It is important to know which of the three α2-AR subtypes  is responsible for the upregulation of TNF-α production. The aim of this study was to determine the contribution of α2A-AR in this process.Norepinephrine (NE) modulates the responsiveness of macrophages to proinflammatory stimuli through the activation of adrenergic receptors (ARs). Being part of the stress response, early increases of NE in sepsis sustain adverse systemic inflammatory responses. The intestine is an important source of NE release in the early stage of cecal ligation and puncture (CLP)-induced sepsis in rats, which then stimulates TNF-α production in Kupffer cells (KCs) through the activation of the α2A-AR was determined. In additional experiments, cultured KCs were incubated with NE with or without BRL-44408 maleate, a specific α2A-AR antagonist, and intraportal infusion of NE for 2 h with or without BRL-44408 maleate was carried out in normal animals. Finally, the impact of α2A-AR activation by NE was investigated under inflammatory conditions . Gene expression of the α2A-AR subtype was significantly upregulated after CLP. NE increased the release of TNF-α in cultured KCs, which was specifically inhibited by the α2A-AR antagonist BRL-44408. Equally, intraportal NE infusion increased TNF-α gene expression in KCs and plasma TNF-α which was also abrogated by co-administration of BRL-44408. NE also potentiated LPS-induced TNF-α release via the α2A-AR in vitro and in vivo. This potentiation of TNF-α release by NE was mediated through the α2A-AR coupled Gαi protein and the activation of the p38 MAP kinase. Treatment of septic animals with BRL-44408 suppressed TNF-α, prevented multiple organ injury and significantly improved survival from 45% to 75%.Adult male rats underwent CLP and KCs were isolated 2 h later. Gene expression of α2-AR stimulation observed in sepsis is primarily due to an increase in α2A-AR expression in KCs. This appears to be in part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the α2A-AR antagonist as a new therapy for sepsis.Our novel finding is that hyperresponsiveness to α In our previous studies, we reported that gut-derived NE upregulates TNF-α production in KCs through the α2-adrenergic pathway 2-ARs are G-protein coupled receptors that mediate the central and peripheral actions of the primary sympathetic neurotransmitter, NE and the adrenal hormone epinephrine through the intracellular suppression of cAMP 2-AR subtypes  is responsible for the upregulation of TNF-α production. The aim of this study was therefore to determine the contribution of α2A-AR in NE-mediated proinflammatory effects in sepsis.Sepsis and septic shock are complications and considered to be major causes of morbidity and mortality in patients with severe trauma, burns, or blood loss ad libitum. The animals were anesthetized with isoflurane inhalation and a 2-cm ventral midline abdominal incision was made. The cecum was then exposed, ligated just distal to the ileocecal valve to avoid intestinal obstruction, punctured twice with an 18-gauge needle, and returned to the abdominal cavity. The incision was closed in layers and the animals were resuscitated with 3 ml/100 g BW normal saline subcutaneously immediately after CLP Polymicrobial sepsis was induced in adult male rats by cecal ligation and puncture (CLP). Briefly, rats were fasted overnight prior to the induction of sepsis, but allowed water 2+ and Mg2+  at 37°C at a rate of 15 ml/min. This was followed by perfusion with 120 ml of HBSS containing 0.02% collagenase  and 100 mM CaCl2 solution at the same perfusion rate. The liver was then removed en bloc, rinsed with ∼25 ml of HBSS, minced in a petridish containing HBSS with collagenase, and incubated for 20 min at 37°C to further dissociate the cells. The cell suspension was then passed through a 150-mesh, stainless steel screen into cold Dulbecco modified Eagel medium , containing 10% heat-inactivated fetal bovine serum and centrifuged (50 g for 2 min at 4°C) to sediment hepatocytes. The remaining cells in the supernatant were collected by centrifugation (450 g for 10 min at 4°C). The cell pellets resuspended in DMEM. After washing twice, cells were centrifuged on a density cushion of Percoll at 1,000 g for 15 min at 4°C. The buffy coat containing the KCs fraction was collected. The cells were further washed twice. Cell viability as determined by trypan blue exclusion was more than 95%. The yield was at 8–12×106 KCs/liver with a purity of at least 90%. The isolated KCs were then cultured in DMEM, containing 10% heat-inactivated fetal bovine serum, 10 mM HEPES, 100 U/ml penicillin and 100 g/ml streptomycin at the concentration of 106 cells/ml overnight with 5% CO2 at 37°C. KCs were allowed to adhere to the bottom of a 24-well plate (Costar) overnight and unattached cells were removed by gentle washing.Kupffer cells were isolated from normal and septic rats as previously described elsewhere with some modifications 2A-AR was assessed by real-time quantitative PCR (Q-PCR). Total RNA was extracted from KCs of CLP and Sham-operated animals as well as from cultured KCs using Tri-reagent . Q-PCR was carried out on cDNA samples reverse transcribed from 2 µg RNA using murine leukemia virus reverse transcriptase (Applied Biosystems). Using the QuantiTect SYBR Green PCR kit , reactions were carried out in 24 µl final volume containing 2 pmol of forward and reverse primers, 12 µl QuantiTect Master Mix, and 1 µl cDNA. Amplification was performed according to Qiagen's recommendations with an Applied Biosystems 7300 real-time PCR. Expression amount of rat GAPDH mRNA was used for normalization of each sample, and analysis of each specific mRNA was conducted in duplicate. Relative expression of mRNA was calculated by the ΔΔCt-method, and results expressed as fold change with respect to the corresponding experimental control. The following rat primers were used: GAPDH (AF 106860): 5′-ATG ACT CTA CCC ACG GCA AG-3′ (forward), 5′-CTG GAA GAT GGT GAT GGG TT-3′ (reverse); rat α2A-AR (NM_012739), 5′-CGT GTT CGT GGT GTG TTG GT-3′ (forward), 5′-GCA GCC GAC CGC TAT GAG-3′ (reverse).Gene expression of α6) from sham and septic animals at 2 h after CLP were incubated with [3H]-yohimbine  with or without 10 µM of unlabeled yohimbine for 30 min at 37°C in an assay buffer  max and Kd were determined by Scatchard analysis after logarithmic transformation.Freshly isolated KCs (106 cells/ml. KCs were then stimulated with NE (20 nM) with or without α2A-AR specific antagonist BRL-44408 maleate  for 4 h. The supernatant was then collected and TNF-α levels were measured by enzyme-linked immunosorbent assay (ELISA) kit specific for rat TNF-α . The assay was carried out according to the instructions provided by the manufacturer. For additional p38 MAP kinase pathway studies, KCs were cultured in DMEM for an 1 h (p38 phosphorylation) or 24 h (TNF-α release) with the following treatments: NE (20 nM), LPS , and the inhibitors BRL-44408 (1 µM), pertussis toxin , or SB203580 (10 µM).KCs isolated from normal animals were cultured overnight in DMEM medium with 10% heat inactive fetal calf serum, 100 U/ml penicillin/streptomycin, 100 mM HEPES and 100 U/ml L-glutamine at the concentration of 10Following anesthesia with isoflurane, a 3-cm midline incision was performed. The small intestine was exposed and a branch of the superior mesenteric vein was cannulated with a PE-10 catheter. It should be noted that this procedure did not cause any apparent gut ischemia. NE  or vehicle was infused into the portal vein at a rate of 13 µl/min for 2 h using a Harvard pump. Since portal blood flow is ∼13 ml/min/liver 6 KCs) TNF-α levels were determined using an ELISA assay kit specific for rat TNF-α . Isolated KCs were also used to determine the TNF-α gene expression by RT-PCR as described previously Plasma, supernatant from KC culture, and cellular , centrifuged at 16,000 g for 10 min, the supernatant was collected, and the protein concentration determined. A total of 10 µg of protein was loaded on a 4–12% Bis-Tris gel  and electrophoretically fractionated in a MES SDS running buffer (Invitrogen). The protein was then transferred to a 0.45-µm nitrocellulose membrane, and blocked with 5% bovine serum albumin in 10 mM Tris saline with 0.1% Tween 20, pH 7.6 (TBST). The membrane was incubated with rabbit anti-phosphorylated p38 MAP kinases polyclonal antibodies (1∶1000) overnight at 4°C, followed by incubation in 1∶20,000 HRP-linked anti-rabbit IgG for 1 h at room temperature. To reveal the reaction bands, the membrane was reacted with ECL Western blot detection system and exposed on X-ray film. The same membranes were stripped and re-blotted with rabbit anti-total-p38 MAP kinases  to determine the ratio of phospho-p38 and total p38. A digital image system was used to determine the density of the bands .Rats underwent CLP as described before, the femoral vein was canulated and connected to a Harvard pump. BRL-44408 (2.5 mg/kg BW) or vehicle was infused for 30 minutes using a Harvard pump. The catheter was tunneled out, and the femoral vein was ligated to stop the bleeding. The animals were allowed to wake up and returned to their cages. 