A multiplicity of vital cellular and tissue level functions are controlled by caveolin-1 and it is considered to be an important candidate for targeted therapeutics. Rac1-cortactin signaling plays an important role in maintaining the functions of the endothelial barrier in microvascular endothelial cells. The activity of Rac1 has been shown to be regulated by caveolin-1. Therefore, the present study investigated the consequences of down-regulating caveolin-1 and the subsequent changes in activity of Rac1 and the endothelial barrier functions in primary rat pulmonary microvascular endothelial cells (RPMVECs). RPMVECs were transfected with a small hairpin RNA duplex to down-regulate caveolin-1 expression. This procedure significantly increased the activity of Rac1. Moreover, down-regulation of caveolin-1 attenuated TNF-α-induced decrease in TER, increase in the flux of FITC-BSA and the disappearance of cortactin from the cell periphery in RPMVEC. Rac1 inhibitors significantly abolished this barrier-protective effect induced by down-regulation of caveolin-1 in response to TNF-α in RPMVECs. In conclusion, our data suggest a mechanism for the regulation of Rac1 activity by caveolin-1, with consequences for activation of endothelial cells in response to TNF-α. Caveolae or “small caves” were originally identified as 50 to 100 nm flask-shaped, non-clathrin-coated invaginations of the plasma membrane. Recent data revealed the implication of caveolae in a variety of cellular processes, including transendothelial vesicular transport Previous studies have proposed and envisaged a working model of EC barrier regulation The Rho GTPase family The cortical actin band spans the entire circumference of endothelial cells and it is composed of F-actin bundles. Cortactin, an actin-binding protein, is a ubiquitously expressed tyrosine kinase and has been implicated in cortical actin assembly and reorganization. Indeed, the functional relevance of cortactin for endothelial permeability was demonstrated by attenuated responses to barrier-protective stimuli following cortactin knockdown In all, considered the important role of Rac1 in controlling cell permeability and the close relationship between Rac1 and caveolin-1, we hypothesized that it was reasonable to explore whether caveolin-1 is involved in regulating endothelial permeability induced by TNF-α through the Rac1 signaling pathway. Our current study provides evidence that TNF-α-induced endothelial barrier breakdown occurs by impairing Rac1 signaling and this process requires caveolin-1 participation. We also found that primary rat pulmonary microvascular endothelial cells (RPMVECs) lacking caveolin-1 were significantly resistant to TNF-α-induced barrier dysfunction by up-regulating Rac1 activity. Therefore, this study describes a novel mechanism by which the down-regulation of caveolin-1 confers cytoprotection to RPMVECs in response to TNF-α.TNF-α was purchased from Peprotech Inc. . Caveolin-1 antibody was purchased from Abcam Inc. . Polyclonal anti-cortactin antibody, rhodamine-conjugated phalloidin, FITC-labeled BSA and unlabeled BSA were purchased from Sigma-Aldrich . NSC-23766 was obtained from Calbiochem/Merck . 8-pCPT-20 -O-Methyl-cAMP (O-Me-cAMP) was from Biolog Life Science Institute . Horseradish peroxidase (HRP)-labeled antibody to rabbit IgG was purchased from Wuhan Boster Biological Engineering Co, Ltd . Transwell clear polyester membrane cell culture chambers  were purchased from Costar . High glucose Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Invitrogen . Fetal calf serum, penicillin, streptomycin, and trypsin were purchased from HyClone Laboratory .2. Experimental data were obtained from cells in their third to fifth generation. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of Anhui Medical University. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.As described previously, the method of isolation and culture primary RPMVECs has been successfully established in our laboratory 6 RPMVEC were seeded in a 6 cm plate. The next day, the medium was replaced with fresh DMEM containing 15% fetal calf serum, lentivirus and the medium with 8 mg/ml polybrene. The supernatant with the lentivirus particles was replaced the following morning with media containing 15% fetal calf serum.Caveolin-1 RNAi lentiviruses were packaged with the use of a four-plasmid system as described previously 2) across the endothelial monolayer according to the manufacture’s protocol as described previously 5 cells/cm2) were seeded on gelatin-coated transwell polyester membranes and grown to confluence for 48 h. Cells were then allowed to stabilize for 24 h, incubated with fresh culture medium in the presence or absence of TNF-α for different time points. The changes of TER across RPMVEC monolayer were recorded.An epithelial volt-ohm meter and STX-2 electrodes  was used to measure transendothelial resistance  and grown to confluence. The monolayers were serum-starved for 1 h then incubated with fresh culture medium in the presence or absence of TNF-α for the different time points, as described below. At the end of the stimulation, FITC-BSA  and an equimolar amount of unlabeled BSA were mixed with phenol red-free DMEM and added to the top chamber and the bottom chamber, respectively, for 1 h at 37°C. Paracellular flux was assessed by obtaining aliquots from both chambers to measure the real-time changes of permeability across the endothelial cell monolayers. The concentration of FITC-BSA was quantified with a fluorescence spectrofluorophotometer as described previously 4 RPMVECs were seeded on 2 cm2 coverslips and grown for 12 hours. The coverslips were washed with phosphate buffer saline (PBS) after drug treatment and then fixed with 4% formaldehyde. After blocking with 2% BSA for 1 h, cells were exposed to the primary antibodies overnight at 4°C. After washing with PBS, cells were incubated with the appropriate secondary antibodies conjugated to immunofluorescent dyes for 60 min. In order to stain for F-actin, fixed cells were incubated with rhodamine-conjugated phalloidin for 1 hour at 37°C,then washed with PBS. Coverslips were mounted on glass slides with 60% glycerol in PBS. Cells were scanned with the laser scanning confocal microscope.5×10Determination of Rac1 activation (Rac1-GTP) was performed with a commercially available kit . Briefly, after stimulation, cell lysates were collected, and GTP-bound Rac1 was captured using a pull-down assay with immobilized PAK1-PBD according to the manufacturer’s protocols. The levels of activated small GTPases and total Rac1 were assessed by Western blot analysis and quantified by scanning densitometry of autoradiography films. The levels of activated proteins Rac1 were normalized to total Rac1 levels.Confluent monolayers were serum-starved overnight before treated with desired agents. The cells were lysed in RIPA buffer at 4°C. The lysates were centrifuged at 14,000×g for 15 min followed by heating at 95–100°C for 5 min. Equal amounts of protein from each sample were electrophoresed by 12% gels and transferred to 0.45 µm nitrocellulose membranes. The membranes were blocked with 5% nonfat milk for 1 hour at room temperature, then probed with different antibodies overnight at 4°C The blots were washed with wash buffer, followed by incubation with HRP-conjugated secondary antibody for 1 h at RT. The blots were visualized using an enhanced chemiluminescence kit. Blots were scanned and quantitatively analyzed by ImageQuant software.t-test or a one-way ANOVA. Significance in all cases was defined at P<0.05.Values were shown as the mean ± SD. Data were analyzed using a standard Student’s 2. Exposure to TNF-α (100 ng/ml) for 1 hour, TER decreased significantly and then dropped to 63±5% of baseline levels after 2 hours. Negative controls showed no alteration of TER  n of TER . Pre-incn of TER  after 1 n of TER . Pre-incn of TER .Immunoﬂuorescent images were generated in order to link the influence of TNF-α on EC TER with cytoskeleton changes. In the negative controls, few stress fibers were observed and cortactin was detected in the cytoplasm as well as along with some parts of the cell membrane . PrimaryPrevious studies demonstrated that the activity of Rac1 was decreased in response to TNF-α and the activation of Rac1 could alleviate the loss of barrier function of microvascular endothelial cell, which was caused by cytokines To determine the role of caveolin-1 in TNF-α–induced hyperpermeability of primary RPMVECs monolayer, we utilized shRNA to specifically down-regulate caveolin-1 expression. As indicated in 2 by 116±10% compared with control shRNA cell monolayers (49.4±3.1Ω*cm2) (Down-regulation of caveolin-1 expression led to an increase in the mean baseline TER of RPMVECs monolayer to 57.8±4.9 Ω*cm.1Ω*cm2) . At the .1Ω*cm2) . When Ca.1Ω*cm2) . Further.1Ω*cm2) . These d.1Ω*cm2) .P<0.001) and after TNF-α stimulation for 2 hours  suggesting that caveolin-1 acts as a negative modulator of Rac1.In our study, we observed that shRNA-mediated caveolin-1 loss enhanced Rac1 activity in the resting state and that the loss of caveolin-1 also attenuated decreased Rac1 activity induced by TNF-α stimulation in primary RPMVECs . CaveoliIn the previous section, we have confirmed that inhibiting caveolin-1 could protect barrier function from injury of TNF-α in Cav-1-shRNA cells. We also observed that this barrier-protective effect induced by knocking down caveolin-1 was abolished by pharmacological Rac1 inhibitor NSC-23766 . The TER of Cav-1-deficient PMVECs monolayers were higher than that of the control shRNA cells,regardless of whether they were pretreated with TNF-α or not. Such TER increase could be inhibited by Rac1 inhibitor NSC-23766. Moreover,NSC-23766 could make the TER decrease more in PMVECs which losing caveolin-1 . HighperInflammatory mediators induce vascular hyperpermeability primarily through the formation of intercellular gaps between endothelial cells, which is a process known as paracellular leak In the present study, we investigated the requirement of caveolin-1 in Rac1 signaling and the role of cortactin in the stabilization of endothelial barrier functions. We focused on cortactin because it is known to be important for endothelial barrier integrity and in previous studies endothelial cell barrier enhancement correlated with activation of cortactin Our experiments demonstrate that TNF-α induced barrier breakdown in cultured primary RPMVEC was a direct result of impaired Rac1 signaling. We observed an increase of central actin stress fibers and the disappearance of cortactin from the cell periphery when exposured to TNF-α. This cellular change was accompanied by decreased Rac1 activity. All these changes were associated with hyperpermeablilty of the endothelial cell monolayer. However, activation of Rac1 could enhance cortical actin rim formation resulting in the protection of endothelial barrier from injury due to TNF-α. In our study, activation of Rac1 induced lamellipodia formation P<0.001) and after TNF-α stimulation , suggesting that caveolin-1 acts as a negative modulator of Rac1 in primary RPMVECs, which was consistent with other data Previous studies have shown that Rac1 associates with the scaffolding domain of Cav-1 through its hypervariable C-terminal domain and that Cav-1 is part of a negative-feedback loop that controls cell polarity, spreading and migration by regulating the degradation of activated Rac1 Mechanistically, we have found that alterations in the signaling of Rac1 and cortactin are responsible for the changes in morphology and permeability in Cav-1-deficient cells. Previous reports have identified that down-regulation of caveolin-1 increases Src activation, which activates Rac1 through the exchange factors Dock180 However, it has to be emphasized that additional mechanisms, such as oxidative stress, may also contribute to the TNF-α-induced breakdown of endothelial barrier functions In summary, our findings provided novel data demonstrating that TNF-α-induced RPMVECs barrier breakdown is, at least in part, mediated by Rac1 inactivation. Secondly, we confirm that down-regulation of caveolin-1 could attenuate permeability increasing induced by TNF-α partially by activation of Rac1-cortactin signaling pathway. Because the molecular basis of acute lung injury is not well understood and there are no specific therapies for it, this study may not only confirm the potential therapeutic value of caveolae in the management of ARDS but also depict a role of Rac1-cortactin signaling in maintainning RPMVECs barrier integrity. We believe that this research will deepen our recognition of lung vascular barrier function and may lead to novel treatments in ARDS.


