The anthrax toxin receptors, ANTXR1 and ANTXR2, act as molecular clamps to prevent the protective antigen (PA) toxin subunit from forming pores until exposure to low pH. PA forms pores at pH ∼6.0 or below when it is bound to ANTXR1, but only at pH ∼5.0 or below when it is bound to ANTXR2. Here, structure-based mutagenesis was used to identify non-conserved ANTXR2 residues responsible for this striking 1.0 pH unit difference in pH threshold. Residues conserved between ANTXR2 and ANTXR1 that influence the ANTXR2-associated pH threshold of pore formation were also identified. All of these residues contact either PA domain 2 or the neighboring edge of PA domain 4. These results provide genetic evidence for receptor release of these regions of PA as being necessary for the protein rearrangements that accompany anthrax toxin pore formation. Bacillus anthracis that is thought to cause many of the symptoms observed in anthrax disease. The toxin consists of a single receptor-binding subunit, protective antigen (PA), and two catalytic subunits: lethal factor (LF), a metalloprotease that cleaves and inactivates MKKs 83), is cleaved by cell-surface furin into a 63 kD form, (PA63), which goes on to heptamerize into a ring, or pre-pore 63, which exists as monomers or multimers in the blood of infected animals Anthrax toxin is a major virulence factor of 20 removal and PA63 oligomerization, domain 1 binds EF and/or LF; domain 2 is involved in pore formation, EF/LF translocation, PA63 oligomerization, and receptor binding; domain 3 in PA63 oligomerization; and domain 4 in receptor binding 2) than typical α-integrin-ligand binding (∼1300 Å2) D = 170 pM) D = 130 nM) PA is comprised of four protein domains with different functions: following PA63 forms pores at neutral pH, while low pH is required for pore formation when the toxin subunit is bound to receptor. Thus it has been proposed that each receptor acts as a molecular clamp to restrict pore formation until the complex encounters an acidic endosomal compartment where low pH induces structural changes in the PA heptameric pre-pore leading to pore formation. In this model, receptor contact with the base (residues 340–348) of the PA domain 2 membrane insertion loop  restrains pore formation until protonation of PA and/or ANTXR2 residues loosens this interaction to allow domain 2 to undergo a large conformational change and form an extended β-barrel pore 4Cl) treatment, which raises endosomal pH In the absence of receptor, PAPrior to this report it was not known which receptor determinants are responsible for dictating the receptor-specific pH thresholds of anthrax toxin pore formation. Here we identify these determinants of ANTXR2 and show that they are involved with binding PA domain 2 and the neighboring edge of PA domain 4.489-EGFP) 6 CHO-R1.1 cells were transfected with 1 or 3 µg plasmid DNA ANTXR1- or ANTXR2-EGFP and 5 or 3 µg pBSII KS(-) carrier DNA using Lipofectamine2000 (Invitrogen). All constructs were confirmed by DNA sequencing. WT PA protein was isolated from the periplasm of E. coliN-DTA  was produced as previously described QuikChange mutagenesis (Stratagene) was performed on ANTXR2-EGFP  to determine the relative EGFP cell viability. WT ANXTR2-expressing cells are normally susceptible to an NH4Cl block to intoxication, while ANTXR1-expressing cells are resistant At 24 hours post-transfection, triplicate samples of ∼7×106 cells were pre-incubated in supplemented F12 media with dGAB cocktail  for 45 minutes at 37°C. Cells were then bound with 10−8 M PA63 (List Labs) in the presence of dGAB for 2 hours at 4°C. Pore formation was induced by incubation for 10 minutes at 37°C with PBS buffered with Tris pH 6.8, Tris pH 6.5, MES pH 6.0, MES pH 5.6, MES pH 5.4 and Sodium Acetate pH 5.0 or alternatively MES pH 6.0, MES pH 5.8, MES pH 5.6, MES pH 5.4, Sodium Acetate pH 5.2, and Sodium Acetate pH 5.0 respectively . Samples were analyzed as previously described At 48 hours post-transfection, ∼2×10We reasoned that non-conserved amino acid residues located at the PA-binding interface were likely to be responsible, at least in part, for the distinct receptor-specific pH thresholds of anthrax toxin pore formation. There are eight such residues in ANTXR2 , which correspond to Leu, His, Arg, Leu, Gly, His, Glu, and Asp, respectively, in ANTXR1 4Cl treatment, resistant to that treatment . By contrast, the G153E and L154D amino acid substitutions weakened the ANTXR2 molecular clamp, allowing PA pore formation at a pH value that was 0.6 units higher than that seen with