20 h later animals sacrificed and blood collected for the measurement of liver transaminases, creatinine, lactate and TNF-α using commercial assay kits (Pointe Scientific). KC TNF-α mRNA expression was also assessed using the methods described above. The liver enzymes AST and ALT as well as creatinine and lactate levels were measured in blood serum by using commercial assay kits . In addition we conducted a 10-day survival study was conducted. 20 animals in each group underwent CLP and received either BRL-44408 (2.5 mg/kg BW bolus iv) or the same volume normal saline (Vehicle). 20 h after CLP, the cecum was removed, which based on our experience normally yields a 10-day survival rate of approximately 50%. In additional groups of normal rats, both the left femoral vein and artery were cannulated with a PE-50 tubing. The femoral vein catheter was connected to a Harvard pump for BRL-44408 (2.5 mg/kg BW) or vehicle infusion as described above. The femoral artery catheter was connected to a blood pressure analyzer  and mean arterial pressure (MAP) and heart rate were recorded continuously.All data that passed the normality test are expressed as mean±SE and compared by Student's t-test or analysis of variance (ANOVA) using Student-Newman-Keul's post-hoc analysis. Data that are of percentile based nature or failed the normality test are expressed as mean±95% confidence, and compared with ANOVA on Ranks. The binding capacity and affinity was estimated using the Scatchard analysis and curves compared by two-way ANOVA. The survival study was analyzed using the Kaplan-Meyer log-rank test. Differences in values were considered significant if P<0.05. All statistical analysis was performed using either the SigmaStat® or PRISM® software.2A-AR was significantly upregulated by 179% at 2 h post-CLP compared to respective sham-operated animals. In contrast, α2B and α2C-AR expression did not show any changes after CLP (Data not shown).Rats were subjected to sepsis by CLP and KCs were isolated 2 h thereafter. As shown in 2-AR expression in sepsis leads to enhanced receptor binding, we incubated KCs isolated from sham-operated or CLP animals with [3H]-yohimbine, a radio-labeled α2-specific AR antagonist. As shown in 2-AR was saturated at approximately 30 nM [3H] yohimbine in both sham-operated and CLP animals. However, maximal binding of the α2-specific ligand was much higher in KCs from CLP animals .To investigate whether increased α animals Fig. 2A.2A-AR blockade by BRL-44408 maleate (Isolated KCs were stimulated with NE (20 nM) with or without BRL-44408 maleate (1 µM) for 4 h. While TNF-α release increased by 440% after NE-stimulation, its increase was completely suppressed after α maleate Fig. 3. 2A-AR is also responsible for TNF-α upregulation in vivo, we administered NE through the portal vein in normal animals for 2 h and isolated KCs for analysis. TNF-α gene expression was upregulated by 4-fold in KCs from animals that were subjected to intraportal infusion of NE as compared to vehicle-treated animals  significantly attenuated TNF-α release by over 60%  and systemic intravenous administration of NE. LPS alone resulted in a 10-fold increase of TNF-α plasma levels after 4 h  and blocked either the α2A-AR with BRL-44408 or its coupled Gαi-protein with pertussis toxin (PTX). Both BRL-44408 (1 µM) and pertussis toxin  prevented the NE-induced phosphorylation of the p38 MAP kinase . Either agent by itself induced the phosphorylation of p38 compared to control, however, the combination of NE and LPS showed the strongest activation (over 7-fold induction) of p38 in cultured KCs  and SB203580  attenuated the NE-mediated increase in TNF-α release by 46% and 55% respectively , kidney (creatinine) and general oxigenation (lactate). As expected, TNF-α mRNA levels in KCs as well as plasma levels were increased 20 h after CLP by 6.6-fold and 2.4-fold, respectively  for 4 h 1, α2, and β, which are then subdivided into α1A, α1B, and α1C, α2A, α2B, and α2C and β1, β2, and β3. Upon binding of β2-ARs for example, epinephrine and high doses of NE through increasing intracellular cAMP levels 2-adrenoceptors are Gi- and G0-protein coupled receptors that decrease intracellular cAMP, open K+ channels, and inhibit voltage gated Ca2+ channels, all of which lead to hyperpolarization of neurons and activation of immune cells 2-adrenoceptors are predominantly presynaptic. They regulate the release of neurotransmitters through a negative feedback. Functional studies of the genetic receptor subtypes have linked the α2B-adrenoceptor to peripheral vasoconstriction and analgesic effects of N2O (nitrous oxide) and other anesthetic agents. The α2A-AR, either alone or with α2C-AR co-activation, is involved in the central inhibition of sympathetic activities, modulation of neurotransmitter release, sedation, and anti-epileptic effects. As we have shown here, the pro-inflammatory action of NE, mediated by the α2A-receptor subtype expressed on hepatic macrophages  can now be added to this list.Adrenergic receptors are subdivided in to three major subtypes α2-AR subtypes responsible for TNF-α upregulation in pulmonary inflammation and none in sepsis itself. In their work by Flierl et al., the authors focused on the phagocyte-derived cathecholamines that boost inflammatory responses via the α2-AR 2A-AR subtype in alveolar macrophages and neutrophils after LPS-stimulation, the role of individual subtypes in the proinflammatory response was not addressed 2A-AR has significantly increased 2 h after CLP, while no significant changes in α2B and α2C-AR could be observed. Kupffer cells stimulated with NE in combination with the α2A-AR inhibitor BRL-44408 maleate reduced TNF-α release, while the α2B-AR inhibitor imiloxan hydrochloride increased TNF-α levels (data not shown). The difference between those two receptor subtypes may lie in the intracellular signaling pathways. While all α2-ARs suppress intracellular cAMP levels through its Gαi coupled protein, they also change intracellular calcium and potassium levels to a different degree, which may influence intricate signaling pathways and eventually cellular response. As opposed to in vivo findings that showed no differences, our in vitro results show that α2B-AR inhibition is able to inhibit TNF-α release from cultured KCs, which is further complicating the role of α2B-ARs in the inflammatory response. In vivo, intraportal administration of NE significantly increased serum and Kupffer cell levels of TNF-α, and only the α2A-AR specific antagonist, BRL-44408 could significantly reduce TNF-α plasma levels and Kupffer cell TNF-α release. Recent reports show that not only sympathetic mediators, but also the cholinergic pathways modulate the systemic inflammatory response. Thus TNF-α release can be reduced by increasing efferent vagus nerve activity and acetylcholine release 7 subunit appears to be responsible for the inhibition of macrophage TNF-α release by acetylcholine 2A-AR in the regulation of vagus nerve activity is a possibility in vivo, but our in vitro results indicate that the TNF-α suppressive effect of BRL-44408 is independent of a parasympathetic influence. Our present work and others' studies also show that this tremendous activation of the sympathetic and parasympathetic nervous system during sepsis is not only a result of these devastating conditions but also influences of inflammatory responses itself by regulating proinflammatory cytokines via adrenergic and cholinergic receptors.So far, however, we could find only one report regarding the α2A-ARs also have effects on the cardiovascular and nervous systems 2A-AR blockade in sepsis is unlikely due to its direct effects on the cardiovascular and nervous systems.α2-AR stimulation observed in sepsis is primarily due to an increase in α2A-AR expression in KCs. This appears to be in-part responsible for the increased proinflammatory response and ensuing organ injury in sepsis. These findings provide important feasibility information for further developing the α2A-AR antagonist as a new therapy for sepsis.In summary, our results suggest that hyperresponsiveness to α


Thus, the physical proximity of activated β2-AR and PI-3K allows the activation of the latter. In contrast, it has been observed that the β1-AR is unable to activate the PI-3K/Akt pathway. We hypothesized that the difference might be due to molecular determinants present in the carboxy termini of the two β-AR subtypes. Using transiently transfected HEK 293 cells expressing either β1- or β2-AR, we also observed that in presence of an agonist, β2-AR, but not β1-AR, is able to activate the PI-3K/Akt pathway. Switching the seventh transmembrane domain and the carboxy tail between the two receptors reverses this phenotype; that is, β1 × β2-AR can activate the PI-3K/Akt pathway whereas β2 × β1-AR cannot. Pretreatment with pertussis toxin abolished the activation of PI-3K by β2- or β1 × β2-AR stimulation. Ligand-mediated internalization of the β2-AR induced by a 15-minute stimulation with agonist was abolished in the presence of a dominant negative of PI-3K or following pertussis toxin pretreatment. These results indicate that the subtype-specific differences in the coupling to PI-3K/Akt pathway are due to molecular determinants present in the carboxy tail of the receptor and further that β2-AR activates PI-3K via a pertussis toxin-sensitive mechanism.An agonist-occupied  The binding reactionswere incubated at room temperature for 90 minutes and terminated by rapidfiltration with ice-cold 25 mM Tris-HCl, pH 7.4, over Whatman GF/C glass fiberfilters preincubed for ≥30 minutes