Thus, acetylcholine antagonists or nitric oxide synthase (NOS) inhibitors failed to inhibit the TNF-α induced activation. In conclusion, TNF-α may play a pivotal role in the protection of lower airways by inducing ciliary activity and increase in particle transport velocity via TNF-R1 and 5-HT2 receptor.The tracheal epithelium prevents via its highly effective clearance mechanism the contamination of the lower airways by pathogens. This mechanism is driven by ciliary bearing cells which are not only in contact with the gas phase; in addition they are also influenced by inflammatory mediators. These mediators can alter the protective function of the epithelium. Since the pro-inflammatoric cytokine tumor necrosis factor-α (TNF-α) plays a pivotal role within the inflammatory cascade, we investigated its effect onto the tracheal epithelium measured by its ciliary beat frequency and the particle transport velocity. In organ explant experiments the ciliary beat frequency and the particle transport velocity were measured under the application of TNF-α using tracheae from male C57BL6J mice. We observed a dose dependent TNF-α induced increase of both particle transport velocity and ciliary beat frequency. Knock out mice experiments made evident that the increase was depended on the expression of tumor necrosis factor receptor 1 (TNF-R1). The increases in ciliary beat frequency as well as the accelerated particle transport velocity were either inhibited by the unspecific serotonin antagonist methysergide or by cyproheptadine a specific 5-HT The mucociliary clearance is an important defence mechanism of the lower respiratory tract. This innate protector clears the airway surface area from debris, infectious particles and pathogens and is propelled by directed function of ciliary bearing cells integrated in the airway epithelium. In addition to the mechanical defence mechanism, a specific composition of mucus with a balanced mixture of salt and water is an important regulation factor for successful airway clearance. A defect of the cystic fibrosis transmembrane conductance regulator (CFTR) gene causes a dysfunction of the CFTR-protein. This protein defect, which regulates the salt water balance, leads to dehydration of the epithelia lining airway surface and results in an ineffective clearance, formation of local inflammation and an increase of chronic airway infections It is generally accepted that the protective beating function of ciliary bearing cells is modulated by different stimuli. In addition to cyclic adenosine monophosphate (cAMP) and intracellular calcium concentration, cyclic guanosine monophosphate (cGMP) seem to be important messengers to activate phosporylation and mediate an increase in cilliary beat frequency Among epithelial cells it has been assumed that other cell types e.g. activated mast cells and platelets affect the mucociliary transport via the endogenous mediator serotonin that can act as an activator of ciliary beat function Beside this constant regulation of cilia bearing cells, further modulators can alter ciliary function depending on physiological or patho-physiological requirements. Among patho-physiological processes inflammation leads to activation of the immune system and early in this mechanism cytokines are generated and released, which then can cause imbalances of organ functions. These cytokines released during inflammation or infection may also influence mucociliary clearance capacity Within in the family of pro-inflammatory cytokines, TNF-α plays a key role in activation of immune cells integrated in the cascade of host response to infections. However, TNF-α has not only an impact on genuine immune cells, it also initiates functions of cells associated to the immune system that provide specific functions in the pro-inflammatory response. Beside cells of the innate and adaptive immune system, many other non-immune cells are also capable of producing TNF-α like e.g. endothelial cells, neuronal cell and cardiac myocytes. The effects of TNF-α are mediated mainly via TNF-R1 which is found on many cell types and TNF-α receptor-2 (TNF-R2) that is exclusively expressed by immune cells. Upon binding, TNF-α upregulates pro-inflammatory gene expression via different signal transduction cascades including NF-κβ or MAP kinase Nevertheless, the entire effects of cytokines onto the clearing system of lower airways are still poorly understood. The tracheal epithelium may be attributed to the immune system first by its passive barrier function to block microbial intrusion, and second by its active clearing mechanism driven by ciliary cells. For this reason, we analysed the short term effects of TNF-α onto the functions of tracheal epithelial cells by recording ciliary beat frequency (CBF) and epithelial particle transport velocity (PTV) in murine tracheae.tm1Mak/J), which are established animals for mice deficient in TNF-R1 with a C57BL6J background strain In the present study we used male C57BL6J (Charles River) mice aged between 12 and 15 weeks (25–35 g). In a further series of experiments we employed C57BL6J TNF-R1 knock out mice  leading to maximal ATP-induced CBF increase. In each experiment tracks of approximately 200 to 400 particles were evaluated at certain pre-defined time points  . Short mViability of the tissue was ensured by adding ATP at the end of each experiment. If the response to ATP failed the experiment was not included into further statistical analysis.The following drugs were administered: ATP (100 μM), methysergide (100 μM), WAY-100635 (1 μM), L-NAME (100 μM), L-NMMA (100 μM), atropine (1 μM) all from Sigma  cyproheptadine (5 μg/ml) .2, 11 mM Glucose, 140 mM NaCl, 1 mM MgCl2, pH was adjusted to 7.4 at 30°C or 4–8°C for tissue preparation using NaOH (4 molar).Application of drugs taken from a stock solution (see above) was performed with a pipette into the organ bath solution; end concentration was achieved by gently mixing the buffer with a pipette. All preparation and experiments were carried out in HEPES solution consisting of : 20 mM HEPES, 4,5 mM KCl, 2,5 mM CaClTNF-α  was taken out of a stock solution  that was freshly prepared for each experimental day.For PCR analysis mRNA was isolated from tracheae that had either been stimulated with TNF-α during PTV/CBF experiments or untreated organs as control. For further positive controls we isolated peripheral blood mononuclear cells (PBMC).The tissue was processed using a bead mill homogenizer . Afterwards the supernatant was transferred into a gDNA eliminator column and then centrifuged with 8000 g for 30 s. Immediately followed by the mixture with 600 μl ethanol 70% and then 700 μl of this mixture was placed in a RNeasy Plus Mini Kit  column. Two further centrifuge steps followed each at 8000 g for 15 s and in between the solution was resuspended with 700 μl RW 1 buffer solution. Thereafter 500 μl RPE buffer was added and the suspension centrifuged by 8000 g for 15 s, followed by a 2 min centrifugation. Eventually the solution was transferred into a 1.5 ml vessel and diluted with 50 μl RNase free water followed by a final centrifugation step at 8000 g for 15 s.Transcription of RNA to cDNA was conducted using the Quantitect RTranscription kit  by transferring 1 μg RNA into 2 μl gDNA Wipeout Buffer and dilution with RNase free water up to a total volume of 14 μl followed by incubation at 42°C for 2 min and further addition of 1 μl reverse transcriptase, 4 μl Quantiscript RT buffer and 1 μl RT primer omitted in control experiments. The transcription was then performed at 42°C for 20 min and stopped by heating up to 92°C for 3 min. The obtained cDNA was stored at –80°C until further PCR experiments.2, 5 μl dTNPs, 5 μl upstream primer and 5 μl downstream primer 1.25 μl DNA polymerase and 158.75 μl nuclease free water.PCR experiments were performed using the Promega-PCR-kit, the master mix consisted of 50 μl flexi buffer, 20 μl MgClATCTGCTGCACCAAGTGCC, as: TGCATGGCAGTTACACACG; TNF-R2 s: TACCAAGGGTGGCATCTCTC, as: TCCTGGGATTTCTCATCAGG. Additional 5 μl of template cDNA was added to the reaction solution, controls were conducted by the application of 5 μl nuclease-free water. The PCR was performed in a Mastercycler gradient  for 40 min. administering the following cycle protocol: 95°C for 2 min followed by 95°C for 1 min then 56°C for 1 min afterwards 72° for 1 min and for 30 cycles, followed once by 72°C for 5 min and eventually cooling to 4°C. PCR products were evaluated by gel electrophoresis using 2% agarose gel, each trace was loaded with 25 μl of the PCR product and then started with 95 V for 20 min . The evaluation was achieved in a chemiluminescence imager (Fusion – SL 3500 WL) employing the Fusion software .The primers used were as follows: TNF-R1 s: Positive controls were performed by using RNA that was harvested from mice PBMC by small canules out of the heart after the animals had been sacrificed with isoflurane. The blood sample was transferred into a small glass tube containing 3 ml Ficoll and then centrifuged at 1500 g at 10°C for 30 min. Afterwards the cell layer containing the PBMC was pipetted into a fresh glas tube that was filled with phosphate buffered saline (PBS) to 10 ml and then centrifuged at 10°C at 6000 g for 10 min. The supernatant was discarded and the cell pellet was resuspended with 10 ml PBS and further centrifuged as mentioned above, the last step repeated twice. Transcription from RNA into cDNA was performed after transfer of the pellet into an Eppendorf cup and the RT protocol was used as described for tracheal transcription (see above).x g for 15 minutes at 4°C. The aqueous phase containing the RNA was removed and directly used for the further extraction using the binding column. RNA was eluted from the column with 30 μl RNAse-free water and stored at -80°C until further analysis.The excised tracheae were homogenized in TRIzol  using a bead mill and subsequent RNA extraction was performed using a combination of guanidinium thiocyanate-phenol-chloroform separation followed by column-based RNeasy Plus Mini Kit  extraction according to the manufacturer’s instructions. Briefly, each trachea was individually homogenized in 1 ml of TRIzol, then 200 μl chloroform were added, mixed and after 3 minutes incubation at room temperature centrifuged at 12.000 ΔCt (ΔCt: Ct 18S – Ct TNFR1a) of each sample.RNA concentration was photometrically measured and 350 ng of each preparation was reverse transcribed using the QuantiTect Reverse Transcription Kit  according to the manufacturer’s instructions including a genomic DNA removal step and a prolonged RT synthesis step to ensure proper transcription of difficult RNA species. Subsequent Real time PCR analysis was performed on a StepOnePlus™ PCR cycler . Following pre-designed TaqMan assays were used: mouse 18S (assay ID Mm03928990_g1) and mouse TNFR1a (assay ID Mm00441883_g1). PCR reactions were set up using the TaqMan Universal PCR Master Mix . All experiments were run in triplicate. Results are interpreted by calculating the 2For immunohistochemistry mice were sacrificed by an overdose of isoflurane, after confirmation of death the tracheae were harvested as described in previous sections. The