 60 and 90 kg in muscle and at 35,60, and 90 kg in back fat. Energy level also significantly increased the expression of C/EBP-fl at 35 and 60 kg in both muscle and back fat （ P 〈 0.05 ）. Higher dieta- ry lysine increased the expression of C/EBP-fl in muscle at 35 and 90 kg （ P 〈 0.05）, but decreased the expression in back fat at 35 （P = 0.03 ） and 90 kg （P = 0.09）. The lysine level increased the expression of PPAR-3~ in muscle at 60 kg only. Energy level and lysine content had no significant effects on promote the activity of PPAR-γ, C/EBP-α, or C/EBP-β either in muscle or in back fat at any level of the body weights tested. Collectively, these data indicated that dietary energy density and lysine level were equally important for lipid deposition in muscle tissue, whereas dietary energy density was more important than lysine level for fat deposition in fat tissue.