wild-type ANTXR2 . Indeed,reatment .Among the mutations tested in PA-domain 4 contact residues all had almost no effect on the pH of pore formation (data not shown) with the exception of the Q88R mutation that weakened the molecular clamp, allowing PA pore formation at pH 5.4 . These d4Cl  shift in threshold of PA pore formation . Consist4Cl . TherefoTo test whether additional β4-α4 loop residues are required to convert ANTXR1 more fully into an ANTXR2-like receptor, ANTXR1 residues 154–159 were replaced with the corresponding amino acids of ANTXR2 . The ANTTo test the additional requirement for ANTXR2 residue Q88, this residue was substituted in the ANTXR1 154–159 protein, generating the ANTXR1 R88Q/154–159 protein. This additional change fully converted ANTXR1 154–159 into an ANTXR2-like receptor that restricted pore formation to pH 5.0 or below . These rAt the PA binding interface, ANTXR2 amino acid residues K51, Y119, H121, E122 and Y158 are absolutely conserved with ANTXR1, and residues S87, R111 and E117 are residues are highly similar  Among the PA domain 2-contact residues, the most important was Y119: changing this residue either to an Ala or Phe significantly weakened the molecular clamp allowing some toxin pore formation at pH 5.6–6.0 . Also, mIn this report, receptor determinants that influence the strength of the ANTXR2 clamp which acts to restrict PA pore formation at neutral pH have been defined. Residues G153 and L154, located on the β4-α4 loop of the receptor I domain which interacts with the flexible 340–380 loop of PA domain 2 Residue G153 permits a tight turn in the β4-α4 loop and L154 participates in hydrophobic contacts with the side-chains of PA residues L340 and T349 . The seqAmong the conserved receptor residues, the key regulator of acid pH-dependent pore formation was Y119 of ANTXR2, which occupies a planar position between domains 2 and 4 of PA  and appeSubstituting other conserved residues E117, H121, E122, and Y158 each gave rise to similar 0.2–0.4 unit changes in the pH threshold of toxin pore formation . E117 isd of the toxin-wild-type receptor complex and a more sensitive FRET-based assay involving the use of AF488 (donor fluorophore)-labeled PA and AF546 (acceptor fluorophore)-labeled ANTXR2 I domain had to be used in that study. Consequently, previous measurements obtained by binding PA to cell surfaces for 1 hour at 4°C It remains to be determined if the residues implicated in this study affect the affinity of PA-ANTXR2 binding at neutral pH; the long half-life of the complex (15 hours) The ANTXR2 residues that form the functional core of the receptor clamp, as well as those that dictate the receptor-specific pH thresholds of pore formation, map to the region that contacts PA domain 2 and the neighboring edge of PA domain 4 . These dTable S1Mutagenesis Primers(0.07 MB DOC)Click here for additional data file.


E. coli periplasm, or into the extracellular fluid has many advantages; therefore naturally occurring signal peptides are selected for proteins translocation. The aim of this study was the production in high yields of a recombinant pectin lyase that is efficiently secreted and the encapsulation of transformed E. coli cells for pectin degradation in a biotechnological process.Production of heterologous proteins in the Bacillus subtilis α-amylase's signal peptide was fused to the N-terminal of an heterologously expressed pectin lyase in E. coli BL21 [DE3]. Thus pectin lyase secretion was achieved into the extracellular growth medium. E. coli cells harboring the recombinant plasmid heterologously express pectin lyase to around 22% of the total cellular proteins, as it was estimated by SDS-PAGE and image analysis. IPTG induces the heterologously expressed enzyme, which is initially distributed extracellularly (7 hour) and later on at the periplasmic (9 hours) or cytosolic fraction (20 hours). No pectin lyase activity was found in the membranes fraction and in the inclusion bodies. Encapsulation of the recombinant strains of E. coli in alginate or alginate/silica beads 1:5 showed that pectin lyase could degrade effectively its substrate, for at least ten operational cycles.The nucleotide sequence of E. coli BL21 [DE3] was achieved in this study. For this purpose the signal peptide of α-amylase from B. subtilis was fused to the N-terminal domain of pectin lyase. Encapsulated E. coli BL21 [DE3] cells harboring pET29c/exPNL were used successfully for pectin degradation up to ten operational cycles indicating that under special conditions this might have biotechnological