organs were transferred into 4% paraformaldehyde (PFA) in phosphate buffer solution pH 7.2 for immersion fixation and stored overnight at room temperature (21°C). After rinsing in phosphate buffer the tissue was cryoprotected using 18% sucrose and then snap frozen in Tissue Tek. Cryosections 10 μm were cut and mounted on glass slides, air dried and subsequently incubated for 1 h using 10% normal horse serum containing 0.5% Tween 20, 0.1% BSA in PBS, pH 7.4. After removal of the blocking solution the sections were incubated with the primary antibody against TNF-R1  overnight at room temperature. Afterwards the antibody solution was rinsed off using phosphate buffer. Secondary antibody was applied for 1 h at room temperature using donkey anti rabbit 1∶2000 linked with Cy3 . Afterwards the secondary antibody was rinsed off in phosphate buffer and then the sections were further fixed in 4% PFA in 0.1 M phosphate buffer for 10 min. and rinsed again in phosphate buffer and finally coverslipped in carbonate buffered glycerol (pH 8.6). The tracheal sections were evaluated using an epifluorescence microscope using a 40x lens  fitted with the appropriate filter sets. Specificity of the primary antibody against TNF-R1 (1:400) was tested by re-absorption with the TNF-R1 peptide  at 8°C for 24 hrs. After this incubation the sample was centrifuged for 1 min at 1000 rpm and then the tracheal sections were incubated at room temperature over night. All other incubation steps were performed identical as described for immunohistochemical staining.Certain pre-defined time points of different experiments were compared using Mann-Whithney test. Statistical significance was assumed when p<0.05.−5 M) at minute 60 and further data sampling over 10 minutes  was used to achieve a steady-state condition before stimulation, which started 60 minutes after confirmed death. Measurements were performed over a period of 60 minutes. Vitality was proofed at the end of each experiment by application of ATP . PTV was arbitrarily set as 100% for minute 0 to normalise for inter-individual variability .Application of 100 ng/ml (p = 0.59) or 250 ng/ml (p = 0.39) TNF-α did not result in an increase of the PTV compared to control. In contrast, higher concentrations with 360 ng/ml (p<0.01) or 1000 ng/ml (p<0.01) TNF-α evoked a significant increase in cilia driven particle transport compared 60 minutes after TNF-α application with the control group .Under control conditions CBF remained stable throughout the entire experimental period. Application of TNF-α (360 ng/ml) led to an increase of the CBF that became significant from minute 48 until minute 60 p<0.01, .TNF-α-induced increase of CBF strongly correlates  with the parallel increase in PTV during the stimulation period and the vitality phase . This suTo further evaluate which receptors are involved in the TNF-α induced mechanism we performed both RT-PCR and qPCR experiments on freshly isolated tracheae and on organs that had previously been stimulated with TNF-α during CBF or PTV experiments. In control experiments using freshly harvested whole tracheae we detected mRNA of the TNF-R1 but not of TNF-R2. TNF-R2 mRNA was neither detected in control nor in TNF-α treated tracheae  mice. In contrast, the same concentration TNF-α could not provoke an increase in CBF in TNF-R1 knock-out mice (–/–) . The con1A) did not alter the response to TNF-α , wereas cyproheptadine  completely inhibited the TNF-α evoked increase in PTV  and a pre-incubations with either a NOS inhibitor (L-NMMA) or a muscarinic receptor blocker (atropine) were performed. Neither atropine (1 μM), nor L-NMMA (100 μM) or L-NAME (100 μM data not shown) could prevent a TNF-α induced increase in the PTV of tracheal epithelium . AdminisIn the present study we provided evidence for an early TNF-α triggered increase in PTV and CBF. The mucociliary clearing system is an important innate lung protective mechanism to disburden the airway surface area from debris and pathogen particles. In addition to the active mechanical clearing, a specific composition of mucus covers as a thin liquid layer the entire epithelium of lower airways and lungs to reduce the surface tension. Additional the surfactant contains ATP, that has been released by pulmonary epithelial cells, which stimulates purinergic receptors P2Y on apical membranes of ciliary bearing epithelial cells Nevertheless, additional different physiological factors like the neuromediators acetylcholine, adrenaline and serotonin are known as constant regulators to maintain the permanent ciliary beating. Beside these classical modulators, under certain circumstances further physiological or patho-physiological conditions can alter ciliary function Correspondent we observed a dose dependent effect of TNF-α on ciliary bearing cells , that beCurrently two TNF-α receptors are known that transpond the effects to its target cells. The main target of TNF-α seems to be the plasma membrane-bound receptor TNF-R1, which is expressed in various cell types. However, the TNF-R2 is assumed to be located only on immune cells, but may also alter organ function, via these activated cells.The mRNA of TNF-R1 was detected in whole trachea using RT-PCR or qPCR. Immunohistochemical staining against TNF-R1 protein revealed the location of this receptor being exclusively located within the epithelium of the trachea under non-stimulated control conditions . In qPCR−/− mice the response to TNF-α was almost completely diminished, confirming a signal transduction pathway via TNF-R1. Additionally the concentration dependent effect to TNF-α application  was extinguished in TNF-R1−/− mice. Activation of TNF-R1 seems to be the main pathway of TNF-α evoked responses in cilia bearing cells which are non immunological cells. Further TNF-R1 plays not only in the tracheal epithelium a pivotal role for cell activation but also in lower airway smooth muscle cells. Despite the coexistence of both TNF-R2 and TNF-R1 in this cell line, the latter plays the key role for activation of nuclear factor-κB transcription factor function or calcium-signal potentiation and contributes to overreaction of bronchial airway muscle during inflammation In experiments using TNF-R12 serotonin receptor antagonist but not WAY-100635 a selective 5-HT1A serotonin receptor antagonist could inhibit the TNF-α induced ciliary activation (2.Ciliary function is conducted continuously by physiological neuromediators released from autonomic nerve fibres or from adjacent cells in a paracrine pattern tivation . This st2B receptor signaling of endothelial cells from pulmonary arteries consequently increasing vascular resistance 2 receptors on parasympathetic cholinergic neurons, which lead to acetylcholine release and airway smooth muscle contraction 2 serotonin receptor blocker. Therefore we assume an early TNF-α induced serotonin dependent action onto the ciliary activity, that is independent from the late increase in CBF as has been reported for isolated and cultivated bovine cilia bearing cells 2 serotonin receptor.Serotonin is a derivative from tryptophan and known as a neurotransmitter released mainly from neuroendocrine cells. It is as well a regulator for various biological processes even outside the central nervous system. It has direct influence on the cardiovascular system, e.g. regulation of the heart rate. Especially in the gastrointestinal tract serotonin plays an important role, here it stimulates intestinal peristalsis and intestinal secretion. In the lung serotonin is involved in the pathogenesis of pulmonary hypertension, during hypoxia the intravascular serotonin concentration increases which then results in a rise in 5-HT2 serotonin receptor on cilia bearing cells. This cascade may function as a protective mechanism during early periods of inflammation or developing pneumonia.In conclusion we assume that TNF-α induces an increase in PTV and CBF by binding to TNF-R1 on tracheal mast cells which then release serotonin that stimulates the 5-HT


Cotton leaf curl Kokhran virus- Dabawali were analyzed in vitro and in vivo by their overexpression in E coli, insect cells and transient expression in the plant system. Purified recombinant V2 and CP proteins were shown to interact with each other using ELISA and surface plasmon resonance. Confocal microscopy of Sf21 cells expressing V2 and CP proteins revealed that V2 localized to the cell periphery and CP to the nucleus. Deletion of the N terminal nuclear localization signal of CP restricted its distribution to the cytoplasm. GFP-V2 and YFP-CP transiently expressed in N.benthamiana plants by agroinfiltration substantiated the localization of V2 to the cell periphery and CP predominantly to the nucleus. Interestingly, upon coinfiltration, CP was found both in the nucleus and in the cytoplasm along with V2. These results suggest that the interaction of V2 and CP may have important implications in the cell to cell movement.The functional attributes of coat protein (CP) and V2 of the monopartite begomovirus,  Plant viruses are challenged by the presence of the “cell wall” and they need to traverse this barrier while moving from an infected cell to an adjacent cell. Hence, they employ the resident communication system, plasmodesmata (PD) which permit direct intercellular exchange of macromolecules Geminiviruses possess a small circular single stranded DNA (ssDNA) as their genome and are the causative agents for decreased yield in many economically important crops. They infect both monocotyledonous and dicotyledonous plants in tropical and subtropical regions Tomato yellow leaf curl virus (TYLCV) and Tomato leaf curl virus (TLCV) have shown that V1 (CP), could replace the function of NSP E. coli expressed proteins and transient expression assays, Rojas et.al., (2001) have proposed a model for TYLCV movement, in which CP mediates the nuclear export of double stranded DNA (dsDNA) for cell to cell and long distance movement within the plant. The export of DNA is further enhanced by CP at the nuclear periphery and the DNA is delivered to C4 at the cell periphery. C4, through its N-terminal myristoylation domain possibly mediates cell-to-cell transport via the PD. Further, V2 was found to be involved in viral spread Tomato leaf curl Java virus-A (ToLCJV-A) Tomato leaf curl New Delhi virus (ToLCNDV) DNA-A and the DNA-β associated with CLCuD have shown that the βC1 is essential for the systemic infection. Further, the heterologous βC1 protein was shown to replace the movement function of the DNA-B of a bipartite begomovirus Cotton leaf curl disease (CLCuD) causing viruses are monopartite begomoviruses having a single genome (DNA-A) and are often found to be associated with DNA-β and DNA 1 satellite molecules Cotton Leaf curl Kokhran virus-Dabawali (CLCuKV-Dab) interact with each other using ELISA and Surface Plasmon Resonance (SPR). Transient expression of these proteins in insect cells and in N. benthamiana showed that CP localized to nucleus whereas V2 localized to cell periphery. Coinfiltration studies in plants revealed that CP is in the cytoplasm along with V2, which suggests that they may interact with each other and play a predominant role in