ina buffer containing 25 mM Tris-HCl, pH 7.4, 0.1% bovine serum albumin, and 0.3%polyethylenimine.Radioligand bindingassays were conducted essentially as described previously  with ~5 μg of cytosolic or membrane protein preparationswere subjected to 10% denaturing polyacrylamide gel electrophoresis aspreviously described. Transfer was performed with a Trans-Blot SD Semi-drytransfer cell (Bio-Rad) on Protran nitrocellulose membrane . Protein transfer efficiency was assessed using Ponceau Sstaining. Membranes were blocked using 5% nonfat dry milk in TBS-T (10 mM Tris(pH 7.4), 150 mM NaCl, and 0.05% Tween 20) and membranes were incubated at 4°C overnight with primary antibody (anti-Phospho-Akt (Ser 473) andanti-Akt from Cell Signaling Technology,  or anti-PI-3K,anti-β1- or anti-β2-AR from Santa-Cruz diluted 1:1000 in 5% nonfat dry milk into TBS-T. Subsequently, membranes werewashed and incubated for 45 minutes at room temperature with the secondaryantibody (diluted 1:5000 in 5% nonfat dry milk into TBS-T) conjugated tohorseradish peroxidase. Membranes were washed and exposed to scientific imagingfilm  or quantified using a KodakImageStation 440CF using enhanced chemiluminescence reagent (Perkin Elmer LifeSciences). Band intensities were analyzed using Kodak 1D v.3.5.5 ScientificImaging Software.Western blotting was conducted as described previously . Brieflyβ-ARwas observedby immunolocalisation. After agonist treatment, cells were washed with PBS andfixed with 3% paraformaldehyde for 15 minutes. After these washes, nonspecificsites were blocked with 0.2% BSA and 0.15% Triton x-100 (blocking solution) for10 minutes. Primary antibody (β1- and β2-AR from Santa-Cruz), prepared in theblocking solution (1:200), was added for 30 minutes at room temperature. Afteranother series of washes, secondary antibody  alsoprepared in the blocking solution (1:500) was added for 30 minutes. After afinal series of washes, slides were mounted and viewed using a Leica epi-illumination microscope.Sequestration of μg of cytosolic and membrane proteins were precipitated withanti-phosphotyrosine antibody conjuged to biotin overnight at 4°C. The immune complex was pelleted (with streptavidinbeads) and washed three times with lysis buffer and twice withphosphate-buffered saline buffer containing 0.1 mM Na3VO4. The immune pellet was then suspended in activation buffer  and incubated at room temperature for 20 minutes. The reaction was stopped with the addition of 100 μL HCl 1 M and 200 μL of chloroform:methanol (1:1). The aqueousphase was then discarded. Eighty μLof HCl:methanol (1:1) were then added before discarding the aqueous phase. Twenty μLof the organic phase containing 32P-phosphatidylinositol wereresolved by thin layer chromatography on K6 Silica Gel plates  in a solvent system containing chloroform:methanol:ammoniumhydroxide (45:35:10). Plates were exposed to film for three to five days(−80°C).PI-3K activity was measured as previously described . BrieflyP < .05 was considered significant.Results are expressed as mean ± SEM and were evaluated usinganalysis of variance adapted for factorial experimental design. Orthogonalization was performed when necessary . P < .05β1-AR Wt, β2-AR Wt,β1 × β2-AR, orβ2 × β1-AR and in some case, p85ΔPI-3K. Forty-eight hours after transfection β-AR expression levels were approximately 500 fmol/mg of proteins(compared to 10–20 fmol/mg inuntransfected cells). Expression of p85ΔPI-3Kdetermined by western blot was increased 3.24 times as compared to wild-typecells .HEK 293 cells were transiently transfected with cDNAs encoding for β1- or β2-AR was measured by in vitro phosphorylation of L-α-phosphatidylinositol. Transfected cells were stimulated withisoproterenol 1 μM for 0, 5, or 15 minutes at 37°C. Stimulation of β2-AR- for 5 or 15 minutes induced asignificant augmentation in PI-3K activity compared to basal conditions activation. Akt activation, as determined by the phosphorylationstatus of Serine 473, was significantly increased after β2-AR-stimulation for 5 and 15 minutesas compared to the basal state .To confirm differences between stimulation of the two al state . Stimulaβ1-AR can recruit PI-3K to the plasmamembrane, immunolocalisation of PI-3K was performed using Western blotting withanti-P110γ antibody. Transientlytransfected cells with β1- or β2-AR were stimulated by isoproterenol1 μM for 0, 5, or 15minutes at 37°C. Both β1- and β2-AR stimulation results in recruitment of PI-3K to theparticulate fraction (P < .05) inmembrane fractions by agonist stimulation of either β1-AR or β2-AR for 5 or 15 minutes. Nosignificant difference was detected between 5 and 15 minutes of stimulation. Thus, either subtype of β-AR can recruit PI-3K to the membrane after agonist stimulation butonly the β2-AR results in PI-3K activation.To determine whether fraction . Compareβγ subunit-dependent, β1- or β2-AR was transiently cotransfected with thecarboxyl-terminal portion (ct) of GRK2 (a sequestering agent for Gβγ) in HEK 293 cells. Cells cotransfected with β1- or β2-AR and ct-GRK2 were stimulated with1 μM isoproterenol for 5minutes. ct-GRK2 when transfected alone had no effect on PI-3K activity. Stimulation of the β2-AR for 5 minutes with isoproterenolin cells cotransfected with ct-GRK2 resulted in a significantly decreased (P < .001) PI-3K recruitment to the particulate fraction  PI-3K recruitment to membranes  as well as the reciprocalreceptor which consisted of β2-AR with the carboxy-terminal tail ofthe β1-AR (β2 × β1-AR). When theβ1 × β2-AR was stimulated by 1 μM isoproterenol for 5 or 15 minutes, PI3-kinase activity wassignificantly increased (P < .05). On the other hand,agonist stimulation of theβ2 × β1-AR for 5 or 15 minutes did notresult in increased PI3-kinase activity. These results suggest that thecarboxy-terminal tail of the β2-AR contains molecular determinantswhich are required for PI3-kinase activation.To determine the importance of receptor-specific determinants in thencreased ; P < .05β-ARs, we againdetermined the activation status of Akt. Chimeric receptors were stimulatedwith 1 μM isoproterenol for 0,5, or 15 minutes at 37°C. Aktphosphorylation was significantly increased (P < .05) in HEK 293 cellsexpressingβ1 × β2-AR , and PI-3K. Indeed, the chimeric receptorβ1 × β2-AR can activate thePI-3K by a pertussis toxin sensitive mechanism whereas the chimeric receptorβ2 × β1-AR is unable to activatePI-3K. However, other regions of the receptor may also be important for fullactivation or regulation of the response by the desensitization machinery.Pretreatment with pertussis toxin results in a loss of PI-3Kactivation by  studies , 12. Theβ1-AR (for up to 15 minutes) cannotactivate PI-3K. These results contrast with those reported in aprevious study in which activation of PI-3K was observed after stimulation of β1-AR transfected into HEK 293 cells [μM isoproterenolalthough sufficient to induce submaximal adenylyl cyclase activity [β1-AR and β2-AR lead to PI-3K and GRK2recruitment; boththe signalling and desensitization outcomes for these events are markedlydifferent. Some studies have also shown that β1-AR, in contrast to the β2-AR, cannot activate Gi [Our results demonstrate that isoproterenol-stimulated activity  cannot ivate Gi  while otβ2-AR internalization is inhibited inthe presence of a dominant negative PI-3K, p85ΔPI-3K, results that are similar to previousstudies [β2-AR sequestration can be abolishedwith pertussis toxin pretreatment. In contrast to β2-AR, we observed that the β1-AR sequestration is minimal with 15minutes of stimulation. This is similarto the 10% sequestration observed in β1-AR expressing cells obtained bySuzuki et al. [β1-AR might be due to the weak PI-3Kactivation induced by the stimulation of this receptor subtype. Resultsobtained with the pertussis pretreatment and p85ΔPI-3K also suggest that activated PI-3K isimportant for receptor sequestration.In this study, we observed that studies . We alsoi et al. . Since pi et al. , 19, it β-AR subtype specific and the difference is due to molecular determinantspresent in the carboxy-terminal tail of the receptors. PI-3K activation ispertussis toxin sensitive and is necessary for the sequestration of the β2-AR.In conclusion, we observed that PI-3K activation is