implementations.Secretion of an heterologously overexpressed pectin lyase in  Pectin lyase PNL, EC 4.2.2.10) belongs to polysaccharide lyase family 1 and cleaves α-1,4 bonds between galacturonic acid residues of pectin. The mode of PNL action is trans-elimination . The enz.2.2.10 bpnl) from Pseudomonas marginalis N6301 in E. coli BL21 [DE3] has previously been reported  competent cells were prepared according to Sambrook et al. [expnl.We have recently reported that on sites . In thissequence  to secreE. coli BL21 [DE3] with the constructed plasmid pET29c/expnl were grown at 37°C, in LB medium containing kanamycin (50 μg/ml) with vigorous agitation. When cells reached an optical density of 0.6 at 600 nm, IPTG (1 mM) was added and the cultures were incubated at 24°C, for 3 hours. B. subtilis was grown at 37°C, in LB medium with vigorous agitation.The recombinant strains of E. coli BL21 [DE3] harboring pET29c/expnl, grown as described above, were harvested by centrifugation at 3,000 × g for 15 min at 4°C. The cell pellet was washed twice with PBS and suspended in 5 × ml of 0.5 M sucrose, 250 mM EDTA and 2 mg/ml lysozyme. The suspension was incubated on ice for 30 min and centrifuged at 20,000 × g for 15 min. The supernatant was kept/saved as the periplasmic fraction. The remaining pellet was suspended in 50 mM Tris-HCI (pH 7.5), 2.5 mM EDTA and then sonicated . After addition of MgSO4 to a final concentration 40 mM, the mixture was centrifuged at 100,000 × g for 40 min. The 100,000 × g supernatant was kept as the cytoplasmic fraction and the 100,000 × g pellet as the membrane fraction with the inclusion bodies. The pellet 100,000 × g was suspended in 1 ml of 50 mM Tris-HCl buffer pH 7.5 [Recombinant strains of r pH 7.5 .-1cm-1.PNL activity was determined spectrophotometrically by monitoring the increase in absorbance at 235 nm of a solution containing 0.1% w/v pectin  in 50 mM citrate/phosphate buffer pH 6.5 at 37°C. The increase in absorbance was measured after 20 minutes of incubation. One unit of PNL activity was defined as the nmoles of unsaturated oligogalacturonides produced per minute, under the above-specified condition . SpecifiSDS electrophoresis was performed in 10% w/v polyacrylamide gels containing 0.1% w/v SDS as described by Laemmli . Protein2, 2.5 mM β-MSH, 0.1% w/v Tween 20  containing 8 M urea, or in 50 mM Tris-HCl pH 8.0, 6 M guanidine hydrochloride, 1 mM DTT to a final concentration of 1 mg/ml. Solubilization of inclusion bodies was proceed at room temperature for 1 hour. In the first case the mixture was centrifuged at 100,000 × g for 20 min at 4°C and then the 100,000 × g supernate was dialyzed gradually against DR buffer containing lower concentrations of urea  and finally overnight just in DR buffer. In the second case the mixture was diluted tenfold with the same buffer and dialyzed overnight against a 100 fold volume of 50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT and 5% glycerol. Dialysis was followed by centrifugation at 50,000 × g for 30 min at 4°C and enzyme activity was determined. In all steps 1 mM PMSF was added.The 100,000 × g pellet containing the inclusion bodies was either dissolved in 10 fold volume of 50 mM Tris-HCl pH 8.0, 5 mM NaCl, 5 mM MgCl2 at a volumetric ratio of 1:50. A spontaneous cross-linking reaction produced spherical hydrogel beads of barium alginate, which remained for 30 minutes at 4°C in order to stabilize the formed network. After stabilization, the beads were washed with 0.9% v/v NaCl (twice) and used immediately. All solutions that were used are sterile.After 2 hours of induction with IPTG, cells were harvested at 3000 × g for 20 min and mixed with sterile water (300 mg cells/ml). Sodium alginate solution (2% v/v) was mixed with the above cell suspension at a volumetric ratio of 9:1 and the mixture was dropped with an insulin syringe into a gently stirred 1% w/v solution of BaCl2 at a final concentration of 0.005% v/v in order to preserve cell viability and network stability. At the end of each cycle, beads were washed with 0.9% v/v NaCl (twice) and placed with fresh LB solution at room temperature under gentle shaking for 1 h. This procedure was repeated after each operational cycle.Pectin hydrolysis was performed in conical flasks at 37°C under gentle stirring and each operational cycle lasts for 1 h. The substrate used was the same as for enzyme assay with the addition of LB solution  and BaClPNL, pectin lyase; exPNL, extracellular pectin lyase; IPTG, isopropylthiogalactosideAll authors contributed equally in this paper.