viral cell to cell movement. A model for cell to cell movement of CLCuKV-Dab is proposed based on these results.We have reported earlier the DNA-A sequences of CLCuD causing monopartite begomoviruses and demonstrated the genetic diversity of begomoviruses associated with cotton leaf curl disease in India E.coli as described in E.coli Origami (DE3) strain at a lower temperature (20°C). The soluble protein was purified using Ni-NTA affinity chromatography. However, many recombinant viral MPs expressed in E. coli are reported to form inclusion bodies that hamper their biochemical and biophysical characterization The V2 gene was cloned and overexpressed in ( lane 2 respectively) and western blotting using anti-V2 and anti GST-CP antibodies ( lane 3 respectively).Similarly, the CP was overexpressed as GST fusion protein and purified as described in the methods section. The purified V2 and GST-CP were analyzed by SDS PAGE . This was confirmed experimentally by far-UV CD analysis of purified recombinant V2, which showed the minima at 209 and 222, indicating that the protein is folded and adopts a largely α-helical conformation . The expected molecular mass of the V2 protein was 19000 Da and it was further confirmed by mass spectrometry to be 1915.338 Da . We have recently shown that Sesbania Mosaic Virus MP is also a helical protein which interacts with its CP Tobacco mosaic virus (TMV) MP A secondary structure prediction was carried out on the amino acid sequence of V2 using the PSIPRED Protein Structure Prediction Server . This wa. This wain vitro by an ELISA based assay. Purified V2 or GST-CP was coated onto ELISA plates and incubated with increasing concentrations of interacting protein. The V2 and GST-CP interaction was assessed by either anti V2 or anti GST-CP specific antibodies. As shown in . The interaction was further found to be concentration dependent .In order to understand the mechanism of movement of CLCuKV-Dab and the role of CP and V2 in the process, we performed direct interaction studies shown in . The int. The kinetic constants were determined using BIA evaluation software 3.0. The global fitting analyzes both association and dissociation data for all concentrations simultaneously using a 1∶1 Langmuir binding model. A random distribution of residuals and a χ2 value for this interaction indicated that this model describes well the experimental data. The estimated ak and dk values of the interaction are 1.03×103 (M–1s–1) and 2.67×10–3 (s–1), respectively. The DK value was calculated to be of 2.6×–6 M. Thus our results clearly demonstrate the direct interaction of CLCuKV-Dab V2 with CP. The interaction of proteins encoded by the viral genome with each other and with many other host proteins Maize streak virus, a geminivirus that infects monocots, interacts with its MP Squash leaf curl virus (SqLCV) was shown to interact cooperatively The V2-CP interaction was quantified using SPR. The V2 was immobilized on the Ni-NTA chip and the experiments were performed as described in the methods section. (a–d) showed that the V2 distribution is outside the nucleus. Z-sections were taken at every 0.36 µm and a representative Z section image is shown in (e). The GFP fluorescence was predominantly seen as punctuate bodies in the cytoplasm and at the cell periphery. In contrast, GFP alone was uniformly distributed throughout the cell . To further confirm that the localization of GFP fusion protein is indeed due to V2 moiety, immunocolocalization studies were performed scoring for both GFP and V2 expression individually. V2 was detected by indirect immunofluorescence using V2 specific antibodies and TRITC-conjugated secondary antibodies. The degree of colocalization was measured by plotting the scattergram as described in methods section. The colocalized pixels are located along the diagonal, while those with no colocalization occupy left and bottom portions . For V2, colocalization and overlap coefficients of 0.76 and 1.0 were obtained, respectively. The overlap coefficient of 1 indicates significant colocalization. The corresponding colocalization percentage was calculated to be 60%.The localization of V2 was monitored by observing the GFP localization in the GFP-V2 fusion protein. Sf21 cells were infected with recombinant baculovirus expressing GFP-V2 as described in the methods section. The cells were fixed, stained for nucleus with DAPI and observed under confocal microscope. The GFP-V2 expressing Sf21 cell and the DAPI staining of the same cell a–d showea–d showe(b). Initially, the distribution of CP was uniform in the nucleus (b) but, at later periods of baculoviral infection, CP was found to accumulate in the nucleus as discrete bodies (c–f). Like in V2, immunocolocalization studies were carried out to confirm that the localization of GFP-CP fusion protein is due to CP moiety using CP specific primary and TRITC-conjugated secondary antibodies. Clear merge (c) of the fluorescence due to GFP (a) and TRITC (b) and the scattergram (d) confirm the CP localization in the nucleus . Colocalization parameters were calculated for CP as described earlier. The colocalization and overlap coefficients of 0.98 and 1.0 respectively, were obtained. Like in V2, the overlap coefficient was 1 indicating significant colocalization. The corresponding colocalization percentage was calculated to be 92%.As shown in http://expasy.org/tools/scanprosite/) and PSORT (http://psort.nibb.ac.jp) tools predicted a putative bipartite localization signal, 1MSKRPADIIISTPASKVRRRINF 23 at the N terminus of CP. When the amino acid sequence corresponding to the bipartite nuclear localization signal was deleted, the expression of CP was restricted to the cytoplasm substantiating the importance of the signal sequence for nuclear localization of CP . Interestingly, the NET- program .Bioinformatic analysis of CP by Expasy ScanProsite . A large part of the mature leaf cell is occupied by the vacuole and due to which the cytoplasmic space appears as lining adjacent to the plasma membrane. Nucleus is stained using Propidium Iodide, PI ( b & e). GFP-V2 remained primarily in the cell periphery, although its presence in the cytoplasm could not be ruled out ( d & f). Similar results were obtained with TYLCV Tomato leaf curl Java virus-A (ToLCJV-A), where V2 was shown to localize to nuclear periphery and cell periphery The subcellular localization of V2 in ytoplasm  a–c. A lytoplasm  a–c. A l( d–f). In contrast, YFP alone appears to be distributed throughout the cell ( a–c). The fact that CP localized to the nucleus even in the absence of genomic DNA suggests that during the life cycle of the virus the CP that is translated in the cytoplasm can enter the nucleus and once in the nucleus it could specifically interact with genomic ssDNA. We have shown earlier that ToLCBV-Ban5 CP The confocal image of YFP-CP depicts that CP is limited to the nucleus. This was confirmed by PI staining which was restricted to the nucleus  d–f. In in vitro as demonstrated by ELISA and SPR studies, it was of interest to examine the localization of these two proteins when expressed together. Therefore, agrobacterium harboring the CP and V2 constructs were coinfiltrated into N. benthamiana plants. As shown in the (b & c), and merged (d) confocal images of CP and V2, and the co-staining of nucleus with PI (a) unveiled that CP was found in the nucleus as well as in the cytoplasm along with V2. V2 was also localized to the cell periphery and nuclear periphery ( b & e). Thus the coinfiltration of CLCuKV-Dab V2 and CP transformants revealed that the localization pattern of CP is altered. These results suggest that the interaction of nuclear localized CP with V2 present at the nuclear periphery might render the complex to move out of the nucleus and hence both proteins are seen in the cytoplasm. Similarly, transient expression studies have shown that AbMV MP can redirect movement of NSP from the nucleus As CP and V2 interact with each other Based on the results presented in this paper, a model for cell to cell movement of CLCuKV-Dab is proposed. The CP translated in the cytoplasm is targeted to the nucleus via its NLS, where it binds to progeny ssDNA and exports the ssDNA out of nucleus acting as a nuclear shuttle protein. V2 present at the nuclear periphery might interact with CP-DNA complex and assist in the nuclear export. The complex together with V2 might be transported to the cell periphery via the interaction of other viral encoded protein such as C4 and other host factors. The complex is then transported from one cell to the neighboring cell via PD. Both C4 and V2 in addition to being involved in movement function have been shown to act as suppressors of gene silencing and as pathogenicity factors E.coli strains used in the study and the affinity purification kits were purchased from Novagen–EMD4Biosciences (USA). Baculovirus expression system was purchased from Invitrogen, Life technologies, (USA).The chemicals and the secondary antibodies were procured from Sigma-Aldrich (USA). The primary polyclonal antibodies used in the study were raised in our laboratory.All the GCTAGCTGGGATCCACTGTTAAATGCATGCCATG 3’ and reverse primer for V2; 5’ CGGAATTCTTACTCGAGGGAACATCTGGACTTC 3’. V2 was cloned into the pRSET C at PvuII site in order to obtain a hexahistidine tag at its N-terminus. The CP gene was PCR amplified from pRCP clone GCTAGCTCGAAGCGACCAGC 3' and reverse primer; 5’ GGATCCTTACTCGAGATTTGTCACGGAATCCG 3'. CP gene was then cloned into pGEX-5X-2 vector (Novagen) at SmaI site and there by a GST tag at the N-terminus of CP was fused. Both the clones were confirmed by DNA sequencing.The total gDNA was isolated from the CLCuKV-Dab infected cotton leaf material and used as template DNA to amplify V2 gene by PCR with gene specific primers: forward primer for V2; 5’ E.coli strain Origami™ B (DE3) and BL21 (DE3) pLysS respectively and the proteins were overexpressed as indicated in the manufacturer's instructions (Novagen). Both the overexpressed proteins were soluble when the cultures were grown initially at 30°C and at 20°C post induction.The pRSETC V2 (pR-V2) and pGEX-CP plasmids were transformed into . The GFP fusion was at the N-terminus of the V2 and CP. The GFP-V2 and GFP-CP fusion constructs were generated by inserting a restriction site Stu1 and a linker sequence corresponding to a stretch of serine and glycine residues in the GFP antisense primer. The primers used were as follows. Forward primer: 5’-ATGGATCCCCAGGTACCGGTCGCCACCATAGTG 3’; reverse primer: AGGCCTTCCGGAGGAGGACTTGTACAGCTCGTCC5′- 3’; the restriction site for Stu1 is underlined. All the clones were confirmed by sequencing .GFP, GFP-V2 GFP-CP and GFP-CP del NLS  were cloned in a donor vector, pFastBac1  individually under Polyhedrin promoter . The GFPStu1 site of pBICP35 vector. V2 and CP were fused to the C terminal of GFP and YFP respectively by cloning at Kpn1 site. The primers used were as follows YFP forward primer: 5’GCCAGTAAAGGAGAAGAACTTTTCACT 3’ and YFP reverse primer: 5’GCCTCTAGAGTCTCCGGCTGGTCCGCCTCCTTCT 3’. All the clones were confirmed by sequencing .GFP, YFP (Yellow Fluorescent Protein), GFP-V2 and YFP-CP were cloned into binary vector pBICP35. GFP