To this end, we exposed CXCR3−/− hepatocytes to CXCL10 or vehicle. Interestingly, CXCL10 also induced KU-60019 order apoptosis in these cells, as evidenced by increased levels of active caspase-3

and caspase-8 (Fig. 6A,B) as well as by prolonged Akt phosphorylation (Fig. 6C and Supporting Fig. 3B). To exclude a contamination of the recombinant CXCL10 by lipopolysaccharide, we preincubated CXCR3−/− hepatocytes with polymyxin B. In fact, this preparation did not change caspase-3 and Akt activation (Supporting Fig. 3C,D), demonstrating a CXCL10-specific effect on hepatocyte apoptosis. Importantly, in contrast to CXCL10, the related chemokine (CXCL9) did not affect hepatocyte apoptosis, as evidenced by measurement of caspase-3 activity (data not shown). Because CXCR3 is not involved in hepatocyte apoptosis, we became interested whether

EPZ-6438 ic50 an alternative receptor could trigger CXCL10-induced apoptosis in hepatocytes. Recently, Schulthess et al.24 identified TLR4 as a receptor for CXCL10 in pancreatic β-cells. First, we confirmed the expression of TLR4 on hepatocytes by PCR analysis (Supporting Fig. 4A). Next, we stimulated TLR4−/− hepatocytes with CXCL10 or vehicle. Indeed, we found no caspase-3 and caspase-8 activation (Fig. 6D,E). These results were confirmed by lack of Akt phosphorylation (Fig. 6F and Supporting Fig. 4B) subsequent to CXCL10 stimulation of these cells. Thus, activation of TLR4 signaling appears essential to trigger CXCL10-induced hepatocyte apoptosis. In light of these in vitro data, we hypothesized that systemic administration of CXCL10 might also induce liver cell apoptosis

in vivo. Indeed, a single injection of CXCL10 led to a low, but increased, number of TUNEL-positive liver cells, compared to vehicle treatment (Fig. 7A). The apoptotic response in CXCL10-treated animals was also reflected by increased caspase-3 and caspase-8 activity within livers of these animals (Fig. 7B and Supporting Fig. 4C). Moreover, treatment with CXCL10 increased AST serum levels (Fig. 7C) and reduced intrahepatic mRNA expression of the antiapoptotic factor, BCL-2 SDHB (Fig. 7D). Importantly, in this experimental setting, TLR4−/− mice were almost completely protected from the proapoptotic effects of CXCL10. In contrast to WT mice, treatment of TLR4−/− mice with CXCL10 neither resulted in augmented cell death (Fig. 7A) nor in caspase-3 or caspase-8 activation (Fig. 7B and Supporting Fig. 4C). In line with these results, lack of TLR4 also triggered no changes in AST and BCL-2 levels after CXCL10 challenge, compared to their vehicle-treated counterparts (Fig. 7C,D), identifying the CXCL10/TLR4 axis as an important chemokine-based apoptotic pathway within the murine liver in vivo. Here, we provide in vitro and in vivo evidence that CXCL10 exerts proapoptotic effects in hepatocytes through its noncognate receptor (TLR4).

We demonstrate that after treatment with sorafenib, SDF-1α increased the survival of HSCs and their alpha-smooth muscle actin and collagen selleck kinase inhibitor I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1+ myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis—despite persistently elevated

hypoxia—in part by reducing Gr-1+ myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. Conclusion: Blocking SDF-1α/CXCR4 or Gr-1+ myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment. (Hepatology 2014;59:1435-1447) “
“Das M, Garlick DS, Greiner DL, Davis RJ. The role of JNK in the development of hepatocellular carcinoma. Genes Dev 2011;25:634-645. (Reprinted with permission.) The c-Jun NH2-terminal kinase (JNK) signal-transduction pathway has been implicated in the growth of carcinogen-induced hepatocellular carcinoma. However, the mechanism that accounts for JNK-regulated tumor growth is unclear. Here, we demonstrate that compound deficiency

of the two ubiquitously expressed JNK isoforms DAPT mouse (JNK1 and JNK2) in hepatocytes does not prevent hepatocellular carcinoma development. Indeed, JNK deficiency in hepatocytes increased the tumor burden. In contrast, compound JNK deficiency in hepatocytes and nonparenchymal cells reduced both hepatic inflammation and tumorigenesis.