Bcl-3 as an oncoprotein is overexpressed in nasopharyngeal carcinoma (NPC). Nedd4 binding protein 2 (N4BP2), which is located in the NPC susceptibility locus, is a Bcl-3 binding protein. This study is aimed to explore the association between N4BP2 genetic polymorphism and the risk of NPC.We performed a hospital-based case-control study, including 531 sporadic NPC and 480 cancer-free control subjects from southern China. PCR-sequencing was carried out on Exons, promoter region and nearby introns of the N4BP2 gene. The expression pattern of N4BP2 and Bcl-3 was also analyzed.We observed a statistically significant difference in haplotype blocks ATTA and GTTG between cases and controls. In addition, three novel SNPs were identified, two of which were in exons , and one was in the intron6 . Moreover, N4BP2 was at higher levels in a majority of tumor tissues examined, relative to paired normal tissues.These data suggest that haplotype blocks ATTA and GTTG of N4BP2 is correlation with the risk of sporadic nasopharyngeal carcinoma in the Southern Chinese population and N4BP2 has a potential role in the development of NPC. AY267013) is a Bcl-3 binding protein, N4BP2 protein contains a polynucleotide kinase domain (PNK) at the N-terminus and a Small MutS Related (Smr) domain with nicking endonuclease activity near C-terminus [Bcl-3 was originally identified as expressed in chronic B cell lymphocytic leukemia . Many ceterminus . Since NN4BP2 gene.NPC is an epithelial tumor with an exceptionally high incidence in southern China, particularly in the Guangdong province ,7. EtiolN4BP2 gene lead to a predisposition to developing NPC. We further hypothesized that the N4BP2 gene plays a role in tumorigenesis. To address these hypotheses, we examined N4BP2 haplotypes among NPC patients from southern China. We also examined mRNA levels of Bcl-3 and N4BP2 in NPC cell lines and tissues.We thus hypothesized that SNPs or other variation in the A total of 531 sporadic NPC patients and 480 unrelated age, sex-, and geographically-matched healthy individuals from southern China were used for our case-control study. NPC patients were recruited from Sun Yat-sen University Cancer Center and the presence of differentiated non-keratinizing NPC or undifferentiated NPC was confirmed by histological analysis. Control subjects were recruited from the People's Hospital of Guangdong Province. The characteristics of cases and controls are shown in Table Genomic DNA was extracted from 5–10 ml peripheral blood using the QIAamp DNA Blood Midi kit .Between 250 and 400 bp of sequence surrounding SNPs sites were submitted to the primer design program . The priLong-distance PCR was performed in a total volume of 15 μl containing 200 ng of genomic DNA, 1.5 μl 10× Buffer, 50 μM dNTPs, 0.3 μM each primer, and 1U Taq DNA polymerase. Samples were amplified with each pair of primers described above as follows: 94°C for 3 min, 10 cycles of 94°C for 30 s, 63°C for 1 min, and 72°C for 1 min; 25 cycles of 94°C for 30 s, 58°C for 1 min, 72°C for 1 min, and a final extension at 72°C for 7 min. First-round PCR products were diluted 5-fold for the second-round of PCR. Round 2 PCR conditions were 94°C for 3 min, 10 cycles at 94°C for 30 s, 65°C for 1 min, and 72°C for 1 min; 30 cycles at 94°C for 30 s, 62°C for 1 min, and 72°C for 1 min, and 72°C for 7 min. PCR products were visualized on a 1.2% agarose gel, stained with ethidium bromide, and visualized by a transilluminator.PCR products were sequence using ABI377 or ABI3730 sequencers (PE Applied biosystem). Base calling, contig assembly contigs, and mutation detection was performed using Polyphred package  . All traUnrelated control samples were selected for analysis using the Hardy-Weinberg Equilibrium (HWE) test using an exact test. Standard EM algorithm was used to infer haplotype and estimate population frequency. Single marker and haplotype association test and significance estimation were performed using a permutation test.LMP1 gene) were cultured in Keratinocyte-SFM medium (GibcoBRL) with Bovine Pituitary Extract and rEGF. C666-1  was grown in RPMI 1640 supplemented with 10% fetal bovine serum . CNE-1 , CNE-2  and Sune were maintained in RPMI 1640 with 10% FBS.NP69  and NP69-LMP1  and Bcl-3 with primers  and β-actin as control with primers (5'-acactgtgcccatctacgagggg-3' and 5'-atgatggagttgaaggtagtttcgtggat-3').A total of 21 tissues were collected from Sun Yat-sen University Cancer Center. Six paired matched tissues from different organs included esophagus, stomach, liver, lung, cervix and breast; nine nasopharyngeal tissues contained 2 chronic nasopharynx inflammation (Inf.), 1 Differentiated Carcinoma (DNK), 4 Undifferentiated Carcinoma (NDNK), 1 low differentiated squamous carcinoma (LDS) and 1 non-Hodgkin's lymphoma (NHL). RNA was extracted using TRIZOL Reagent , and reverse transcription was performed using the TaKaRa RNA PCR kit (AMV) Ver.3.0 . PCR to detect N4BP2 locus, four of which were upstream of the N4BP2 gene and eight which were within N4BP2 gene  combined haplotype Block 2 ATTA and GTTG exhibited notable difference between case and control groups Table . Permuta-7-9-10 cBcl-3 and N4BP2 were detected in all cell lines examined  declare that they have no competing interests.YXZ and HDQ were responsible for the design of study. MZZ carried out the experiments and drafted the manuscript. HDQ participated in data analysis. XJY and RHZ performed sequencing. LZC and QSF helped in cell culture and tissue collection.