was amplified from pEAQGFP vector and YFP from pDH5YFP vector and were inserted into Agrobacterium tumefaciens strain EHA105 by electroporation. Transformants were selected on 25 mg/ml Rifampicin and 50 mg/ml Kanamycin plates. Agroinfiltration was done as described elsewhere 2, 10 mM MES, pH 5.9, and 150 µM Acetosyringone) and incubated at room temperature for a minimum of 3 to 5 hours. Bacterial cultures  were infiltrated by gently pressing the end of a 3-ml syringe loaded with appropriate culture to the leaf and exerting gentle pressure to flood the interstitial areas within the leaf.Clones of interest were mobilized into E.coli cells were harvested and re-suspended in buffer  and lysed by sonication. The cell lysate was then spun down at 10,000 rpm for 10 minutes. The supernatant was used for purification of V2 using His-Bind resin (Novagen), according to the manufacturer's instructions.The IPTG induced GST and GST-CP were purified as described in the instruction manual (Novagen). In brief, culture supernatant obtained after sonication was mixed with glutathione sepharose (GSH) beads, pre-equilibrated with extraction buffer  pH 7.4, 1 mM DTT and 0.1% Triton X-100) and incubated for 2 h at 4°C to allow the protein to bind to the beads. The beads were then packed in a column and washed with 50 and 20 bed volumes each of washing buffers I (1x PBS pH 7.4 and 1 mM DTT) and II (50 mM Tris-HCl pH 7.5 containing 1 mM DTT and 0.1% Triton-X 100) respectively. GST-CP was eluted with 50 mM Tris HCl pH 8.0, 200 mM NaCl and 20 mM Glutathione (reduced). The eluted fractions were checked for the presence of the protein on SDS-PAGE and by western blotting using anti-GSTCP antibodies.Spodoptera frugiperda, Sf-21 cell lines (Invitrogen Corp.) were cultured at 27°C in insect cell medium  with 10% serum supplement . Recombinant baculoviruses were transfected using cellfectin and propagated in Sf21 cell lines as indicated in the instruction manual.2O2 was used as substrate. Wells were washed three times with PBS (pH 7.2) and PBST (containing 0.05% (v/v) Triton X-100) between incubations. Interactions were quantified by reading the absorbance at 450 nm using a Spectramass 340PC ELISA reader (Molecular devices). All the experiments were done in triplicates and standard deviation was calculated.Interaction of V2 with CP was tested by ELISA as described previously with minor modifications using either GSTCP or V2 specific polyclonal antibodies 2+ by washing it with 500 µM NiCl2 (20 µl at 20 µl/min) followed by immobilization of purified His-tagged V2 (2 µl/min) up to 350 response units (RU) in eluent buffer. Unbound V2 was removed by passing the buffer at the flow rate of 100 µl/min. The binding reactions were carried out in a continuous flow of running buffer A . The buffer for the sample pump was dispenser buffer . Regeneration was achieved by washing the flow cell with regeneration solution . Various concentrations of GST-CP (100 nM– 800 nM) in the running buffer were injected at a flow rate of 20μl/min and the interaction was monitored for 90 sec. Dissociation was achieved with the running buffer containing 1 M NaCl. The specific changes in the experimental sensorgram were measured by subtracting the values of the reference cell containing no protein. The binding data were analyzed using a 1∶1 Langmuir binding model in BIAcore evaluation software, version 3.0The binding kinetics of V2 and CP was determined by SPR using the BIAcore 2000 optical biosensor  operated at 25°C. The Nitrilotriacetic acid chip (NTA) was first saturated with NiCD measurements were recorded on a Jasco-815 spectropolarimeter  at 25°C. The CD spectrum was monitored from 200 to 250 nm using 0.3 mg/ml protein in a 0.2 cm path length cuvette with a bandwidth of 1 nm and response time of 1 s. The data were expressed as molar ellipticity. The spectra were corrected with the respective buffer control.For mass spectrometric analysis, purified V2 was extensively dialyzed to remove the salts and thereafter subjected to matrix-assisted laser desorption ionization-mass spectrometry analysis using a Ultraflex MALDI TOF/TOF  mass spectrometer equipped with a nitrogen laser (337 nm).Sf21 cells were grown on coverslips and infected with recombinant baculovirus encoding GFP, GFP-V2, GFP-CP and GFP-CP NLS deletion mutant separately. 60 h of post baculoviral infection, the cells were washed with PBS and fixed with 1.5% paraformaldehyde for 30 min at RT and washed again with PBS. For direct fluorescence experiments the cells were then incubated with 1 µg/ml DAPI for 2 min., mounted with fluorescence preserver, and the samples were examined for GFP expression by confocal microscopy (Carl Zeiss LSM 510 META) and the image was processed using LSM 5 image examiner. Indirect immunofluorescence was performed by incubating the cells with respective primary and TRIT C conjugated secondary antibodies. For the colocalization analysis, the optical section of the image was chosen. Images captured at different wavelengths were superimposed, and the intensity of expression for each fluorochrome in the field was then plotted in a scatter gram. The colocalization coefficient and the correlation coefficient were obtained by LSM Colocalization software (Carl Zeiss). The background thresholds are determined by considering the optimal intensities of both red and green according to LSM localization software.Leaf samples were examined under confocal microscopy (Carl Zeiss LSM). For detection of GFP fluorescence, excitation filter 365 nm and emission filter 420 nm were used. For YFP detection excitation filter 520 nm and emission filter 535 nm were used.4) containing 3% paraformaldehyde, 0.05% Triton X−100, 0.25% DMSO, 50 µM PMSF and incubated for 1 h. After the incubation, leaf samples were washed three times each for 5 min in PBS. They were then transferred to freshly prepared PI solution  in PBS and incubated for 1 h in the dark. PI solution was decanted, the leaf samples were washed four times, each for 30 min duration with PBS, dried on Whatmann #1 paper and mounted on a glass slide with anti-fading agent Elvanol to observe the fluorescence Leaf samples were stained with Propidium Iodide (PI) as described earlier Figure S1GFP expression in insect cells. The Sf21 cells expressing GFP alone was fixed and observed under the confocal microscope (a). Graph plotted by quantifying the intensity of the GFP across the cell (red line) as a function of distance (in µm) further confirmed the distribution of GFP throughout the cell (b).(TIF)Click here for additional data file.Figure S2Multiple alignment of the deduced amino acid sequence of CLCuKV-Dab CP with representative begomoviral CP. The crucial amino acid residues predicted by ScanProsite (http://expasy.org/tools/scanprosite/), PSORT (http://psort.nibb.ac.jp) and NetNES (http://www.cbs.dtu.dk/services/NetNES/) for putative nuclear localization signal (A) and nuclear export signals (NES) (B) of the CP sequences are shown in bold letters. Names of the viruses used for the analysis are given as abbreviations and their corresponding NCBI accession numbers are also mentioned.(DOC)Click here for additional data file.


The proinflammatory cytokine tumor necrosis factor (TNF) has been shown to contribute to the pathogenesis and development of pulmonary edema. However, some recent studies have demonstrated surprisingly that TNF can also promote alveolar fluid reabsorption in vivo and in vitro. This protective effect of the cytokine is mediated by the lectin-like domain of the cytokine, which is spatially distinct from the TNF receptor binding sites. The TIP peptide, a synthetic mimic of the lectin-like domain of TNF, can significantly increase alveolar fluid clearance and improve lung compliance in pulmonary edema models. In this review, we will discuss the dual role of TNF in pulmonary edema.—tumor necrosis factor (TNF); acute lung injury (ALI); acute respiratory distress syndrome (ARDS); positive end-expiratory pressure (PEEP);epithelial sodium channel (ENaC);neural precursor cell-expressed developmentally downregulated (gene 4) protein (Nedd4-2);serum and glucocorticoid dependent kinase (Sgk-1);insulin-like growth factor 1 (IGF-1);Protein Kinase C (PKC);reactive oxygen species (ROS);myosin light chain (MLC);pneumolysin (PLY);listeriolysin (LLO);interleukin (IL);bronchoalveolar lavage fluids (BALF);Bacillus Calmette-Guerin (BCG);TNF receptor type 1 (TNFR1); TNF receptor type 2 (TNF-R2); Pulmonary edema, defined as an increase in lung water content, occurs when the rate of fluid movement out of the lung’s microvasculature exceeds the capacity of the lymphatics to clear the fluid from the lung’s interstitium+/K+-ATPase on the basolateral surfaceThe alveolar–capillary barrier is comprised of capillary endothelium and of alveolar epithelium. Pulmonary alveoli, the primary sites of gas exchange with the blood, are composed of a thin alveolar epithelium (0.1–0.2 µm) that covers 99% of the airspace surface area in the lung and contains thin, squamous type I cells and cuboidal type II cellsBesides pulmonary epithelia, endothelial barrier function is also a key component for maintenance of the integrity of the vascular boundaries in the lung, particularly since the gas exchange surface area of the alveolar-capillary membrane is largeAlthough many diseases cause pulmonary edema, they do so by means of one or a combination of the following three processes:An increased capillary pressure in the lungs;An increased permeability or disruption of the alveolarepithelial-endothelial barrier (permeability edema);+/K+-ATPase.A dysregulated expression or function of crucial ion channels in type II alveolar epithelial cells (type II AEC) implicated in lung liquid clearance, such as the apically expressed epithelial sodium channel ENaC and the basolaterally expressed NaAs a consequence, a reduction of lung compliance and an impaired gas exchange may occur, leading to hypoxemia and respiratory acidosis. Patients with pulmonary edema show typical symptoms, including shortness of breath, lung-crackling sounds, pink-stained sputum, cough, anxiety, breathing difficulty, restlessness and wheezing.Pulmonary edema is differentiated into two categories: cardiogenic and non-cardiogenic edemaCardiogenic pulmonary edema is defined as pulmonary edema due to increased capillary hydrostatic pressure, secondary to elevated pulmonary venous pressureNon-cardiogenic pulmonary edema occurs due to changes in permeability of the pulmonary capillary or alveolar epithelial membranes, as a result of either a direct or an indirect pathological process and is therefore also known as permeability pulmonary edemaAs indicated above, in the clinics, hydrostatic edema is mostly a consequence of heart failure, whereas non-cardiogenic pulmonary edema is mostly a consequence of acute lung inflammation. ARDS is a medical emergency, characterized by the sudden failure of the respiratory system. In 2007, the NHLBI estimated that approximately 190,000 Americans are affected by ARDS annuallyApart from strategies to optimize ventilation procedures, currently no standard therapy exists for permeability edema. Moreover, viral and bacterial