These data indicate that JNK plays a dual role in the development of hepatocellular carcinoma. JNK promotes an inflammatory hepatic environment that supports tumor development, but also functions in hepatocytes to reduce tumor development. The c-Jun NH2-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK)-signaling pathway, which are involved in regulating differentiation, proliferation, migration, immune reaction, and cell death in response to a diverse range of extracellular stimuli.1, 2 Targets PI-1840 of JNK signaling include members of the activating protein-1 (AP-1) transcription factor group, such as c-Jun and JunD,3 which play part of a regulatory network, suggesting that JNK plays a key role in growth regulation. Indeed, in vitro studies with fibroblasts lacking the JNK genes, JNK1 and JNK2, revealed major defects in cell proliferation.3 Furthermore, JNK1 appears to mediate the majority of proliferation, because JNK1-deficient cells display reduced c-Jun phosphorylation and decreased proliferation.

Patients with M1-MCA occlusion shown on CT angiography or by conventional angiography were chosen for the study. Patients who had associated intracranial internal carotid artery (ICA), anterior cerebral artery (ACA) or M2 were excluded. Patients without follow-up scans within 48 hours were excluded. We measured lengths of thrombotic clots depicted as arterial hyperdensities documented on admission (HMCAS) nonenhanced CT images with 5 mm slice width by placing CTA images side-by-side and confirming the site of M1 MCA occlusion. CTA source images or maximum intensity projection images were used to confirm the

location of the thrombus (Fig 1). Volumes of HMCAS was done using volume estimation Quantomo software[8] (Fig 2). Similar measurements were performed on the follow-up CT brain performed within the next 48 hours. Patients were treated in clinical routine with learn more intravenous and/or endovascular thrombolytic therapy (tPA and/or mechanical Ixazomib nmr thrombectomy) or conservatively at the discretion

of the attending stroke neurologist and according to current standards of care. Interobserver reliability of the thrombus length and volume was assessed from the interpretation of three independent stroke neurologists. Patients with HMCAS were divided into three groups based on lengths of HMCAS (Group 1. <10mm, Group 2. 10-20 mm, Group 3. >20 mm). Thrombus length as predictor of resolution of hyperdense sign at follow-up was assessed using receiver-operator Branched chain aminotransferase curve characteristics analysis and by trichotomizing thrombus length at the 25th and 75th percentiles. A total of 114 patients

with acute MCA stroke and hyperdense MCA sign, confirmed with CT angiography or conventional angiogram to be a M1-MCA occlusion were studied. Ten patients were excluded due to unavailable or uninterpretable follow-up scans; half (5/10) had symptomatic hemorrhage. Baseline characteristics are shown in Table 1. Good interrater reliability was shown among three different readers for length (intraclass correlation coefficient = .99), volume of hyperdense sign (intraclass correlation coefficient = .88), and ability to detect disappearance on follow-up NCCT brain (intraclass correlation coefficient = .72). Among 104 patients, 28 patients were treated conservatively and 76 with thrombolysis (41 intravenous tPA alone, 35 endovascular). Disappearance of the HMCAS on the follow-up scans was noted in 43 (41%) patients and was length dependent with thrombus length <10mm showing nearly 70% resolution (P < .001) and volume dependent (P < .002) (Table 1). In all treatment groups, shorter thrombus length and smaller volumes were associated with a greater probability of resolution at follow-up (Table 1). Thrombus length was a good predictor of resolution of thrombus at follow-up with a c-statistic of .77 (Fig 3).