During human pregnancy, the placental villi produces high amounts of estradiol. This steroid is secreted by the syncytium, which is directly in contact with maternal blood. Estradiol has to cross placental foetal vessels to reach foetal circulation. The enzyme 17beta-hydroxysteroid dehydrogenase type 2 (17beta-HSD2) was detected in placental endothelial cells of foetal vessels inside the villi. This enzyme catalyzes the conversion of estradiol to estrone, and of testosterone to androstenedione. It was proposed that estradiol level into foetal circulation could be regulated by 17beta-HSD2.We obtained placentas from 10 to 26 6/7 weeks of pregnancy from women undergoing voluntary termination of pregnancy, term placentas were collected after normal spontaneous vaginal deliveries. We quantified 17beta-HSD2 mRNA levels in mid-gestation and term human placenta by RT-QPCR. We produced a new anti-17beta-HSD2 antibody to study its spatio-temporal expression by immunohistochemistry. We also compared steroid levels  and 17beta-HSD2 mRNA and protein levels between term placenta and endometrium.High 17beta-HSD2 mRNA and protein levels were found in both mid-gestation and term placentas. However, we showed that 17beta-HSD2 mRNA levels increase by 2.27 fold between mid-gestation and term. This period coincides with a transitional phase in the development of the villous vasculature. In mid-gestation placenta, high levels of 17beta-HSD2 were found in mesenchymal villi and immature intermediate villi, more precisely in endothelial cells of the stromal channel. At term, high levels of 17beta-HSD2 were found in the numerous sinusoidal capillaries of terminal villi. 17beta-HSD2 mRNA and protein levels in term placentas were respectively 25.4 fold and 30 to 60 fold higher than in the endometrium. Steroid levels were also significantly higher in term placenta than in the endometrium.The spatial and temporal expression of 17beta-HSD2 in the placenta during pregnancy and the comparison of 17beta-HSD2 expression and steroid levels between placental villi and endometrium are compatible with a role in the modulation of active and inactive forms of estrogens. Our observations strongly support the hypothesis that 17beta-HSD2 acts as a barrier decreasing estradiol secretion rates in the foetal circulation. During human pregnancy, the placenta produces and secretes high amounts of estradiol E2) and estrone E1) . E and estr17β-hydroxysteroid dehydrogenases (17βHSDs) catalyze oxidoreduction at position C17 of C18 steroids such as E1 and E2, and C19 steroids  and at position C20 of progestins. The human placenta expresses mainly two 17βHSDs, types 1 and 2. Type 1 almost exclusively reduces E1 in E2 ,15, wherIn human placenta, 17βHSDs type 1 and type 2 are abundantly expressed ,19. All During normal placental development, villous trees expand extensively by sprouting . BetweenIn the present study, we have further evaluated the mRNA and protein expression of the key steroidogenic enzyme 17βHSD2 in various structures of human placenta obtained at mid and term pregnancy. The levels of 17βHSD2 mRNA in several human placentas collected during this pregnancy interval have been precisely determined by real-time quantitative PCR using several housekeeping genes for data normalization. The protein has been detected by immunohistochemistry using a novel and very sensitive anti-17βHSD2 antibody, while the mRNA was localized by in situ hybridization. 17βHSD2 is recognized to play a role in the control of E2 levels in the endometrium ,29. To fCentre Hospitalier Universitaire de Québec . Gestational age was determined based on bi-parietal and abdominal diameters, and cephalo-caudal and femur lengths. Tissues were collected on ice. To avoid contamination of the villi by decidua or foetal membranes, a 0.5 cm slice of tissue was removed on each side of the placentas. Then, small pieces of villi (0.5 cm3) were prepared and extensively rinsed in PBS. Several pieces were kept frozen until RNA or protein extraction, while others were fixed in 4% paraformaldehyde for 24 hours prior processing for routine paraffin wax embedding. Five μm tissue sections were used.Human placentas from weeks 10 to 24 6/7 of pregnancy (n = 25) were obtained from women undergoing voluntary pregnancy termination. Term placentas  were collected after normal spontaneous vaginal deliveries. No placenta had