AR) with androgenetic alopecia (AGA) remains unidentified, but Copy Number Variation (CNV) might be relevant. CNV involves changes in copy number of large segments of DNA, leading to the altered dosage of gene regulators or genes themselves. Two recent reports indicate regions of CNV in and around AR, and these have not been studied in relation to AGA. The aim of this preliminary case-control study was to determine if AR CNV is associated with AGA, with the hypothesis that CNV is the functional AR variant contributing to this condition.The functional polymorphism that explains the established association of the androgen receptor (AR exons and a conserved, non-coding region upstream of AR in 85 men carefully selected as cases and controls for maximal phenotypic contrast. There was no evidence of CNV in AR in any of the cases or controls, and thus no evidence of significant association between AGA and AR CNV.Multiplex Ligation-dependent Probe Amplification was used to screen for CNV in five AR locus is unlikely to predispose to AGA.The results suggest this form of genomic variation at the  AR; NCBI NM_000044) in the development of androgenetic alopecia  is now well established. Our initial observation AR with AGA has been replicated in three independent studies AR coding sequence polymorphisms such as triplet repeats are not associated with AGA The importance of the gene encoding the androgen receptor (Copy Number Variation (CNV) has been recognised recently as a common form of genomic variation ARAR seen in men predisposed to AGA.CNV has been reported in the region of AR CNV could be associated with AGA. Therefore, our approach was to first search for evidence of CNV at the AR locus and then, if present, to test for association of CNV with AGA.We hypothesised that The study was approved by the Ethics Review Committee of the Alfred Hospital Melbourne, and informed consent was obtained from each participant. Subjects were drawn from the population-based Victorian Family Heart Study cohort AR. Six probes were selected to test AR exons and one upstream non-coding region for the presence of CNV  was performed to detect the presence of CNV around AR test probes in all case-control subjects. The standard deviation for each of the six AR test probes was within 15%, except for probes 2 and 6 which were slightly higher, possibly suggesting less reliable performance of the probes. Nevertheless, across the remaining samples, good coverage of the AR region was achieved by the higher performing probes, and the results provide no evidence of CNV in this region, either as deletion or duplication, in cases or controls.AR and their potential relationship with AGA. CNV may contribute to differences in gene expression through duplications or deletions of genes or gene regulatory elements AR that we examined. Insofar as our original hypothesis, we could therefore find no evidence of association of CNV with AGA.This study examined CNV in and around AR gene was well covered by the remaining probes. The lack of evidence for CNV at this locus supports the findings of a very recent study by McCarroll et al which has demonstrated a lack of CNV in the AR coding regions Although data generated by probes 2 and 6 showed relatively high standard deviations, casting doubt on their reliability, the AR CNV in DNA derived from lymphocytes, we cannot rule out CNV around the AR in DNA contained in the cells of the hair follicles themselves.This study examined a locus on chromosome × in males, meaning that if changes in CNV dosages were detected, they would be expected to be at either a 0 (deletion) or 2 (duplication) level. AR. However, we focussed on a region that we have already shown to be strongly associated with AGA and it would be likely that causative CNV, if they exist, would be in the region examined. Sample sizes used were relatively small, however power is augmented by the fact that association of AGA with AR is well-established and that the cases and controls were selected to represent high phenotypic contrast.Our preliminary investigation did not thoroughly investigate the downstream non-coding flanking region and other more distant sites from AR CNV involvement in AGA, other forms of non-coding AR sequence variation, and also epigenetic mechanisms at this locus, should be considered in clarifying the role of AR as a genetic risk locus for AGA.In the absence of evidence of


Arabidopsis thaliana mutants defective in seed ABA synthesis (aba1-1) or perception (abi1-1). Even though imbibed mutant seeds showed no dormancy, they exhibited changes in global gene expression resulting from dry AR that were comparable with changes occurring in wild-type (WT) seeds. Core gene sets were identified that were positively or negatively regulated by dry seed storage. Each set included a gene encoding repression or activation of ABA function , thereby suggesting a mechanism through which dry AR may modulate subsequent germination potential in WT seeds. Application of exogenous ABA to after-ripened WT seeds did not reimpose characteristics of freshly harvested seeds on imbibed seed gene expression patterns. It was shown that secondary dormancy states reinstate AR status-specific gene expression patterns. A model is presented that separates the action of ABA in seed dormancy from AR and dry storage regulated gene expression. These results have major implications for the study of genetic mechanisms altered in seeds as a result of crop domestication into agriculture, and for seed behaviour during dormancy cycling in natural ecosystems.After-ripening (AR) is a time and environment regulated process occurring in the dry seed, which determines the germination potential of seeds. Both metabolism and perception of the phytohormone abscisic acid (ABA) are important in the initiation and maintenance of dormancy. However, molecular mechanisms that regulate the capacity for dormancy or germination through AR are unknown. To understand the relationship between ABA and AR, we analysed genome expression in  Seed survival following shedding from the mother plant is a key component in the life history of flowering plants. Under natural conditions, seed survival in the soil and cycling through states of dormancy are major ecological characters determining entry and persistence in ecosystems , and seeMeasurement of water contents of unimbibed seeds suggests that cellular environments are compatible with the operation of complex biochemical processes, and that AR rate is affected by seed water content , providiArabidopsis thaliana. Mutation of these loci range from subtle changes in germination potential and dormancy capacity, to extreme conversion of embryo characteristics into those of seedlings, as in severe abi3, lec and fus3 mutants  or hypersensitive  in their germination response to applied ABA , these processes may be uncoupled, i.e. changes in the dry seed that would lead to the promotion of germination (AR) in WT seeds may still occur, even though mutant seeds have no dormancy. The advantage of this as an experimental tool would be that dry-stored mutant seeds, although exhibiting the same germination potential as freshly harvested mutant seeds, would have gone through the dry AR process. In this way, AR-specific gene sets could be identified as being shared between mutant and WT imbibed seeds either before or following dry storage. Because ABA has been shown to have a profound effect on germination potential, we chose to address this possibility using two mutants with impaired synthesis (aba1-1) or perception (abi1-1) of ABA, which have been shown to have constitutive effects and not to be restricted to a specific stage of development.Previous studies have defined AR as a widening or increased sensitivity of perception of seeds to environmental conditions promoting germination, at the same time as a narrowing or decrease in sensitivity of perception of conditions repressing germination . This deaba1-1 and abi1-1, were compared for AR capacity and germination potential (er and Cvi). Both aba1-1 and abi1-1 freshly harvested (F) seeds were able to complete germination  seeds, suggesting that these seeds are similar at the transcriptome level. Conversely, comparisons of aba1 and abi1 F and S samples showed these to be very different, suggesting that changes occur in these non-dormant mutant seeds under the same conditions that promote AR in D seeds, and therefore that AR may occur in these non-dormant mutant seeds. This observation was confirmed through principal component analysis (PCA) of the datasets   annotation, because it provides more useful information related to seed biology . This anuishable . Using tT AR > D .LPP2 that removes sensitivity to ABA during germination) and 103 common to F-D sets . The expression patterns of representative members of each set was measured in WT and mutant seeds from 0–48 h of imbibition using quantitative (Q)RT-PCR RT-PCR . For eacer and Cvi revealed very similar average ratios of expression of each gene set. Ratios of expression for the genes ABA1 and LPP2 were consistent with their placement in ‘AR-downregulated’ and ‘AR-upregulated’ sets, respectively.In order to further examine the robustness of expression characteristics for these gene sets, we also analysed global gene expression in WT accession Cvi. Comparison of transcriptomes of AR and D Cvi seeds showed large changes in expression between the two physiological states at 24 h of imbibition (Table S1). To compare expression properties of the AR-associated and AR-independent gene sets, we derived the average ratio of expression levels of each gene set, comparing either different physiological states or different genetic backgrounds . CompariRD29B (AR downregulated) was upregulated by ABA at 48 h of imbibition, as has previously been shown for this ABA-inducible gene  may provide materials to analyse the molecular mechanisms regulating AR. It is also important to note that transcriptional changes have also been observed in ‘dry’ seeds  represent downstream genes that are ABA responsive and may contribute to dormancy maintenance. Interestingly the ‘ABA-responsive’ gene RD29B is also part of the ‘AR-downregulated’ set, which may suggest that it is an early target of increased ABA1 function  or reduction in the AR dependence of seeds. These are two distinct molecular processes occurring in two different developmental pathways. By defining gene sets that are AR regulated it may now be possible to distinguish between the contributions of these two