00 ± 387.53, which were not significantly different (p > 0.05) from those of groups B (B1 = 993.20 ± 327.19, B2 = 1471.00 ± 311.68, B3 = 1408.40 ± 295.07), or group PD98059 molecular weight C (C1 = 1326.80 ± 785.30, C2 = 1322.20 ± 285.33, C3 = 1348.40 ± 527.21). SEM images of the fractured crowns showed that the origin of the fracture appeared to be located at the occlusal surfaces of the crowns, and the crack propagation tended to extend from the occlusal

surface towards the gingival margin. Conclusions: Implant abutment angulations of 0°, 15°, and 30° did not significantly (p > 0.05) influence the fracture resistance of overlaying Ceramage single crowns constructed with or without reinforcing fibers. The two types of fibers used for reinforcement (Connect and Interlig) had no effect (p > 0.05) on the fracture resistance of overlaying Ceramage single crowns. SCH772984 purchase “
“Purpose: To evaluate the effect of three commonly used bond primers on the bending strength of glass fibers and their bond strength to maxillofacial silicone elastomer after 360 hours of accelerated daylight aging. Materials and Methods: Eighty specimens were fabricated by embedding resin-impregnated

fiber bundles (1.5-mm diameter, 20-mm long) into maxillofacial silicone elastomer M511 (Cosmesil). Twenty fiber bundles served as control and did not receive surface treatment with primers, whereas the remaining 60 fibers were treated with three primers (n = 20): G611 (Principality Medical), A-304 (Factor II), and A-330-Gold (Factor II). Forty specimens were dry stored at HAS1 room temperature (23 ± 1°C) for 24 hours, and the remaining specimens were aged using an environmental chamber under accelerated exposure to artificial daylight for 360 hours. The aging cycle

included continuous exposure to quartz-filtered visible daylight (irradiance 760 W/m2) under an alternating weathering cycle (wet for 18 minutes, dry for 102 minutes). Pull-out tests were performed to evaluate bond strength between fiber bundles and silicone using a universal testing machine at 1 mm/min crosshead speed. A 3-point bending test was performed to evaluate the bending strength of the fiber bundles. One-way Analysis of Variance (ANOVA), Bonferroni post hoc test, and an independent t-test were carried out to detect statistical significances (p < 0.05). Results: Mean (SD) values of maximum pull-out forces (N) before aging for groups: no primer, G611, A-304, A-330-G were: 13.63 (7.45), 20.44 (2.99), 22.06 (6.69), and 57.91 (10.15), respectively. All primers increased bond strength in comparison to control specimens (p < 0.05). Primer A-330-G showed the greatest increase among all primers (p < 0.05); however, bonding degraded after aging (p < 0.05), and pull-out forces were 13.58 (2.61), 6.17 (2.89), 6.95 (2.61), and 11.72 (3.03). Maximum bending strengths of fiber bundles at baseline increased after treatment with primers and light aging in comparison with control specimens (p < 0.05), and were in the range of 917.72 to 1095.25 and 1124.

Real-time ECAR (as a measure of lactic acid production) and accumulated lactate levels of hepatocytes were measured as

previously established in our laboratory20, 21 with alterations detailed in the Supporting Materials and Methods. These assays were performed as described.11, 17 The details are described in the Supporting Materials and Methods. For statistical analyses, the two-tailed Selleck LY2109761 Student’s t-test was used. Significance was defined as P < 0.05. Two murine models of HCC were established and examined in this study. First, de novo HCC formation was induced in C57/BL6 WT and Cd39-null mice. Although all mice developed liver cancers, the tumor burden in Cd39-null mice was markedly increased when compared to WT controls (Fig. 1A-C). Incidence of tumors greater than 5 mm in diameter was 69% (9/13) in null livers and 6.7% (1/15) in WT livers (Supporting Table S1A, P < 0.001). Using a morphological classification of “mouse liver tumors” (MLT),22 30% of null tumors ABT-199 solubility dmso had high-grade malignancy (MLT type III and IV) (Fig. 1C, lower), whereas tumors arising in WT livers were of low-grade malignancy (chiefly MLT type I and II) (Fig. 1C, upper). We next studied HCC