histopathological abnormality. Endometrial tissues in mid-late secretory phase were obtained by biopsy. For all patients, informed consent was obtained according to the policies for the Human Studies and the Institutional Review Board of the Northern blot analysis were performed as described . Briefly3 placental samples and then purified on CsCl gradients. A 4 μg aliquot of total RNA was treated with DNase , and reversed transcribed  according to the protocol of the manufacturer, using hexameric random primer (pd(N)6, Invitrogen) in a 20 μl final volume. LightCycler-FastStart DNA Master SYBR Green I kits (Roche) were used for real-time PCR. Reactions were performed according to the protocol of the manufacturer with 0.5 μM of each primer , 3 mM MgCl2, and an amount of cDNA samples corresponding to 100 ng of total RNA input in a 20 μl final volume. After enzyme activation , 40 PCR cycles were performed: 0 sec, 95°C; 5 sec annealing temperature (see below); 20 sec, 72°C; 5 sec, temperature of fluorescence intensity reading (see below). At the end of each run, samples were heated to 95°C with a temperature transition rate of 0.2°C/sec to construct dissociation curves. Several PCR reactions were tested on a 2% agarose gel, and amplicons were subjected to DNA sequencing to confirm the specificity of the PCR reactions. The primers selected for 17βHSD2 encompass more than one intron. Gene/GenBank accession number/5' oligonucleotide/3' oligonucleotide/length of amplicon/annealing temperature/temperature of fluorescence intensity reading:17βHSD2/NM_002153/TGTCAGCAGCATGGGAGGAG/CGCCAAGATAGCATGCTGGA/346 nt/70°C/84°C; GAPDH/NM_002046/GAAGACTGTGGATGGCCCCTC/GTTGAGGGCAATGCCAGCCCC/358 nt/58°C/88°C; YWHAZ/NM_003406/AGACGGAAGGTGCTGAGAAA/GAAGCATTGGGGATCAAGAA/127 nt/59°C/76°C; TBP/NM_003194/GAACCACGGCACTGATTTTC/CCCCACCATGTTCTGAATCT/157 nt/58°C/80°C; HPRT1/NM_000194/TGACACTGGCAAAACAATGCA/GGTCCTTTTCACCAGCAAGCT/94 nt/59°C/78°C. A standard curve for real-time PCR was prepared for each gene using specific amplicons previously obtained by PCR, sequenced, and calibrated by electrophoresis on an agarose gel. The program supplied by the manufacturer  was used to import the standard curves and calculate the amount of PCR products.RNA extraction, cDNA synthesis, and real-time quantitative PCR were performed as described previously . BrieflyTo ensure a more accurate evaluation of the relative expression of 17βHSD2 mRNA in mid-gestation and term placentas, we assessed the expression stability of four housekeeping genes , based on the approach proposed by Vandesompele . HousekeService de Protéomique de l'Est du Québec (CHUL Research Center) using standardized protocol, purified by HPLC, and coupled to keyhole limpet hemocyanin. A polyclonal anti-17βHSD2 antiserum was developed by repeated immunization of rabbits. The antiserum was purified using a sulfoLinK coupling gel .A synthetic peptide (LEKDILDHLPAEVQ) corresponding to amino acids 279 to 292 of 17βHSD2 was synthesized by the g for 10 min to remove cell debris and again at 105,000 × g for 1 h. Supernatants were saved as cytosol, and pellets as microsomes. Ten micrograms of each protein sample (cytosol and microsomes) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-RAD). Membranes were blocked overnight at 4°C with 5% (wt/vol) fat-free dry milk powder in PBS containing 0,05% (vol/vol) Tween 20. These membranes were then incubated with our anti-17βHSD2 antibody (1:1000) for 2 hours at room temperature. After washing, bound antibodies were visualized with horseradish peroxidase-conjugated goat anti-rabbit antibodies using a Western Lightning kit (Perkin Elmer). An aliquot of the 17βHSD2 antiserum was pre-incubated at 4°C for 24 h with an excess (10-6M) of 17βHSD2 synthetic peptide used previously to develop the antibody. This adsorbed antiserum was used as negative control. The absence of primary antibody was also tested as negative control.Villous tissues from term pregnancy were first fractionated in cytosol and microsomes as described previously ,18. Brieg. The pellet was re-suspended in 0.5% Nonidet P-40 in PBS. After a 15 min centrifugation at 300 × g, 9 volumes of acetone were added to the supernatant. After a 2 h incubation at -20°C, an aliquot was used for protein determination, and then 350 μg of proteins were centrifuged 15 min at 300 × g. The pellet was air dried and then dissolved in 7 M urea, 