mechanisms, for example through the identification of molecular markers or transcriptome developmental signatures that can be used to differentiate crop varieties with differing D/AR characteristics. As a lack of dormancy can be a major agronomic problem (for example in the disorder pre-harvest sprouting in wheat and barley), such approaches may provide the tools to deliver improved AR and dormancy characteristics (Loss of the capacity for seed dormancy is one of the major traits acted on by evolutionary processes during the introduction of wild plant species into agriculture (the ‘domestication syndrome’) . There afus3-8, rdo2 and abi3-4 were provided by Prof. M. Koornneef ; Ler, abi1-1, aba1-1 were obtained from NASC (http://arabidopsis.info). All mutants are in the Ler background.Wild-type accession Cvi and mutant seeds er seeds completely after-ripen within 60 days , was included in media at a concentration of 10 μm.Arabidopsis WT and mutant plants were grown as previously described ( 60 days . All seehttp://www.roche.com), with primers and probes for each gene assay designed using the Universal ProbeLibrary Assay Design Center (Roche Applied Science) as previously described (http://affymetrix.arabidopsis.info/narrays/experimentbrowse.pl). Gene expression levels were measured using the microarray suite 5.0 software , and further analysis was performed with genespring 7.3 software  using a twofold differential expression between samples  to define differentially regulated genes. Normalized expression values from microarray datasets were used to obtain ratios of expression. Developmental signatures were obtained using the TAGGIT workflow within Microsoft Excel (http://www.microsoft.com) (All experiments analysing RNA expression levels were carried out using three replicates of seed materials, obtained from three independent seed lots, from plants grown under controlled-environment conditions. Total RNA was extracted from seed material and real-time quantitative PCR was carried out using the Universal ProbeLibrary system (Roche Diagnostics,


Rhizobium genus, which provide them with available nitrogen. Nodulation factors (NFs) produced by Rhizobium initiate legume root hair deformation and curling that entrap the bacteria, and allow it to grow inside the plant. In contrast, legumes and non-legumes activate defense responses when inoculated with pathogenic bacteria. One major defense pathway is mediated by salicylic acid (SA). SA is sensed and transduced to downstream defense components by a redox-regulated protein called NPR1.Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from Arabidopsis mutants in SA defense pathway to test the role of NPR1 in symbiotic interactions. Inoculation of Sinorhizobium meliloti or purified NF on Medicago truncatula or nim1/npr1 A. thaliana mutants induced root hair deformation and transcription of early and late nodulins. Application of S. meliloti or NF on M. truncatula or A. thaliana roots also induced a strong oxidative burst that lasted much longer than in plants inoculated with pathogenic or mutualistic bacteria. Transient overexpression of NPR1 in M. truncatula suppressed root hair curling, while inhibition of NPR1 expression by RNAi accelerated curling.We used nodulin expression. Our results also show that basic plant responses to Rhizobium inoculation are conserved in legumes and non-legumes.We show that, while NPR1 has a positive effect on pathogen resistance, it has a negative effect on symbiotic interactions, by inhibiting root hair deformation and  Rhizobium genus, which provide plants with a source of available nitrogen. Symbiosis is regulated by complex mutual interactions between the organisms. Symbiosis between legumes and Rhizobium is initiated by specific nodulation (Nod) factors (NFs) that are secreted into the soil by the bacteria. In response to compatible NFs the legume root hairs begin to curl, entrapping the bacteria. The cell wall within the curl undergoes a local hydrolysis, allowing bacteria to enter the root hair and form an intracellular infection thread from the curled region to the root cortex RhizobiumPlants continually interact with soil micro-organisms that are broadly divided into pathogenic, saprophytic or symbiotic. While the pathogenic and saprophytic interactions are common to all plant species, symbiosis with the nitrogen-fixing rhizobacteria is a relatively recent evolutionary development that is restricted to plants of the legumes family. Legumes are unique in their ability to establish symbiotic interaction with rhizobacteria from the NPR1 overexpression cause enhanced disease resistance (edr1) phenotype in heterologous plant species, suggesting an evolutionary conserved SA-mediated signaling pathway in different plants nim1/npr1, as well as mutations that reduce SA production (pad4 or eds1) suppressed the edr phenotype in all of the edr mutants edr phenotype was also suppressed by expression of SA hydroxylase NahG transgene, which converts SA to catechol, resulting in rapid SA decomposition In contrast to symbiotic interaction with rhizobacteria, plants mount a defense response when challenged with pathogenic bacteria. Several signaling pathways that mediate local and systemic plant responses to pathogens have been identified. One of the major signaling pathways induced during pathogenic interactions, including local and systemic defense responses to pathogens is mediated by salicylic acid (SA); for reviews: M. truncatula during the first stages of symbiotic interaction with Rhizobium showed a reduction in the amount of SA M. truncatula by the NahG transgene resulted in increased rhizobial infection and nodulation. Furthermore, inoculation of incompatible strains of S. meliloti on alfalfa (Medicago sativa) roots led to accumulation of SA, and exogenous application of SA to alfalfa plants inhibited nodule formation S. meliloti infection threads Lotus japonicus with no persistent meristem) 2O2 accumulation in soybean infected with avirulent pathogens Compared to the pathogenic interactions only a few studies addressed the involvement of SA in symbiotic interactions. SA measurements in M. truncatula and Rhizobium. We also show that npr1 mutants in non-legume A. thaliana respond to S. meliloti by activating root hair deformation and induction of early and late nodulin genes. Interestingly, both M. truncatula and npr1 mutant A. thaliana responded with an extremely strong oxidative burst to S. meliloti inoculation, which lasted beyond the restoration of redox after inoculation of Pseudomonas putida or Pseudomonas syringae.Here, we show that SA and NPR1 negatively affect the symbiotic interactions between Rhizobium interaction, involving root hair deformation that precedes hair curling in legumes. Root hair deformation is one of the first steps in interaction with compatible rhizobacteria Rhizobium-induced root hair deformation we first pretreated M. truncatula seedlings with SA prior to inoculation of S. meliloti, which resulted in the inhibition of root hair deformation  gene that is regulated by SA in tomato, tobacco and Arabidopsis thalianaalpha-Dox gene expression in M. truncatula roots was reduced during the first day after S. meliloti inoculation , which are mutated in cyclic nucleotide-gated ion channel ndr1-1 (nonrace-specific disease resistance), which encodes a plasma membrane protein with unknown function nim1 (noninducible immunity) npr1 (nonexpresser of PR genes) To explore if the root hair deformation is a typical legume response to compatible A. thaliana seeds were germinated on nitrogen poor medium, and inoculated in the root elongation zone with S. meliloti eight days later. No difference in root hair behavior was seen in dnd1  nim1/npr1 seedlings . No deformation was seen also in plants inoculated with P. putida (.p). Interestingly, quantitative analysis of A. thaliana nim1/npr1 mutants showed a similar percentage of deformed root hairs .In addition, we inoculated SA-deficient im1/npr1 . Also, iA. thaliana, we applied purified nod factor from the S. meliloti strain used above to seedlings' roots, without the bacteria. The NF treatment induced root hair deformation exclusively in the SA-insensitive nim1/npr1 mutants, or in SA-deficient NahG transformants, in agreement with data observed in plants treated with intact S. meliloti  that act in accommodating the rhizobial bacteria, and late nodulins that are thought to be involved in the nodule functioning. ENODs are induced within one or few days after inoculation, while late nodulins take several days A. thaliana are shown in ENOD homologs, representing early  and a late  nodulins, that are expressed 2–5 and 7 days post infection, respectively, in M. truncatulaA. thaliana ENOD20 homolog, At5g57920, also called early nodulin-like protein in the Arabidopsis TAIR database, shares 38% identity and 56% similarity with the M. truncatula protein, while the At5g07050, also called nodulin-related protein, contains 64% identity and 78% similarity. Both genes were induced by the inoculation of S. meliloti in the nim1/npr1 background, but not in wild-type plants , which leads to hypersensitive cell death S. meliloti, or with P. putida, or P. syringae bacteria, using 2′,7′-dichlorofluorescin diacetate, which reports ROS production inside the cells M. truncatula roots already 5 hours after inoculation with either bacterium  by diphenyleneiodonium (DPI), implicating activation of NADPH oxidase RIP1At5g57920. Ten days-old wild-type and nim1/npr1 Arabidopsis mutants were pretreated with DPI, as described in S. meliloti. The expression of At5g57920 was tested by quantitative real-time RT-PCR four days after inoculation  brings out similar responses in both plant species. The NPR1 protein may be involved in reducing intracellular ROS, possibly by inducing antioxidants. This suggestion is supported by analysis of NPR1-dependent expression of multiple genes Our data indicates that ROS induction during the symbiotic interaction is regulated by NF perception. We also show that in legume NPR1 is the founding member of a small gene family that contains several NPR1-related or NIM1-like proteins, all of which share the BTB–POZ and the ankyrin-rich repeats domains NPR1-like homolog of M. truncatula (TC102752) in the public EST database (MtDB2.0). The NPR1-like proteins in M. truncatula also form a family . Since normally after two days root hairs show only swelling, and root hair curling occurs around 4 days after S. meliloti inoculation To further analyze the role of NPR1 in root hair curling, we silenced the pression , and resM. truncatula following S. meliloti inoculation with gene expression in A. thaliana infected with pathogenic bacteria, we selected several defense genes that were shown to be regulated by NPR1 in A. thalianaM. truncatula orthologs of the Arabidopsis LRK (Lectin Receptor Kinase), ARP (Ankyrin repeat-containing protein) and WAK  genes were identified by BLAST analysis of the M. truncatula genome project database (http://www.medicago.org/genome/). All of the genes showed constitutive expression in M. truncatula roots. However, inoculation of S. meliloti caused a window of transcriptional downregulation, starting at 4 hours post inoculation (p.i.) and culminated at 9 hours p.i. . A. thaliana NPR1 construct was a gift from Xinnian Dong . P. syringae were grown as described in A. thaliana seeds were grown on agar plates containing 1/60 MS medium. S. meliloti were inoculated on the roots in zone 1 at a concentration of 107 cells. NF was prepared from the S. meliloti strain 1021, according to http://align.genome.jp/). Protein sequences. The A. thaliana genes were downloaded from The Arabidopsis Information Resource (TAIR) website (http://www.arabidopsis.org/) and were uploaded to the Kyoto ClustalW website. The M. truncatula genes were from public EST database (MtDB2.0).Phylogenetic N-J tree of the plant genes was constructed by using the Kyoto University ClustalW multiple sequence alignment website,  and transcribed into cDNA using oligo dT as a primer with SuperScript II reverse transcriptase (Invitrogen). cDNA was amplified by PCR using Taq polymerase and the following primers: NPR1: forward TGACTTGTTTTACCTTGAGAA and reverse, AATTATTTTATAGAGAGGAGA. α-dioxygenase: forward, GAAGTTTTGGACAAAGTGAGGACT; reverse TGTCAGTTTTAAGAAGCTCCACAG. At5g57920: forward, TAACGAATGGGCTCAAAAGG; reverse CTGGACCGTCGAACTCAGAT. At5g07050: forward, TGGGATTGTGGCATCAAGTA; reverse CCCCTTCCGAGATTTTCATT. LRK: forward CAACTCATTTGGTTGGAACTGTAG reverse GGATAAGACAAAGGAAAGTCCTCA. TCTTCTCCATTTCCTCAATTTCA, reverse TTATTAAGAGCAGCCCACTGAAG.ARP: forward WAK: forward CAGGAGGTTGTCATAAACAAGATG reverse, AAGTGTAACCCGTTGCTAACAAAT. EF1a gene, forward TCACATCAACATTGTGGTCATTGGC; reverse, TTGATCTGGTCAAGAGCCTCAAG. EF1a and Actin2 were used to normalize RNA amounts in M. truncatula and A. thaliana respectively.Total RNA was extracted from roots before and after m 2′,7′-dichlorofluorescin and ROS levels were quantified with ImagePro Plus analysis package  as described in Seedlings were taken out of agar plates after 3 days, washed, and transferred to new plates with nitrogen-free medium. ROS in Arabidopsis roots were detected by 10 µNPR1 (TC102752) gene were selected, and used to design complementary oligonucleotide primers: forward: 5′- ATCTCTGCCGGAATCAACAC-3′, and reverse: 5′-TCTGATGCACAAGCTCCGTTTTTC-3′. The segment was amplified by PCR and cloned into pENTR using Invitrogen TOPO10-cloning kit and transformed at room temperature for 5 min, then on ice for 30 min, 42°C for 45 sec, and spread on LB solid medium with 50 µg/ml kanamycin. Clones were selected one day later, and sequenced. NPR1- pENTR plasmid was used for the LR recombined reaction using Invitrogen's Gateway LR Clonase II enzyme mix and transformed to TOPO10 competent cells as described above, only the LB contained 100 µg/ml spectomycin and 300 µg/ml streptomycin. Clones were selected one day later, and sequenced using the forward and reverse primers from upstream and downstream sequences of the antisense insertion.Silent sites from the Medicago A. tumefaciens GV3101 by freezing in liquid Nitrogen for 5 min and spread on TYNG solid medium containing 50 µg/ml rifampicin, 25 µg/ml Gentamicin, 100 µg/ml spectomycin and 300 µg/ml streptomycin. Monoclonal colonies were selected two days later, and analyzed by PCR for identification. Bacteria were shot with the addition of 1∶1000 M/V carborundum into 7 day-old M. truncatula roots, using Bim-LAB apparatus , essentially, as described in NPR1-RNAi cloned plasmids were transfected into Figure S1Salicylic acid inhibits root hair curling in M. truncatula. (A-F) Three day old M. truncatula seedlings were transferred to plates supplemented with SA (D–F) or replanted on new plates with N-free medium (A–C). The roots of the seedlings were inoculated in zone 1 after 24 h with S. meliloti  or treated with NF . SA was applied by dispersing 1 ml of 500 µM SA on top of the plates for 6 days. Pictures were taken 4 days after inoculation, or 2 days after NF treatemnt. At least one hundred root hairs within zone 1 were scored. The percentage of deformed and curled root hairs is indicated on the bottom of each image . The ± number indicated the standard deviation. (G) M. truncatula seedlings were germinated on N-free medium and inoculated with S. meliloti three days later. Total RNA was extracted 24 hours after Rhizobium inoculation. The amount of RNA in the samples was normalized according to the EF1a gene expression. The primers to alpha-dioxygenase gene were selected by BLAST analysis using the A. thaliana gene sequences (At3g01420). All experiments were repeated at least three times with very similar results.(0.78 MB TIF)Click here for additional data file.Figure S2The effect of S. meliloti inoculation on A. thaliana dnd1 and nim1/npr1 mutants. A. thaliana seedlings were grown as described in (0.15 MB TIF)Click here for additional data file.Figure S3Treatment of M.truncatula with purified nod factor. Five day old M. truncatula seedlings were exposed to nod factor (+NF) or left intact (−NF). Roots were photographed 3 days after treatment under bright light. Roots of six seedlings of each treatment were analyzed. Bar = 35 µm(0.10 MB TIF)Click here for additional data file.Figure S4http://align.genome.jp/). Right panel shows the unrooted version of the tree. The genes were chosen using the BLAST program: http://www.arabidopsis.org/wublast/index2.jsp, to the amino acid sequence of TC114239 (ENOD20) at: http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gireport.pl?gudb=medicago (B) Alignment of the Arabidopsis homologs of the M. truncatula ENOD20 (TC114239). The alignment was done using ClustalW2 multiple sequence alignment website from the European Bioinformatics Institute .Phylogenetic tree of ENOD20. (A) Phylogenetic N-J tree of the Arabidopsis homologs of the M. truncatula early nodulin ENOD20 (TC114239). The tree was constructed using the Kyoto University ClustalW multiple sequence alignment website, ((0.07 MB PDF)Click here for additional data file.Figure S5http://align.genome.jp/). Right panel shows the unrooted version of the tree. Genes were selected with the BLAST program: http://www.arabidopsis.org/wublast/index2.jsp to the amino acid sequence of TC117774 (MtN21) at: http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gireport.pl?gudb=medicago (B) Alignment of the Arabidopsis homologs of the M. truncatula MtN21 (TC114239) late nodulin gene. The alignment was done using ClustalW2 multiple sequence alignment website from the European Bioinformatics Institute .Phylogenetic tree of MtN21. (A) Phylogenetic N-J tree of the Arabidopsis homologs of the M. truncatula MtN21 (TC117774) late nodulin gene. The tree was constructed using the Kyoto University ClustalW multiple sequence alignment website, ((0.09 MB PDF)Click here for additional data file.Figure S6http://align.genome.jp/). The right panel shows the unrooted version of the tree. Genes were selected using the BLAST program at http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gireport.pl?gudb=medicago. The proteins were aligned to the At1g64280 (NPR1) available at: http://www.arabidopsis.org/wublast/index2.jsp. (B) Alignment of the M. truncatula homologs of the Arabidopsis NPR1. The alignment was done with ClustalW2 multiple sequence alignment website from the European Bioinformatics Institute .Phylogenetic tree of the NPR1 protein. (A) Phylogenetic N-J tree of the M. truncatula genes that are homologous to the Arabidopsis NPR1. The tree was constructed using the Kyoto University ClustalW multiple sequence alignment website, ((0.04 MB PDF)Click here for additional data file.Table S1List of mutants and their putative signaling pathways(0.06 MB PDF)Click here for additional data file.