infections can induce a change in the expression or function of epithelial sodium channel (ENaC). Therefore, the search for substances able to reduce the endothelial hyperpermeability and/or restore the sodium uptake in type II AEC is important.The surface expression of ENaC is mainly regulated via the neural precursor cell-expressed developmentally downregulated (gene 4) protein (Nedd4-2), which leads to ubiquitinylation and subsequent degradation of the sodium channel. Specific kinases, such as the cell volume stress-activated serum and glucocorticoid dependent kinase (Sgk-1) and Akt1 (protein kinase B), both part of the insulin and insulin-like growth factor 1 (IGF-1) signaling pathway, were recently proposed to control the surface expression of ENaC, by means of phosphorylating Nedd4-2 and subsequently reducing its binding to ENaC15Streptococcus pneumoniae, lacks catalase and therefore secretes H2O2 as a virulence factorAlternatively, Sgk1 has been shown to phosphorylate iNOS in type II alveolar epithelial cells, as such reducing NO production, which inhibits Na+ transportWithin endothelial cells, three primary signaling pathways are initiated by the binding of vasoactive factors and leukocyte adhesion: Rho GTPases, reactive oxygen species (ROS), and tyrosine phosphorylation of junctional proteins. These pathways converge to regulate junctional permeability, either by affecting the stability of junctional proteins or by modulating their interactions. The regulation of junctional permeability is mediated by dynamic interactions between the proteins of the adherens junctions, which represent 80% of the tight junctions in endothelial cells and the actin cytoskeleton2+ has been proposed to be the initial pivotal signal preceding endothelial cell contraction, since it can activate key signaling pathways, that mediate cytoskeletal reorganization (through myosin light chain (MLC)-dependent contraction) and disassembly of VE-cadherin at the adherens junctions. Rho (Ras homologous) GTP-binding proteins, which comprise multiple members of the Rho, Rac and Cdc42 subfamilies, are involved in the regulation of a variety of cellular processesA rise in cytosolic Ca2+-dependent PKC isoform, PKC-α, was suggested to play a critical role in initiating endothelial cell contraction and disassembly of VE-cadherin junctions33The CaS. pneumoniae) and its homologous toxin listeriolysin  can be considered as model toxin for G+ infection-associated acute lung injury and permeability edema.Death in severe bacterial pneumonia can occur days after initiation of antibiotic therapy, when tissues are sterile and the pneumonia is clearing and correlates with the presence of bacterial toxins2+ and diacylglycerol levelsStreptococcus pneumoniae with endothelial cells represents a crucial step in its pathogenesis. Intravascular PLY was shown to cause a significant dose-dependent increase in pulmonary vascular resistance and in lung microvascular permeability. By immunohistochemistry, PLY could be detected mainly in endothelial cells of pulmonary arterial vessels, which concomitantly displayed strong vasoconstriction and the toxin moreover increased permeability of HUVEC monolayersThese toxins bind to cholesterol, followed by oligomerization and membrane pore formation, resulting in a rapid increase in intracellular CaIt has been known that inflammation plays an important role in the pathogenesis of pulmonary edema. Once a systemic inflammatory response is triggered, circulating monocytes and alveolar macrophages and neutrophils can secrete cytokines and chemokines, including TNF, interleukin-1β (IL-1β), IL-6, IL-8, IL-12, interferon-γ and IL-8Up to date, many studies have shown that TNF can contribute to the pathogenesis and development of pulmonary edemaBy contrast, new evidence has emerged suggesting that TNF can stimulate lung liquid clearancein vitro and in vivo6266TNF was first identified in 1975 as a cytokine with anti-tumor effects TNF is a potent proinflammatory cytokine produced by many cell types, including monocytes, macrophages, lymphocytes, endothelial cells and fibroblasts60Two specific receptors interact with TNF on the cell surface: TNF-R1  and TNF-R2 77N, N’-diacetylchitobiose, a specific binding oligosaccharide of this lectin-like domain, can block this trypanolytic effect whereas it leaves the cytotoxic activity of TNF in cancer cells lines unaffectedAlthough it is generally assumed that cytokines solely exert their activities upon activating their respective receptors, this does not seem to be completely true in the case of TNF. Indeed, in contrast to Lymphotoxin-alpha, which has a highly homologous 3-D structure as TNF and which is able to bind to both TNF receptors, TNF was shown to exert a lytic activity in purified long slender bloodstream forms of African trypanosomes by means of a lectin-like interaction with oligosaccharide residues on the surface of the parasites79Moreover, a circular seventeen amino acid peptide, named the TIP peptide, (a derivative of the tip region of TNF), mimics the functional structure of this lectin-like domain and has similar lytic effect on African trypanosomes+ in the lung. Na+ ions in the alveolar lining fluid were shown to passively diffuse into fetal distal lung epithelial and alveolar epithelial type II cells through nonselective cationic channels and Na+ selective, amiloride-sensitive channels, the most important of which is the epithelial sodium channel ENaC, located in the apical membrane, consisting of at least 4 subunits, i.e. alpha, beta, gamma and delta+ influx is maintained by the basolaterally expressed, ouabain-sensitive Na+/ K+-ATPase that transports Na+ into the interstitial space+ channels represent the rate-limiting step in Na+ absorption85Type II alveolar epithelial cells and small airway epithelial cells represent the primary sites for reabsorption of Na+/K+-ATPase mRNA expression. Moreover, in the same study, TNF decreased amiloride-sensitive sodium uptake in a dose-dependent mannerDuring inflammatory processes in the lungs, proinflammatory substances such as TNF are produced locally. Indeed, it has been demonstrated that there is an elevation of the TNF level in the BALF from patients with ALI/ARDSOne prominent feature of acute lung injury syndromes is the disruption of the vascular barrier, which can result in permeability pulmonary edema formation and subsequent respiratory dysfunctionin vivo as well as in vitroin vivoTNF can trigger endothelial cell activation and barrier dysfunction, which are both implicated in the pathogenesis of pulmonary edemaMore recent studies have demonstrated that TNF can cause microtubule rearrangement in monolayers of human pulmonary artery endothelial cells+ channel and barrier dysfunction, it was also reported that TNF can induce pulmonary edema by means of augmenting reactive oxygen speciesBesides the direct deleterious effects on NaIntriguingly, while many studies have already shown that TNF contributes to the formation of pulmonary edema, other researchers have found that this pro-inflammatory cytokine can actually increase the clearance of alveolar liquid, mainly in infection models.Pseudomonas aeruginosa increased alveolar liquid clearance by 43% over 1 h, as compared to control rats. Moreover, when an anti-TNF neutralizing antibody was instilled into the lungs 5 min before the bacteria, alveolar liquid clearance was significantly decreasedIn 1997, Rezaiguia et al revealed that the instillation of TNF in rats infected with 104, Glu106, and Glu109 with alanines in the lectin-like domain of the TNF trimer. As such, TNF lost its lectin-like affinity, essentially retained its TNF receptor 1-mediated activities, but displayed a 50-fold- reduced TNF receptor 2-mediated bioactivity in vitroIn 1997, a triple-mutant murine TNF was generated upon replacement of the crucial residues ThrIn isolated endo/exotoxin-treated perfused rabbit lungs, Vadasz et al., recently demonstrated that the TNF-derived TIP peptide significantly lowered vascular permeability, as assessed by capillary filtration coefficient and fluorescein isothiocyanatelabeled albumin flux across the alveolocapillary barrierListeria monocytogenes can cause severe lung complications, which can result in permeability edema, characterized by an extensive capillary endothelial hyperpermeability, which requires harsh therapeutic measures and often has a fatal outcomeListeria monocytogenes, induces a dose-dependent hyperpermeability in monolayers of human lung microvascular endothelial cells in vitro. In our recent study, The TNF-derived TIP peptide, which mimics the lectinlike domain of the cytokine, was shown to blunt LLO-induced hyperpermeability in vitro, upon inhibiting LLO-induced PKC-α activation, ROS generation and MLC phosphorylation and upon restoring the RhoA/Rac 1 balance. These results indicate that the lectin-like domain of TNF has a potential therapeutic value in protecting from LLO-induced pulmonary endothelial hyperpermeabilityInfections with the G+ bacterium in vivo, whereas the inactive mutant TIP peptide didn’t have this effect. Using Ussing chamber experiments of primary type II rat pneumocytes, we concluded that the primary site of action of the peptide is on the apical side of these cells, since only apical, but not basolateral stimulation of the monolayers with the TIP peptide resulted in increased amiloride-sensitive transepithelial currentsIn a recent study, we could demonstrate that the TIP peptide, which mimics the lectin-like domain of TNF, is able to blunt ROS production in pulmonary artery endothelial cells under hypoxia and reoxygenation, and reduces ROS content in the transplanted rat lungs + and chloride channels, but also by means of the basolaterally expressed Na+/K+-ATPase in normal and injured lungs+/K+-ATPase activity 1.6-fold by promoting its exocytosis to the alveolar epithelial cell surface and increased amiloride-sensitive sodium uptake, resulting in a 2.2-fold increase in active Na+ transport, and hence improved clearance of excess fluid from the alveolar space+/K+-ATPase in these effects, upon a previous stimulation of ENaC.Active Na+ transport across the alveolar epithelium is regulated via not only apically expressed NaPulmonary edema is still one of the most common medical emergencies, with no standard therapy available for the permeability-induced form of the pathology. As an important cytokine being involved in this pathogenesis, TNF is an example of a “moonlighting protein”, with differential activities mediated by its receptor-binding versus its lectin-like domains, which opens the possibility to design and develop more sophisticated therapeutic regimens to overcome the deleterious fluid accumulation in some major lung pathologiesIn our opinion, the studies mentioned above can generate important advances in our understanding of the complexity of the TNF effects in pulmonary edema. However, in the future, more research is needed in order to reveal the underlying mechanisms of TNF’s protective versus deleterious effects. This research can potentially make the lectin-like domain of TNF an attractive therapeutic option in patients with pulmonary permeability edema.