arising spontaneously and in an autochthonous manner in mice. The incidence of liver neoplasia in aged (18-24 months old) Cd39-null mice (70%, 19/27) was significantly higher than that of WT mice (29%, 2/7) (Table S1B; P = 0.04; Fig. 1D,E). Accordingly, the mouse tumor biology database at Jackson Laboratories indicates that the frequency of spontaneous liver tumor varies from 8.8% to 30% in aged WT C57/BL6J mice (tumor.informatics.jax.org). Phospholipase D1 Hematoxylin-eosin (H&E) staining of liver tumor sections from Cd39-null mice confirmed that tumors were of the hepatocellular type with curiously marked hypervascularity

(Fig. 1E). Cd39-null mice developed spontaneous hepatic necrosis concomitantly accompanied by adjacent hepatocellular dysplasia at a young age (as early as 5 weeks old) (Fig. 2A). These mutant mice, in comparison to WT controls, exhibited significantly increased liver-to-body weight ratios, (Fig. 2B; P < 0.0001). Next we used RT-PCR to characterize the complement of P2 receptors expressed by WT hepatocytes. Mouse hepatocytes expressed messenger RNA (mRNA) transcripts for P2X4, P2X5, P2X7 (weaker), P2Y1, P2Y2, and P2Y12-14 (Supporting Fig. S1). We then examined the impact of Cd39 deletion on hepatocyte proliferation. First, Cd39-null cells exhibited a heightened baseline proliferation rate compared to WT cells (Fig. 2C). This occurred regardless of the culture conditions and appeared indicative of prior “set-points” in null cells (considered as possible “preprogramming”). Second, insofar as ATP has differential effects on cell proliferation at different concentrations, we evaluated the responses of WT cells to increasing concentrations of ATP.

Further studies which precise and determine the molecular mechanism involved in the regulation of TH development and drug therapy in order to design therapeutic options that become applicable to improve the prognostic in these patients. Disclosures: The following people have nothing to disclose: Jorge A. López-Velázquez, Varenka J. Barbero-Becerra,

Vicente Sánchez- Valle, Ylse Gutiérrez-Grobe, Norberto C. Chavez-Tapia, José M. Ramírez-Jaramillo, Fredy Chablé-Montero, Misael N. Uribe-Esquivel, Nahum Méndez-Sanchéz “
“We used concanavalin A (Con A)-induced liver injury to study the role of galectin-3 (Gal-3) in the induction of inflammatory BIBW2992 concentration pathology and hepatocellular damage. We tested susceptibility to Con A–induced hepatitis in galectin-3-deficient (Gal-3−/−) mice and analyzed the effects of pretreatment with a selective inhibitor of Gal-3 (TD139) in wild-type (WT) C57BL/6 mice, as evaluated by a liver enzyme test, quantitative histology, mononuclear cell (MNC) infiltration, cytokine production, intracellular staining of immune

cells, check details and percentage of apoptotic MNCs in the liver. Gal-3−/− mice were less sensitive to Con A–induced hepatitis and had a significantly lower number of activated lymphoid and dendritic cells (DCs) in the liver. The level of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin (IL)-17 and -4 in the tuclazepam sera and the number of TNFα-, IFNγ-, and IL-17- and -4-producing cluster of differentiation (CD)4+ cells as well as IL-12-producing CD11c+ DCs were lower, whereas the number of IL-10-producing CD4+ T cells and F4/80+ macrophages were significantly higher in livers of Gal-3−/− mice. Significantly higher percentages of late apoptotic Annexin V+ propidium-idodide+ liver-infiltrating MNCs and splenocytes were observed in Gal-3−/− mice, compared to WT mice. Pretreatment of WT C57BL/6 mice with TD139 led to the attenuation of liver injury and milder infiltration of IFNγ- and IL-17- and -4-producing CD4+ T cells, as well as an increase in the total number of IL-10-producing CD4+ T cells