How do people respond to others' accidental behaviors? Reward and punishment for an accident might depend on the actor's intentions, or instead on the unintended outcomes she brings about. Yet, existing paradigms in experimental economics do not include the possibility of accidental monetary allocations. We explore the balance of outcomes and intentions in a two-player economic game where monetary allocations are made with a “trembling hand”: that is, intentions and outcomes are sometimes mismatched. Player 1 allocates $10 between herself and Player 2 by rolling one of three dice. One die has a high probability of a selfish outcome, another has a high probability of a fair outcome, and the third has a high probability of a generous outcome. Based on Player 1's choice of die, Player 2 can infer her intentions. However, any of the three die can yield any of the three possible outcomes. Player 2 is given the opportunity to respond to Player 1's allocation by adding to or subtracting from Player 1's payoff. We find that Player 2's responses are influenced substantially by the accidental outcome of Player 1's roll of the die. Comparison to control conditions suggests that in contexts where the allocation is at least partially under the control of Player 1, Player 2 will punish Player 1 accountable for unintentional negative outcomes. In addition, Player 2's responses are influenced by Player 1's intention. However, Player 2 tends to modulate his responses substantially more for selfish intentions than for generous intentions. This novel economic game provides new insight into the psychological mechanisms underlying social preferences for fairness and retribution. Jon and Matt are brothers who share two traits: poor marksmanship and a quick temper. Neither has committed a crime before, but when a rival insults their family name, both decide to take action. Jon pledges to kill the rival with a bullet to the heart, but his shot misses and the rival is unharmed. Matt just wants to spook the rival with a bullet over the shoulder, but he accidentally hits the rival's heart and kills him. In our home state of Massachusetts, Jon can expect a sentence of roughly five to ten years in prison for attempted murder with a firearm. Matt's poor marksmanship, however, earns him second degree murder—and a mandatory life sentence.Cases like this have drawn the attention of philosophers and legal scholars Games in which one player makes an allocation of money and another player rewards or punishes her by adding or subtracting money depending on that allocation have become a standard method of research in behavioral economics Different economic models of responder behavior in allocator/responder games have approached this problem in different ways. According to one family of theories, we assign punishment and reward on the basis of others' intentions an independent, random event happens to kill his rival?”). In other studies, experimenters have restricted the range of allocations available to the allocator so that the allocation is forced to be ‘stingy’ Matt was forced to pull the trigger?”). These experiments find that responders punish stingy allocations substantially less when the ‘allocator’ cannot control them. Such results demonstrate that the distributional effect cannot fully account for responder behavior in allocator/responder games: after all, distributions are just as unequal when made by a computer, a human, or any other mechanism. Something extra must explain the punishment of controllable allocations. It has been argued that the something extra is allocator intent; that is, the punishment of stingy allocations is largely a response to stingy intentions A handful of studies have tested these two models by comparing situations in which money is allocated under full control to situations in which money is allocated by an independent mechanism, such as computerized random assignment intention to make a stingy allocation, but rather because the allocator had no control over the allocation whatsoever. In the real world, we are required to judge individuals act with partial control over their actions, but still bring about unintended consequences—individuals like Jon and Matt, who fire their shots with a trembling hand. Why do we punish Matt severely for killing his rival, even though he intended no physical harm? Possibly, because the rival's death was caused by a behavior under Matt's partial control: the firing of the shot. We refer to this model of punishment as the “control” model: when people have at least partial control over the outcomes of their behavior, they are held accountable for those outcomes, whether specifically intended or not.Evidence from psychological research Testing the reward and punishment of monetary allocations under partial control requires an experimental paradigm where (1) an allocator can attempt to choose between different allocation amounts, (2) these choices exert partial, but imperfect control over the actual outcome of the allocation, and (3) the responder has the opportunity to respond to either the intended outcome, the actual outcome, or some combination. We therefore integrated intentions and outcomes into a single experimental condition, giving respondents the opportunity to weight the importance of each; we refer to this as a “trembling hand” game.In this game, Player 1 allocates $10 between himself and Player 2 by choosing to roll one of three die: A, B or C. If she chooses Die A and rolls a 1, 2, 3 or 4, she receives all $10 ; if she rolls a 5 the money is divided $5/$5 , and if she rolls a 6, Player 2 receives all the money . Thus, Die A has a high probability of a selfish allocation. By contrast, Die B has a high probability of a fair outcome, and Die C has a high probability of a generous outcome. When Player 1 chooses which die to roll, her intentions are transparent: she will choose the die with the highest probability of yielding the desired outcome . However, any die could result in any of the three possible outcomes. Thus, Player 1 has partial but imperfect control over the allocation of the $10.Player 2 is given the opportunity to respond to Player 1's allocation by attempting to increase or decrease Player 1's payoff by any amount up to $9. Both punishments and rewards are costless to Player 2, having no effect on his payoff. Focusing particularly on cases where Player 1's intended allocation does not match the outcome of her allocation , we can assess the degree to which Player 2 assigns punishment and reward on the basis of intent, outcome, or an interaction of both factors.In our experiment, as both players are aware, Player 2's attempt to reward or punish Player 1 is enforced by the experimenter in only 1/10 of games, selected at random. Thus, Player 1 was able to ‘get away’ with selfish allocations most of the time, without fear of punishment. This feature was introduced to convince Player 2 that ‘fair’ or ‘generous’ allocations by Player 1 were the product of genuinely prosocial motivations, rather than strategic attempts to maximize reciprocated payoffs. .What patterns of punishment and reward might we expect from the responder in the trembling hand game? The intention effect would produce responses determined exclusively by the choice of die , and not by accidental outcomes of the roll. By contrast, the distributional effect and the control effect would produce responses determined exclusively by the outcome of the roll. In order to isolate the distributional effect from the control effect, it is necessary to test a case in which allocations are made with no control. In our ‘no control’ condition, Player 1 was forced to roll a single die with equal chances of yielding all three allocations, leaving the allocation entirely to chance. The distributional model predicts that the responder will continue to add or subtract in this condition in order to equate outcomes, while the control model predicts that the responder will not reward or punish because the allocator lacks control. Thus, we can use the results of the no control condition to estimate the effect of outcomes due to the distributional model, and then test whether any additional effect of outcomes is evident in the trembling hand condition, as predicted uniquely by the control model.We also included a ‘full control’ condition, in which Player 1 directly stipulates the amount of the allocation, without any probabilistic error. This allows us to compare the response to fully intentional allocations in the trembling hand game to the response to fully intentional allocations in a more standard allocator/responder paradigm, testing whether our results can be generalized to this large body of research.Research was conducted with the approval of the Committee on the Use of Human Subjects in Research at Harvard University. Written consent was obtained from every subject prior to testing.Subjects were recruited through the online study pool at Harvard University, and comprised both students and local non-students. We report data from 30 subject pairs in each of the three conditions, totaling 180 subjects. Data from two Player 2s were excluded and replaced because matched Player 1 data was not recorded, due to experimenter error. The subjects ranged from ages 15 to 69.All subjects were guaranteed a baseline payment of $5 for their participation in the study, supplementing the money awarded during the game. Subjects interacted anonymously with two experimenters in separate rooms. In some instances where Player 2's response could not affect Player 1's payoff, data was collected in advance from Player 1.In the trembling hand condition, Player 2 was asked to commit to a response for each of the nine possible combinations of die chosen (Player 1's intention) and actual allocation (the outcome) before knowing which die Player 1 chose, or what she rolled. Similarly, in the control conditions Player 2 was asked to commit to a response in advance for each of the three possible outcomes. This response was described by the experimenter in terms of “additions and subtractions”, but never in terms of punishment and reward, in order to minimize demand characteristics of the game.After Player 1's behavior was known, the experimenter then selected the appropriate response from Player 2's advance commitment (applying it with 1/10 probability and failing to apply it with 9/10 probability). This method of data collection, known as the strategy method, greatly increased our statistical power and afforded the necessary sample size of responses to low-probability events . The strategy method has been widely employed in allocator-responder games, and two studies that directly compare the strategy method to sequential responses report no significant effect on responder behavior Player 2's response had no effect on Player 2's monetary outcome—that is, punishment and reward were costless to Player 2. This choice served to minimize the complexity of the game: it was not necessary to introduce an additional source of variability in Player 2's payoff, to specify a ratio of cost to reward/punishment, or to explain how costs would be calculated in the 9/10 chance that reward and punishment were not imposed by the experimenter. .Subjects were provided with a written description of the rules of the game. They then participated in a structured interview with the experimenter to ensure that they fully understood the rules of the game. Player 2 used a worksheet to specify his advanced commitments to all possible moves by Player 1. After each selecting their own move, but before learning of the other player's move and the outcome of the game, both players filled out a brief questionnaire. Written instructions, response sheets, debriefing forms and a script of the structured interview are provided as supplementary information see .In the trembling hand condition, subjects in the role of Player 2 readily responded by adding or subtracting from Player 1's payoff; not a single subject left Player 1's allocation unchanged for all nine possible moves (three possible intentions×three possible outcomes). Five subjects responded uniformly to the nine moves (each awarding $9 for every possible move). Among the remaining 25 subjects, 11 assigned punishments and rewards on the basis of the outcome alone—that is, they modified their responses according to the three different outcomes, but not according to the different intentions. No subjects assigned punishments and rewards on the basis of intent alone. 