A microgrid is composed of distributed power generation systems (DGs), distributed energy storage devices (DSs), and loads. To maintain a specific frequency in the islanded mode as an important requirement, the control of DGs’ output and charge action of DSs are used in supply surplus conditions and load-shedding and discharge action of DSs are used in supply shortage conditions. Recently, multiagent systems for autonomous microgrid operation have been studied. Especially, load-shedding, which is intentional reduction of electricity use, is a critical problem in islanded microgrid operation based on the multiagent system. Therefore, effective schemes for load-shedding are required. Meanwhile, the bankruptcy problem deals with dividing short resources among multiple agents. In order to solve the bankruptcy problem, division rules, such as the constrained equal awards rule (CEA), the constrained equal losses rule (CEL), and the random arrival rule (RA), have been used. In this paper, we approach load-shedding as a bankruptcy problem. We compare load-shedding results by above-mentioned rules in islanded microgrid operation based on wireless sensor network (WSN) as the communication link for an agent’s interactions. Especially, the main energy sources of the microgrid are composed of solar and wind power facilities, micro-turbines and fuel cell-based combined heat and power (CHP) systems, and battery devices. The use of these energy sources have been encouraged in many counties because as clean energy sources they have a close relationship with Climate Change. Research and demonstration projects for microgrids have been pursed ,2.Since the microgrid is composed of small-scale power systems and devices, autonomic and autonomous microgrids based on the multiagent system have been studied for their economical and efficient operation and control –6. The mOne of most important requirements of a microgrid operation is to maintain a specific frequency such as 50 Hz or 60 Hz. In the islanded operation mode, which is an operation mode when the microgrid is electrically isolated from power grids, regulation of DGs, charge and discharge actions of DSs, and load-shedding are generally used. Especially, load-shedding, which intentionally reduces electricity use when power supply is short, is a critical problem because the load-shedding is inconvenient to consumers. Therefore, efficient schemes for handling load-shedding are required.Meanwhile, the bankruptcy problem deals with how to divide an insufficient estate among all claimants and is used in finance, economics, and engineering areas as a subarea of game theory as well as mathematics. To solve bankruptcy problems, various methods such as division rules and cooperative game theories have been studied –10.In this paper, we approach load-shedding in a multiagent-based islanded microgrid as a bankruptcy problem because the purpose of load-shedding is to reduce loads intentionally and divide the reduced amount of electricity among consumers (loads). We consider popular division rules such as the constrained equal awards rule (CEA), the constrained equal losses rule (CEL), and the random arrival rule (RA). Especially, we deal with operation planning of the islanded microgrid, unlike previous works dealing with control of the islanded microgrid .The remainder of this paper is structured as follows. Section 2 describes multiagent-based islanded microgrid operation. Section 3 explains load-shedding schemes based on the bankruptcy problem and division rules. Section 4 shows design and construction of an experimental multiagent-based islanded microgrid. Following this, we test the feasibility of suggested load-shedding schemes based on the divide rules and discuss the feature of the applications in Section 5. Finally, Section 6 summarizes our study results and future plans.2.2.1.The microgrid is composed of distributed generation systems (DSs), such as solar and wind power facilities, micro-turbines and fuel cell-based CHP systems, and distributed energy storage devices (DSs), such as battery systems and flywheel energy storage devices, and loads.The microgrid can be operated in the the grid-connected mode and the islanded mode. A Microgrid Operation & Control Center (MGOCC) manages microgrid operation ,6. One oIn the case of one-owner operation or fully authorized operation from all participants in the microgrid, load-shedding is performed according to a priority order based on load importance. However, in the case of an energy market environment having multiple independent participants, load-shedding should be performed by some reasonable scheme excluding critical loads agreed by all participants. In this paper, we assume that microgrids are operated in two steps: planning and implementation, as shown in 2.2.An agent is able to sense changes of the environment and to act autonomously according to the intension of design purpose. Multiagent system is a system or society composed of multiple agents. In the multiagent system, agents communicate with an agent communication language (ACL) and share knowledge for cooperation. 2.3.MGOCCAg is the MGOCC agent, DGAG is a set of DG agents (DGAg), DSAG is a set of storage device agents (DSAg), and LAG is a set of load agents (LAg). The MOGCC agent manages the entire operation of the microgrid. Each agent controls and operates his/her DG, DS or load.A multiagent-based microgrid is a microgrid operated by autonomous agents. A multiagent system for microgrid operation is defined as follows ,6:(1)Ag3.3.1.lc, Pa), where Pa is available power and lc =  is the vector of load claims. Pa and lc are described as:The bankruptcy problem deals with how to divide an insufficient estate among all claimants. In this paper, we approach the load-shedding problem as a bankruptcy problem. The load-shedding problem can be defined as a pair (la*) of each load is defined as:The vector of allocated power (ls*) is defined as:Finally, the vector of load-shedding of each load  When there are more two digits below the decimal point, the exceed digits are truncated and added to a consumer requiring the largest load amount.Finally, load-shedding is decided by the above-mentioned 4.4.1.MGOCCAg and DSAG, LAGInformation Exchange Protocol for interactions between MGOCCAg and DGAG.Modified Contract Net Protocol (CNP) for interactions between A multiagent system, which is defined by above-mentioned We employ a modified version of the Knowledge Query and Manipulation Language (KQML) as the agent communication language (ACL) and use the following message format for cooperation among agents :(<perfoT) for effective task management as follows:We use a state function , the Interactive Design Environment for Agent Designing Framework (IDEA), and the Java, where the DASH is a multiagent platform, the IDEA is a GUI-based interactive environment for the DASH platform, and the Java is used for user-defined functions ,13–15.5.5.1.- Agents: the MGOCC agent, three DG agents, a DS agent, ten Load agents.- Capacity of DS: 10 [kWh]- Initial charged amount of DS: 5 [kWh]- L1: 30 [kWh]- L2: 50 [kWh]- L3: 60 [kWh]- L4: 100 [kWh]- L5: 110 [kWh]- L6: 120 [kWh]- L7: 150 [kWh]- L8: 200 [kWh]- L9: 230 [kWh]- L10: 250 [kWh]- Total load amount: 1,300 [kWh]- Supply shortage by three DGs: from 1,100 [kWh] to 100 [kWh] with decrease by 200 [kWh] in 6 casesIn this experiment, we compare the feature of load-shedding based on the CEA, the CEL, and the RA. For this, the following multiagent-based islanded microgrid is constructed.In the CEL application, as shown in In the RA application, as shown in 5.2.The CEA has a merit on operational convenience because load-shedding is only required to some loads having a lot of power claim when we consider that exact regulation of load by a control reference plan is not easy.The CEL and the RA have a merit on fairness of load-shedding because of load-shedding is required to all loads evenly and proportionally, respectively.In this experiment, we can see different patterns of load-shedding by the CEA, the CEL, and the RA. To select best rule for load-shedding is not easy because the CEA has the merit of operational convenience and the CEL and the RA have the merits of fairness of load-shedding as mentioned above. The choice of a load-shedding scheme for an islanded microgrid should depend on an agreement by all participants considering the above-mentioned features according to the three load-shedding schemes in principle. Realistically, if we consider that the precise regulation of load amount is not easy, the CEA can be recommended, even though the CEA is less fair than either the CEL and the RA schemes.6.Load-shedding is approached as a bankruptcy problem.In order to decide load-shedding, the CEA, the CEL, and the RA are considered as candidate division rules.Load-shedding schemes based on the above-mentioned three division rules are proposed.The load-shedding schemes are tested in the multiagent-based microgrid based on wireless sensor network (WSN) as a communication link for agents’ interactions of the islanded microgrid.The feature of load-shedding schemes is illustrated and discussed. From the experiment, the CEA has merit in operational convenience and the CEL and the RA have the merit of fairness of load-shedding.This paper presents load-shedding schemes based on the bankruptcy problem and division rules in islanded microgrid operation. The contributions of this paper are stated as follows:In our work, we considered three well-known division rules for load-shedding applications. As a further work, we are considering game theory approaches for load-shedding in islanded microgrid operation.


POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1), in lemma/palea increased grain length and weight in transgenic rice. PGL1 is an atypical non-DNA-binding bHLH and assumed to function as an inhibitor of a typical DNA-binding bHLH through heterodimerization. We identified the interaction partner of PGL1 and named it ANTAGONIST OF PGL1 (APG). PGL1 and APG interacted in vivo and localized in the nucleus. As expected, silencing of APG produced the same phenotype as overexpression of PGL1, suggesting antagonistic roles for the two genes. Transcription of two known grain-length-related genes, GS3 and SRS3, was largely unaffected in the PGL1-overexpressing and APG-silenced plants. Observation of the inner epidermal cells of lemma revealed that are caused by increased cell length. PGL1-APG represents a new grain length and weight-controlling pathway in which APG is a negative regulator whose function is inhibited by PGL1.Grain size is a major yield component in rice, and partly controlled by the sizes of the lemma and palea. Molecular mechanisms controlling the sizes of these organs largely remain unknown. In this study, we show that an antagonistic pair of basic helix-loop-helix (bHLH) proteins is involved in determining rice grain length by controlling cell length in the lemma/palea. Overexpression of an atypical bHLH, named  GS3, encodes a protein with four different domains including the plant-specific organ size (OSR) domain. This domain is necessary and sufficient to function as a negative regulator of rice grain length Small and round seed 3 (SRS3) encodes a kinesin13 protein. Constitutive expression of SRS3 rescued and complemented the short grain phenotype of an srs3 mutant, suggesting a positive role for SRS3 in rice grain length qSW5, was shown to control cell numbers of the lemma and palea, and identified to be functional nucleotide polymorphisms (FNP) for a putative nuclear protein GW2, encodes a RING-type ubiquitin E3 ligase and controls cell numbers of the lemma and palea Grain size is an important yield component, and thought to be partly controlled by sizes of glumes; the lemma and palea Phytochrome Interaction Factors 3, can bind to the G-box motif (CACGTG) in the promoter region of target genes and is involved in light signaling Basic helix-loop-helix (bHLH) proteins are the second largest class of plant transcription factors Increased Leaf Inclination (Ili) and ILI1 binding bHLH (OsIBH1), controls cell length in the lamina joint and leaf bending in rice. Likewise, an Ili homolog of Arabidopsis, Paclobutrazol Resistance1 (PRE1), and AtIBH1 regulate cell elongation Activation-tagged bri1 suppressor 1-Dominant (atbs1-D) and ATBS1 interaction factors (AIFs) regulate leaf cell size through a brassinosteroid signaling pathway Recent studies have revealed antagonistic roles of HLH/bHLH proteins in various plant organ sizes. For instance, an antagonistic pair of bHLH proteins, POSITIVE REGULATOR OF GRAIN LENGTH 1 (PGL1) in rice lemma/palea and found increases in the length and weight of the grain. We identified a typical bHLH protein named ANTAGONIST OF PGL1 (APG) as an interaction partner of PGL1 and the complex of them is localized in the nucleus. Silencing of APG by RNAi resulted in the same grain phenotype overexpression of PGL1. Our results suggest that PGL1 and APG antagonistically regulate rice grain length and weight by controlling cell elongation in lemma/palea through heterodimerization.It is predicted that there are 167 bHLH genes in Arabidopsis, 177 in rice, 99 in poplar, 190 in moss and 13 from five algae species Style2.1, a gene that controls style cell length in tomato Style2.1, Ili1, showed that it is involved in cell length in determining lamina joints of rice Ili1 in the rice genome, and called one of them, Os03g0171300, Ili6POSITIVE REGULATOR OF GRAIN LENGTH 1 , and analyzed its