2 M thiourea (Sigma-Aldrich), 4% CHAPS (Sigma-Aldrich), 0.8% IPG buffer , bromophenol blue, and 10 mg/ml dithiothreitol (DTT). Denatured proteins were first separated according to their isoelectric point using 13 cm Immobiline DryStrip with a pH range of 3–10 with IPGphor isoelectric focusing system (Amersham Biosciences). Isoelectric focusing was done in four steps: re-hydration at 30 V for 10 h; 500 V for 1 h; 1,000 V for 1 h, and; 8,000 V to reach 17,500 Vh final. After these steps, strips were equilibrated 15 min in 50 mM Tris-HCl, 6 M urea, 30% (vol/vol) glycerol, 2% (wt/vol) SDS, and bromophenol blue. Then, each strip  was loaded on a 12% SDS-PAGE. After migration, proteins of one gel were silver stained while proteins of the second gel were transferred to a nitrocellulose membrane and 17βHSD2 protein was detected with our anti-17βHSD2 antibody as described above.Total protein extracts were prepared from small pieces of term villi. Tissues were homogenized in PBS and then centrifuged 15 min at 250 × -6 M) of the synthetic peptide instead of primary antibody. Anti-KDR control experiments were performed by replacing the primary antibody with 0.01 M PBS.Tissue sections were deparaffinized in xylene and rehydrated through graded ethanol. Tissue sections were incubated in 3% hydrogen peroxide in methanol for 30 min to block endogenous peroxidase activity and rinsed in distilled water. Antigen retrieval was carried out in sodium citrate buffer  by boiling slides in a microwave oven for 2 min at maximum power and then cooling down at room temperature. Slides were rinsed once in distilled water for 5 min and once in PBS for 5 min before the blocking step. Unspecific binding was blocked by incubation of tissue sections in 10% goat serum in PBS containing 0,1% BSA and 0,4% Triton X-100 for 30 min. The tissue sections were then incubated with primary antibodies overnight at 4°C in a humidified chamber. The anti-17βHSD2 antibody was used at 1:200 dilution, the anti-KDR  was used at 1:50 dilution. KDR/VEGF-R2  was used as an endothelial cell marker. Expression of KDR decreases as pregnancy progress and becomes barely detectable near term. Following the incubation with the primary antibody, the slides were washed 3 times in PBS and incubated with biotinylated goat anti-rabbit  for 17βHSD2 and goat anti-mouse  for KDR for 45 min at room temperature. The slides were washed 3 times in PBS and the antigen-antibody complex was revealed using avidin-biotin peroxidase reaction method using the ABC Vectastain kit  and AEC (3-Amino-9-ethylcarbazole) as chromagen. The immunoreactivity was visualized as red color. Control experiments for the 17βHSD2 were performed on adjacent sections by substitution of the antibody by the pre-immune rabbit serum or by the 17βHSD2 antibody adsorbed with an excess  using Image Pro-Plus software . Red staining intensities in IIV and SV from mid-gestation placentas or SV and TV from term placentas were quantified as follows using Image Pro-Plus. Briefly, IIV, SV or TV were delimitated using the "New AOI" command. The created areas of interest were measured using measurement commands. The "Select Color" command was used to specify the range of intensities/colors that defined the section analysed. The selected range color was applied for each measurement. The ratio of the mean intensity/density values was calculated for each type of villi from the same slide. Twenty-three IIV and 17 SV structures were analysed from 13 mid-gestation placentas. For the analyses on term placenta, 10 TV and 9 SV structures were analysed from 5 placentas.Eco RV fragment of 17βHSD2 [Bam HI and Xho I, antisense and sense 17βHSD2 RNA probes were synthesized using (35S)-UTP . Probes were synthesized using the Riboprobe Combination System kit , and riboprobes with less than 1.4 × 109 dpm/μg DNA matrix were discarded. After deparaffinization and re-hydration, tissue sections were fixed in 4% paraformaldehyde  for 20 min, treated with proteinase K  for 25 min at 37°C, acetylated in 37.5 mM triethanolamine solution (Sigma) containing 0.25% (vol/vol) anhydric acid (Sigma) for 10 min, dehydrated in graded alcohol solutions, and air-dried. Hybridization (2 × 106 dpm/100 μl/slide) was performed overnight at 58°C in 50% formamide, 0.3 M NaCl, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 1× Denhardt's , 1% dextran sulphate, 10 mM DTT, and 