and F4/80+ CD206+ alternatively activated macrophages and prevented the apoptosis of liver-infiltrating MNCs. Conclusions: Gal-3 plays an important proinflammatory role in Con A–induced hepatitis by promoting the activation of T lymphocytes and natural killer T cells, maturation of DCs, secretion of proinflammatory cytokines, down-regulation of M2 macrophage polarization, and apoptosis of MNCs in the liver. (HEPATOLOGY 2012;55:1954–1964) Concanavalin A (Con A)-induced liver injury is a well-established murine model of T-cell-mediated hepatitis. Intravenous (IV) injection of Con A induces acute liver injury and systemic immune activation in mice that resembles the pathology of immune-mediated hepatitis in humans.1 Activated T cells have a critical role in Con A–induced liver damage.

Further studies which precise and determine the molecular mechanism involved in the regulation of TH development and drug therapy in order to design therapeutic options that become applicable to improve the prognostic in these patients. Disclosures: The following people have nothing to disclose: Jorge A. López-Velázquez, Varenka J. Barbero-Becerra,

Vicente Sánchez- Valle, Ylse Gutiérrez-Grobe, Norberto C. Chavez-Tapia, José M. Ramírez-Jaramillo, Fredy Chablé-Montero, Misael N. Uribe-Esquivel, Nahum Méndez-Sanchéz “
“We used concanavalin A (Con A)-induced liver injury to study the role of galectin-3 (Gal-3) in the induction of inflammatory Barasertib pathology and hepatocellular damage. We tested susceptibility to Con A–induced hepatitis in galectin-3-deficient (Gal-3−/−) mice and analyzed the effects of pretreatment with a selective inhibitor of Gal-3 (TD139) in wild-type (WT) C57BL/6 mice, as evaluated by a liver enzyme test, quantitative histology, mononuclear cell (MNC) infiltration, cytokine production, intracellular staining of immune

cells, PI3K inhibitor and percentage of apoptotic MNCs in the liver. Gal-3−/− mice were less sensitive to Con A–induced hepatitis and had a significantly lower number of activated lymphoid and dendritic cells (DCs) in the liver. The level of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), and interleukin (IL)-17 and -4 in the ifenprodil sera and the number of TNFα-, IFNγ-, and IL-17- and -4-producing cluster of differentiation (CD)4+ cells as well as IL-12-producing CD11c+ DCs were lower, whereas the number of IL-10-producing CD4+ T cells and F4/80+ macrophages were significantly higher in livers of Gal-3−/− mice. Significantly higher percentages of late apoptotic Annexin V+ propidium-idodide+ liver-infiltrating MNCs and splenocytes were observed in Gal-3−/− mice, compared to WT mice. Pretreatment of WT C57BL/6 mice with TD139 led to the attenuation of liver injury and milder infiltration of IFNγ- and IL-17- and -4-producing CD4+ T cells, as well as an increase in the total number of IL-10-producing CD4+ T cells

and F4/80+ CD206+ alternatively activated macrophages and prevented the apoptosis of liver-infiltrating MNCs. Conclusions: Gal-3 plays an important proinflammatory role in Con A–induced hepatitis by promoting the activation of T lymphocytes and natural killer T cells, maturation of DCs, secretion of proinflammatory cytokines, down-regulation of M2 macrophage polarization, and apoptosis of MNCs in the liver. (HEPATOLOGY 2012;55:1954–1964) Concanavalin A (Con A)-induced liver injury is a well-established murine model of T-cell-mediated hepatitis. Intravenous (IV) injection of Con A induces acute liver injury and systemic immune activation in mice that resembles the pathology of immune-mediated hepatitis in humans.1 Activated T cells have a critical role in Con A–induced liver damage.