expression by RT-PCR. The result showed that it is expressed in the pistil, lemma/palea, young panicle, and predominantly in root but not leaf  is an atypical bHLH gene that is not included in the predicted 177 bHLH genes of rice not leaf . To anall length .0 PGL1:OX lines are larger than those of wild types. We examined if the expression level of PGL1in lemma/palea correlates with the size of grains. Four representative T0 PGL1:OX lines from both backgrounds were selected for analysis. As expected, expression of PGL1 in lemma/palea was increased and correlated to grain size in the T0 transgenics (0) accumulating 170-fold more of the PGL1 transcript in lemma/palea showed a 43% increase in 1000-grain weight, while line Ni1 with a 13-fold increase in PGL1 showed 3% increase in grain weight (0) plant with the largest grain was self-pollinated and ten segregated plants were randomly selected for further analysis. The Ni9 (T1) plants which were T-DNA-positive remained long whereas the T-DNA negative plant showed a grain length comparable to the wild type  using the HLH region of PGL1 as a query revealed that PGL1 is highly homologous to Arabidopsis KIDARI  which has 77% and 75% identity with PGL1 for the whole amino acid sequence and HLH domain, respectively. KDR was reported to interact with HFR1  http://rapdb.dna.affrc.go.jp/) and found several bHLH proteins as candidates for interaction partners of PGL1. Proteins with E-values of <4e−12 were selected for analysis; Os12g0610200, Os01g0286100, Os05g0139100, and Os04g0618600. Except for Os04g0618600, all candidates contained amino acids conserved in the basic domain required for binding to DNA ANTAGONIST OF PGL1 , and deposited the cDNA sequence derived from lemma/palea in DDBJ/Genbank/EMBL under accession number AB667900.PGL1 is an atypical bHLH and lacks the basic domain required for DNA-binding, suggesting that it would heterodimerize with other DNA-binding bHLH proteins and abolish their functions as in the case of human Inhibitor of DNA binding (Id) proteins S-transferase (GST) or thioredoxin (Trx) to express soluble recombinant proteins in E. coli for the in vitro pull-down assay. We found that MBP-APG co-precipitated with GST-PGL1, suggesting the interaction of these proteins in vitro , glutathione in vitro . Co-precin vitro .in vivo, we performed a bimolecular fluorescent complementation (BiFC) assay. Agrobacterium harboring constructs for expression of the N-terminal half of enhanced yellow fluorescent protein (EYFP) fused to PGL1 (YN-PGL1) and C-terminal half of EYFP fused to APG (YC-APG) were co-infiltrated into Nicotiana benthamiana leaves. The YFP fluorescence was observed in the nucleus  or N-EYFP (YN) and YC-APG  gene and agro-infiltrated into N. benthamiana leaf epidermal cells. Fluorescent signal was observed in the nucleus for GFP-APG and both the cytoplasm and nucleus for GFP-PGL1  and PGL1:OX (Ni9 and Ni23) to compare their lemma inner epidermal cells to those of wild type. Confocal microscopic observations revealed that the longer grain is caused by enhanced cell length . Transgepponbare  and Kitapponbare . Observapponbare .Grain filling rate is another major factor determining grain weight. We obtained three sterile PGL1:OX lines  which are unable to fill the grain. However, these lines had large lemma/palea, suggesting that the elongated grain in PGL1:OX is not related to grain filling rates . This isSRS3GS3SRS3 and GS3 to be largely unaffected in the selected APG RNAi lines (Ri-1 and Ri-12) and PGL1:OX lines (Ni9 and Ni37)  .BRASSINOSTEROID UPREGULATED1 (BU1), an atypical bHLH gene, increased leaf inclination PGL1 in lemma/palea  showed obvious leaf bending . This isRice grain size is a major yield component, and controlled by at least four factors; grain width, length, thickness and the ability to fill the grain GS3 and a kinesin 13 protein gene SRS3srs3 mutant showed significantly decreased cell length in lemma but only slightly affected cell width (not significant at p<0.01) compared to the wild type GS3 mechanism, the gene negatively regulates grain length and has a small positive effect on width GS3 and SRS3 in the PGL1:OX and APG RNAi lines was largely unaffected, suggesting that the genetic pathway through which PGL1 and APG regulate grain length is independent of these genes, although the possibility of PGL1 and APG being downstream of these genes can not be excluded. We also showed that the PGL1 and APG proteins interact in vitro and in vivo, and the complex of them is localized at the nucleus. bHLH proteins are reported to function either as transcription activators PGL1 and APG represents a novel genetic pathway controlling rice grain size by regulating cell length in the lemma and palea.To date, two grain length-controlling genes have been reported, an OSR domain-containing protein gene PGL1 was previously classified in the Ili gene family consisting of seven rice genes, and named Ili6Ili homologs of Arabidopsis were named the PRE gene family Ili and PRE gene families were grouped to subfamily 16 of the 32 plant bHLH subfamilies Ili1 and BU1 control cell elongation in rice laminar joints ATBS1  and PRE1 regulate cell expansion in Arabidopsis leaves through brassinosteroid signaling Style2.1 controls cell elongation in the developing tomato style PGL1 (Os03g0171300) in Arabidopsis resulted in increased rosette leaf size http://ricefox.psc.riken.jp/, rice FOX Arabidopsis line R90261). Clarification of the protein-protein interaction networks of subfamily 16 bHLH proteins and downstream genes regulated by these proteins would show how the members of subfamily 16 control cell elongation in different organs and species through interaction with different classes of proteins.Based on sequence homology, PGL1 didn't affect pistil length even though a high level of transcript was accumulated in the organ , a DELLA protein, to regulate cell elongation in Arabidopsis APG was grouped to subfamily 24 together with PIFs Zm-CYP-1 increased grain yield by enhancing grain filling in rice OsBRI1/D61, or the BR signaling gene RGA1/D1 resulted in small grains BU1 also increased grain size, although most of the OX lines were sterile BR-related genes are reported to be important in the regulation of rice grain size Oryza sativa L.) cv Nipponbare and Kita-ake were used for transformation as described previously Rice  extracted from lemma/palea at the pre-anthesis stage  was usedPGL1 (Os03g0171300), and inserted into a binary vector pPZP2H-Lac to create chitinase::PGL1 APG (Os05g0139100), 262 bp, was amplified from Nipponbare lemma/palea cDNA and subcloned into pHANNIBAL at EcoRI/KpnI and ClaI/BamHI BamHI and subcloned into the binary vector pBI101H-Ub to create Ubi::APG RNAi Agrobacterium tumefaciens strains EHA101 and EHA105, respectively.The 1685 bp upstream region of a rice chitinase gene (AB012855), hereafter refered as the chitinase promoter PGL1 fragment was subcloned into pGEX-4T.1  to generate GST-PGL1  The open reading frame of each gene was amplified from Nipponbare lemma/palea cDNA by gene specific primers . The PGLAll PCR products were sequenced before further cloning by BigDye terminator ver. 3.1 (Applied Biosystems).Escherichia coli strain BL21(DE3 pLysS) (Novagen). MBP and MBP-APG proteins were purified using amylose resin beads (New England BioLabs). GFP-APG was transiently expressed in N. benthiamiana leaves under the control of the 35S promoter by Agro-infiltration. The expressed GFP-APG protein was extracted by binding buffer . Binding was carried out as described previously The protein expression constructs were introduced into PGL1 and APG were amplified from Nipponbare lemma/palea cDNA and inserted into binary pBiFC vectors . The same cDNA fragments were cloned into the binary vector pBINPLUS A. tumefaciens COR308 N. benthamiana at an OD600 ratio of 0.7∶0.7∶1.0 and 1.0∶1.0, respectively, for YFP/GFP localization. The plants were kept for 48 hours after infiltration under continuous light at 26°C prior to observation. Yellow fluorescence protein (YFP) and the GFP signal were visualized by confocal microscope  or Leica DMR fluorescent microscope.http://rsb.info.nih.gov/ij/) for cell length and width.Ten pre-anthesis florets were randomly selected. The inner epidermal layers were stained using 1 M Tris-HCl pH 9.0 with 0.1 mg/L of calcofluor  and images taken under the Leica confocal microscope . A totalFigure S1Homologs of PGL1 and overexpression of PGL1 in rice pistil. a) Amino acid alignment of PGL1 (Os03g0171300) homologs from rice BU1 (Os06g0226500), ILI1 (Os04g0641700); Arabidopsis KDR (AT1G26945), ATBS1 or TMO7 (AT1G74500), PRE1 (AT5G39860) and tomato Style2.1 (NM001247361) using GENETYX-MAC software. The dotted line indicates the basic region, solid lines indicate helix and curve line indicates a loop region. b) RT-PCR analysis of PGL1 in Nipponbare wild type. G, genomic DNA; R, root; L,leaf; P, pistil; L/P, lemma/palea and YP, young panicle. c) Pistil phenotypes of T0 transgenic and wild type plants (bar = 1 mm). d) Comparison of pistil lengths of T0 transgenic and wild type plants, . ns denotes no significant differences between wild type and transgenic plants as determined by Student's t tests. e) RT-PCR analysis of PGL1 in pistils of T0 transgenic compared with wild type plant. f) Pistil and grain phenotypes of Nipponbare WT and the sterile line Ni10 (bar = 2 mm). g) Quantitative PCR analysis of PGL1 in lemma/palea normalized by OsActin. Error bar indicates ±sd over three biological replicates.(PPT)Click here for additional data file.Figure S2Overexpression of PGL1 increased grain size in Kita-ake. a) Quantitative PCR analysis of PGL1 in lemma/palea of Kita-ake T0 plants compared with wild type plants (WT = 1) normalized by OsActin. Error bar indicates ±sd over three biological repeats. b) Comparison of grain length and width between Kita-ake transgenic T0 and T4 (Ki3(T4)) plants and the wild type . Asterisks denote a significant difference from the wild type as determined by Student's t tests . c) Lemma inner epidermal cells of Kita-ake wild type (KiWT) and transgenic plants overexpressing PGL1 Ki3 transgenic T4 (Ki3(T4) (bar = 100 µm). d,e) Distribution of the number of cells by cell length (d), and cell width (e); KiWT, Kita-ake wild type cyan color; transgenic T0 overexpressing PGL1 line Ki3, red; Ki7 green; T4 line Ki3(T4), purple. Triangles represent average values.(PPT)Click here for additional data file.Figure S3Inner epidermal cells observed by confocal microscopy. A) Palea inner epidermal cells of NiWT and transgenic PGL1:OX (Ni9) and APG RNAi (Ri-12) (bar = 150 µm). B) Distribution of the number of cells at various cell lengths. C) Distribution of the number of cells at various cell widths; NiWT, Nippobare wild type cyan; T0 transgenic PGL1:OX line Ni9, red; Ri-12, green. Triangles represent average values of the respective lines.(PPT)Click here for additional data file.Figure S41.T-DNA segregation and phenotype of Ni9 T a) Grain phenotype of T-DNA positive (+) and negative (−) plants compared to wild type and Ni9 T0 plants. b) RT-PCR analysis of PGL1 in lemma/palea of Ni9 T1 segregated plants compared to wild type and Ni9 T0 plants.(PPT)Click here for additional data file.Figure S5In vitro interaction between PGL1 and other candidates revealed by pull-down assay. a) In vitro interaction between GST-PGL1 and MBP-Os01g (Os01g0286100) detected by pull-down assay. Amylose resin–bound MBP-Os01g or MBP was incubated with an equal amount GST-PGL1. Proteins co-precipitated with amylose resin were detected by immunoblotting using anti-GST antibody. b) In vitro interaction between GST-Os04g (Os04g0618600) and MBP-PGL1. Amylose resin–bound MBP-PGL1 or MBP was incubated with an equal amount GST-04g. Proteins co-precipitated with amylose resin were detected by immunoblotting using anti-GST antibody. c) In vitro interaction between GST-PGL1 and Trx-Os012g (Os012g0610200). Glutathione beads bound to GST-PGL1 or GST-GFP were incubated with equal amounts Trx-Os12g. Proteins co-precipitated with glutathione beads were detected by immunoblotting using anti-His antibody.(PPT)Click here for additional data file.Figure S6Genomic and amino acid structure of APG and RT-PCR analysis of APG in different tissues of Nipponbare wild type. a) Genomic structure of the APG gene, the underline indicates the fragment used for the RNAi construct and bHLH protein domain. b) bHLH domain based alignment of APG1, PIF3 and PIF4. The dotted line indicates the basic region, solid lines indicate helix regions and curve line indicates a loop region. Asterisks (*) indicate conserved His 9, Glu 13 and Arg 16 required for binding G-box (CACGTG). c) Alignment of the N-terminal (1 to 50) amino acid sequence of APG, PIF3 and PIF4. The line indicates the APB (active phytochrome binding) motif which is required for PIF3 and PIF4 to bind to phytochrome. d) RT-PCR analysis of APG (upper), and control OsActin (lower). L, leaf; R, root; P, pistil; YP, young panicle (∼10 cm); L/P, lemma/palea; and g, genomic DNA.(PPT)Click here for additional data file.Figure S7Effect of Brassinolide on PGL1 and APG expression. Expression of PGL1, APG and BU1 of two weeks old shoot (without root) from Nipponbare treated with 10 µm of BL or mock, Error bar indicates ±sd over three independent experiments.(PPT)Click here for additional data file.Table S1List of primers used in this study.(DOC)Click here for additional data file.Methods S1Supporting Information materials and methods.(DOC)Click here for additional data file.