14 subjects assigned punishments and rewards on the basis of both outcome and intention. The statistical analyses below include data from all 30 subjects. Mean responses to each possible outcome of the experiment are charted in In order to analyze the responses of all 30 Player 2s to the full range of 9 possible moves by Player 1, we use a linear estimator (OLS) with robust standard errors.We include individual fixed effects since each subject responds to every possible move by Player 1 see . The dep1), “generous” outcome (β2), “stingy” intent (β3), “generous” intent (β4), the interaction between stingy outcome and stingy intent (β5), and an interaction between generous outcome and generous intent (β6). Thus, our regression treated fair intent coupled with fair outcome as the baseline, with the constant term expressing the mean response to this condition, and all other coefficients expressed mean deviations from this response. The dummy for each individual Player 2 is We use six independent variables to code for “stingy” outcome  and of generous outcome , compared to fair outcome. There was a significant effect of selfish intention  compared to fair intention, but no significant effect of generous intention  and no significant interaction between intention and outcome . We then re-estimated the model including only significant regressors , all of which remained significant at p<.01. The coefficients derived from the re-estimated model indicate that, on average, the response to stingy intentions is $1.47 less than to fair intentions, and $4.29 less for stingy outcomes compared to fair outcomes. The average response to generous outcomes is $2.54 greater than for fair outcomes.Regression analysis revealed a significant effect of stingy outcome , and the remainder chose to roll the fair die. Subjects in the role of Player 2 were asked to guess which die Player 1 would roll; 22 out of 30 guessed the selfish die, and the remainder guessed the fair die. Each Player 2 was asked to provide a written explanation of their guess regarding Player 1's choice of die, and 28/30 subjects explained their prediction in terms of the most probable outcome of the die. This suggests that nearly all subjects in the role of Player 2 understood the probabilistic weighting of the die, and the sense in which the choice of die reflected the intended allocation of Player 1.some sensitivity intentions—that is, whose responses were not uniform across all outcomes. There was a significant effect of stingy outcome  and of generous outcome . There was a significant effect of selfish intention , but no significant effect of generous intention , and a marginally significant interaction between stingy intent and stingy outcome  but no significant interaction between generous intention and generous outcome . Thus, even this subset of subjects in the role of Player 2, who clearly assessed intentions, exhibited substantial sensitivity to accidental outcomes.To further safeguard against the possibility that some subjects failed to consider intentions because they did not understand the probabilistic weighting of the die, we performed a selective analysis of the 14 subjects whose individual patterns of response exhibited at least In summary, the results of the trembling hand condition reveal that Player 2 weighted the actual outcome of Player 1's allocation more heavily than Player 1's intended allocation when deciding whether to add to or subtract from Player 1's payoff. Moreover, only selfish intentions—and not generous intentions—exerted a significant influence on Player 2's behavior (comparing each to fair intentions). The response to selfish outcomes was also substantially larger than the response to generous outcomes (comparing each to fair outcomes).1 = −2.17, t = −3.07, p<0.01. Player 2's response was also significantly higher for “generous” allocations compared to “fair” allocations: β2 = 2.53, t = 3.46, p<0.001. In order to test whether Player 2's response was affected by the allocation amount in the no-control condition, we used the following OLS regression model with robust standard errors:These coefficients provide an estimate of the size of the distributional effect on Player 2 responses. Can subjects' reliance on outcomes in the trembling hand condition be fully attributed to this distributional effect observed in the no-control condition? In order to provide a statistical test of this account, we conducted an OLS regression that pooled data from both the no control and trembling hand conditions. Specifically, we modeled the no control condition as a baseline, and used a dummy variable to identify the trembling hand condition. We included the three significant regressors from the analysis of the trembling hand data . Finally, we included two interaction dummy variables: the interaction between condition (no control vs. trembling hand) and stingy vs. fair outcome, and the interaction between condition and generous vs. fair outcome. These interaction terms are the key regressors of interest, testing whether subjects exhibit a sensitivity to outcome in the trembling hand condition that exceeds the baseline ‘distributional’ effect observed in the no control condition. As above, we used robust standard errors and individual fixed effects. This model can be represented as:4 = −2.12, t = −2.75, p<0.01). However, the interaction of condition with generous vs. fair outcome was not significant . . Thus, subjects were significantly more sensitive to stingy outcomes when behaviors were performed under partial control (in the trembling hand condition) compared to when behaviors were performed without control (in the no control condition). However, the response to generous outcomes performed under partial control was not significantly greater than the response to generous outcomes performed without control.In this pooled regression, the interaction of condition with stingy compared to fair outcome was significant , a “distributional effect” (Player 2 adds or subtracts money from Player 1 in order to equate their relative payoffs), and a “control effect” . Empirical estimates of these effects are summarized in In summary, we find that people punish accidental outcomes in allocator/responder games in a manner that cannot be explained by a pure distributional effect (i.e. aversion to inequitable outcomes). Below, we consider possible interpretations and implications of these results. First, however, we address several potential methodological concerns.One possible explanation for the reliance on outcomes in the trembling hand condition is that subjects did not understand the probabilistic weighting of the three dice and therefore failed to consider Player 1's intentions at all. However, over two thirds of subjects in the role of Player 2 guessed that Player 1 would choose the selfish die, one third guessed that Player 1 would choose the fair die, and none guessed that Player 1 would choose the generous die. Twenty-eight of 30 subjects specifically explained their prediction about Player 1's behavior by reference to the most probable outcome of the die roll. This evidence suggests that nearly all subjects understood the probabilistic weighting of the three dice to reveal Player 1's intention to bring about a particular outcome. Furthermore, accidental outcomes played a strong role even for the subset of Player 2s whose responses clearly indicated a sensitivity to intentions.A second possible explanation for the reliance on outcomes in the trembling hand condition is that the range of punishments available to Player 2 was restricted in the case of fair and generous allocations. When Player 1 was allocated $0, there was no money available for Player 2 to subtract from Player 1—similarly, when Player 1 was allocated $5, the maximum amount Player 2 could subtract was $5. There are reasons to doubt that this feature of the game is responsible for the dominance of outcomes in the trembling hand condition, however. First, in the trembling hand condition not a single subject subtracted the full $5 in response to a fair allocation, even when Player 1 had selfish intentions, suggesting that this value was not perceived as a limiting floor. Second, this “floor” on punishment was consistent across all conditions, and thus cannot explain the substantially different sensitivity to outcomes exhibited in the no control condition compared to the trembling hand condition. Third, in cases of selfish allocations, where the principal difference between conditions was observed, the full range of punishments and rewards was available to subjects.additional sensitivity to outcomes exhibited in the trembling hand condition, from which the “control effect” is inferred. Second, in our study reward and punishment were costless to Player 2. Although it is not obvious a priori why adding a cost to punishment and reward would selectively affect Player 2's sensitivity to intentions versus outcomes, or would selectively affect the trembling hand condition versus the no-control condition, these are important issues for further investigation.Two additional methodological features of the present study are worth noting, and point towards directions for future research. First, punishment and reward were imposed by the recipient of Player 1's allocation, rather than a neutral third-party observer. Might Player 2's responses be additionally sensitive to outcomes because he experienced those outcomes himself? Possibly, but this hypothesized effect should be consistent across the trembling hand condition and no control condition—Player 1 directly experiences negative outcomes in both. Therefore, it is unlikely to explain the We now turn to possible interpretations and implications of our findings, focusing on two issues in particular: (1) the distinction between the “intention effect” and “control effect” on Player 2 responses, and (2) responses to stingy versus fair behaviors, as compared to generous versus fair behaviors.As noted above, past studies have compared experimental conditions similar to our “no control” and “full control” conditions in order to assess the importance of allocator intentions in determining punitive responses Why might the factor of controllability matter? We conjecture that sensitivity to behavioral control has value in repeated social interactions. That is, it may be worthwhile to ‘teach a lesson’ to an accidental harm-doer if you are likely to meet her again, but only if she can exert at least partial control over the occurrence of future harms in similar circumstances. Thus, in the context of repeated play, teaching Player 1 that stingy outcomes will be punished only makes sense if Player 1 can influence the probability of a stingy, fair or generous outcome—as was the case the “trembling hand” condition, but not in the “no control” condition. Additionally, it might be worthwhile to focus on the outcomes of another's behavior rather than their apparent intentions to protect against the possibility of deception.Of course, neither of these considerations are justified in our experimental design: subjects interacted in an anonymous, one-shot game with no possibility of deception. However, psychological mechanisms underlying the control effect may have been shaped in contexts where repeated interactions and the possibility of deception were typical . These psychological mechanisms may be automatically deployed by subjects in our experimental setting even though, strictly speaking, they are not justified.In addition, it is notable that the intention effect and the control effect were only operative when comparing selfish to fair behaviors, and not when comparing fair to generous behaviors. Consistent with these findings, Offerman finds that punishment but not helping behavior is driven by the attribution of intentions Taking the results of the present study at face-value, why might subjects not have exhibited an intention effect or control effect for generous, versus fair, behavior? From a strictly quantitative, economic perspective, this distinction is hard to explain. The quantitative difference between stingy and fair behavior is just the same as between fair and generous behavior: $5 less for the allocator, and $5 more for the responder. However, psychological evidence suggests important qualitative distinctions in the way that people judge harmful versus helpful behaviors A prodigious family of studies in behavioral economics has investigated how people respond to selfish, fair and generous allocations of resources, with the consistent finding that subjects tend to diminish the payoffs of selfish allocators and increase the payoffs of generous allocators. Relatively few studies have directly investigated whether these responses are guided by a sensitivity to the actual distribution of resources or the intended distribution of resources, however. We test these factors in direct competition within a single experimental condition. The results suggest that responses to events under partial control are subject to a outcome bias: specifically, people punish ‘unlucky’ selfish allocations when performed under partial control. This effect can be dissociated from two different effects that we also confirm: an intention effect (people punish stingy intent) and a pure distributional effect . These findings open new directions for research into the psychological mechanisms that underlie our social behavior.Supplement S1(0.04 MB DOC)Click here for additional data file.