5B). These findings support the possibility that PBGs and the epithelial network may serve as a reservoir of epithelial cells either to differentiate into or repopulate the mucosa during the regenerative response of the bile duct unit after an injury. To directly examine the proliferative

potential of PBGs, we quantified cellular proliferation by BrdU uptake in two models of cholestasis. First, we counted the number of BrdU+/CK19+ cells after IP administration of RRV into newborn mice. Infection of RRV soon after birth is a well-established injury model of the biliary epithelium and shares phenotypic features of human biliary atresia, the most common cause of chronic cholestatic liver disease in children.[19] Olaparib solubility dmso We found an unexpectedly high baseline number of BrdU+ cells in age-matched controls (receiving

saline rather than RRV), both in CK-19+ cells of PBGs and the peribiliary epithelium and in CK-negative cells in the submucosal compartment of the duct along the entire length of EHBDs (Fig. 7). After RRV, the percentage of BrdU+/CK19+ epithelial cells did not change from controls (12% ± 3% versus 12% ± 2%; P = 0.8), neither did PBG cells (7% ± 3% versus 10% ± 5%; P = 0.25) at day 3, but increased in both the epithelium (18% ± 6% versus 8% ± 3%; P = 0.009) and PBGs (20% ± 8% versus 7% ± 6%; P = 0.01) Volasertib molecular weight at day 4. These findings are also reproduced when the results of BrdU+ cells are expressed for all epithelial cells together (mucosa plus PBGs; Fig. 7). We were unable to quantify BrdU+/CK19+ cells reproducibly beyond day 4 because of the widespread epithelial loss that typically begins on day 5 after RRV (data not shown). To investigate whether the high baseline BrdU uptake in control newborn

mice was the result of a normal growth phase of postnatal development, we compared the BrdU uptake by CK19+ cells in ducts of unchallenged neonatal and adult mice. We found that baseline BrdU uptake decreased in adult Phosphatidylinositol diacylglycerol-lyase mice in both epithelial cells (10% ± 3% neonate versus 1% ± 1% adult; P < 0.0001) and PBG cells (9% ± 6% neonate versus 1% ± 1% adult; P = 0.0004; Supporting Fig. 3). To assess cellular proliferation in adult mice, we quantified BrdU+/CK19+ cells after surgical ligation of the CBD. Though the percentage of BrdU+/CK19+ cells remained low at baseline levels at 6 and 12 hours after BDL, BrdU+/CK19+ cells of PBGs and the epithelium underwent a dramatic surge to 39% in PBGs and 33% in the epithelium at 24 hours (P = 0.002 and P < 0.001, respectively, when compared to sham operation) or 39% for ligation and 1% for sham when analyzing all cell types collectively for the entire duct (P < 0.001; Fig 8).

Travel destinations were grouped as follows: Africa, Asia, Europe, Latin America/Caribbean, and the USA. A “Multiple/other” category was used for cases that traveled to more than one destination group or that traveled to other parts of the world (eg, Australia). Raf inhibitor The data variables used for the analysis included age; gender; onset date; disease; symptoms

(abdominal pain, abdominal bloating, chills, dehydration, diarrhea, bloody diarrhea, greasy diarrhea, chronic diarrhea, dizziness, fatigue, fever, headache, loss of appetite, weight loss, muscle soreness, nausea, vomiting, and weakness, depending on the disease); hospitalization; recovery date; travel destination; resort accommodation; mode of transportation; and departure and return dates. Where data were available, disease duration was computed as recovery

minus onset dates, and travel duration as return minus departure dates. The TRC were broken down by illness and then further described by hospitalization, symptoms, disease duration, age, gender, travel duration, travel destination, mode of transportation, and resort accommodation. When available, onset, travel departure, and return dates were plotted by month. For each of these three dates, a Poisson regression model of the monthly count was used to test the differences Wnt inhibitor between both years and months at the same time. The year was defined as a successive 12-month period starting from June and ending in May the following year; eg, 2005 to 2006 encompassed June 2005 to May 2006 click here inclusively.

Multiple correspondence analysis (MCA) was used to probe the existence of travelers’ subgroups within the data and, if these existed, to explore any association between these subgroups and illness. MCA is a descriptive statistical technique designed to explore and visualize the relationships between three or more categorical variables (see Appendix 1 for more details).23 MCA was conducted on age (<5, 5–14, 15–24, 25–39, 40–59, and 60+ y), gender, travel duration (<8, 8–28, and 29+ d), travel destination, and accommodation in resort. In addition, disease was the supplementary variable used to assess any relationship between the subgroups identified and the illnesses. Finally, TRC and DC were overall described by, and tested for differences in, gender, age, illness distribution, and hospitalization. For every disease with at least 30 TRC, TRC and DC were compared for gender, age, hospitalization, disease duration, the various symptoms relevant to the disease, and the delay of reporting (difference between onset and report dates). Campylobacter species and Salmonella serotypes were also compared. The Kruskal–Wallis test was used for continuous data and the bilateral Fisher exact test or the Chi-square test for categorical data. The p-value threshold was set at 0.01 for all statistical tests.

Results in Fig. 4b show that in the absence of a plasmid encoding MalI, as expected, these insertions have but small effects on MelR-dependent repression of the melR promoter. However, with plasmid pACYC-malI, which encodes MalI, there is a clear small significant relief of repression with the TB334I-1 and TB334I-2 selleck products fragments carrying one or two MalI operator elements, but no relief with the control TB31, TB33 or TB334 fragments. The expression of many transcription repressors is autoregulated by repression (Browning & Busby, 2004). Kahramanoglou et al. (2006) proposed a two-state model for MelR in which, in the absence of its ligand, melibiose, MelR acts as an autorepressor of

its own production by repressing the melR promoter. Samarasinghe et al. (2008) showed that this repression was due to the formation of a nucleoprotein complex involving four MelR subunits. Here, we report that it is possible to construct simpler derivatives of the melR promoter where only two MelR targets are needed for efficient repression (Fig. 1), and there are clear parallels between this and AraC-dependent repression at the araC–araBAD intergenic region, where repression is dependent on interaction between two AraC subunits bound to targets separated by 210 base

pairs (Schleif, 2010). An explanation for the observed repression with the TB33 fragment is that MelR subunits bound at the upstream and downstream DNA targets interact and result in loop formation, as for AraC. However, there appears to be more flexibility in how the Proteasome inhibitor two DNA sites for MelR Non-specific serine/threonine protein kinase can be juxtaposed, compared to AraC. Hence, AraC-dependent repression is disrupted by +5 base pair insertions (Lee & Schleif, 1989), whilst MelR-dependent repression is not (Fig. 2). The simplest explanation for this would be that the linker joining the N- and C-terminal domains is more flexible in MelR than in AraC. This flexibility is underscored by the experiment in Fig. 4 where MalI binding failed to completely disrupt repression. This experiment also argues that the mechanism of MelR-dependent repression with TB33 is different to the mechanism operating at the more complex

wild type melibiose operon regulatory region in TB22 (Fig. 1), where repression depends on the formation of a nucleoprotein complex. In the new constructs described here, efficient repression of the melR promoter by MelR requires interaction between MelR bound immediately adjacent to the transcript start and upstream-bound MelR, and this can be subverted by the insertion of a supplementary DNA site for MelR (Fig. 3). Hence, efficient repression results from two, but not from three, DNA sites for MelR. Our experiments underline the diversity of protein–DNA architectures that can be responsible for transcription repression. This work was supported by the UK BBSRC with a project grant to S.J.W.B. and a summer studentship to D.D.