500 μg/ml transfer RNA. After hybridization, slides were treated with ribonuclease A for 30 min and washed under high stringency conditions in 0.1× SSC  and 1 mM DTT at 58°C for 30 min. After defatting, tissues were coated with NTB-2 emulsion  and kept at 4°C for 5 to 10 days. Slides were developed with D-19 solution (Eastman Kodak Co.), counterstained with 0.25% thionine (Sigma), dehydrated, and mounted with Permount (Fisher Scientific).Synthesis of RNA probes and in situ hybridization were performed as described previously ,33 with  17βHSD2  insertedPlacental and endometrial tissues were homogenized in ice-cold buffer containing 10% SDS, 10 mM Tris-HCL pH 7.5, 1 mM DTT and 1 mM PMSF. Proteins were precipitated with methanol-chloroform, solubilized in SDS-PAGE sample buffer containing 1% SDS, 62.5 mM Tris-HCL pH 6.8, 1% 2-mercaptoethanol, 10% glycerol and 0.025% bromophenol blue, heated at 95°C for 3 min then stored at -20°C until use. Protein concentration of samples was determined by BCA protein assay after precipitation with trichloro acetic acid. 50 μg of endometrial proteins and 50, 2.5, 1.0, 0.5, 0.25, and 0.1 μg of placental proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Bio-RAD) for Western blot analysis. Membranes were treated as described above. After washing, bound antibodies were revealed by chemiluminescence with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies using a SuperSignal WestDura kit (incubation 5 min) and Kodak XAR films (exposition 30 sec).Steroid hormones were extracted as follows. Frozen tissues (200 mg) were pulverized, weighed, and transferred into glass tubes. After suspension in 0.5 ml PBS pH 7.0, 1 ml of ethanol:acetone  was added. Samples were homogenized and centrifuged to pellet tissue debris. Supernatants were exposed to nitrogen gas flow to remove organic solvents and the volumes were measured by a gas chromatographic mass spectrometric method developed to measure steroid hormone levels in rat and monkey sera . Brieflyp value of less than 0.05 was considered a significant difference.Statistical analyses were performed usingt-test function of GraphPad Prism 3.0  and SPSS 10.0.5 softwares . A two-tailed 17βHSD2 mRNA levels were determined by Northern blot analysis in 5 samples of villi collected at different positions relative to the insertion point of the umbilical cord. Two placentas obtained at term pregnancy were studied. 17βHSD2 mRNA levels in placental samples are quite similar, and independent from the sampling site . No significant variation was observed in the expression of the 17βHSD2 among samples collected around mid-gestation , immature intermediate villi (IIV) and stem villi (SV). These three types of villi are shown in Figure At term, the placental villi is mainly composed of terminal villi (TV) and stem villi (SV). TV is the most abundant type of villi at term, with more than 50% of its stromal volume occupied by the foetal capillaries. All these capillaries strongly expressed the 17βHSD2. Again, the syncytium was negative. In the SV Figure  and 5E, In mid-gestation placentas, 17βHSD2 mRNA was abundant in the IIV and in the PC network of the SV Figure  and 6C. Quantification of 17βHSD2 staining using the Image-Pro Plus software showed that in mid-gestation placenta, 17βHSD2 was more abundant in the IIV than in the SV and that in term placenta, 17βHSD2 was more abundant in the TV than in the SV (Figure As shown in Table in utero to environmental components with estrogenic activity could also results in reproductive disorders [During pregnancy, E2 plays an important role in the maturation of some foetal organs such as the lungs, skin and HPA axis -7,36. Hoisorders -47. Elevisorders -50. A stisorders . It was isorders ,20,22. Iisorders ,10,12,1317βHSD2 mRNA level was measured in different parts of term placenta to evaluate if there is a spatial variation in expression (Figure We have precisely determined the level of 17βHSD2 mRNA by real-time quantitative PCR in different samples of mid-gestation and term placentas (Figure We have characterized the distribution of 17βHSD2 in placental villi at different developmental stages using immunohistochemistry Figures  and 5. QWith respect to E2 inactivation, it is interesting to compare 17βHSD2 mRNA and protein, and steroid levels between the placenta and the endometrium. In endometrium, 17βHSD2 is expressed in glandular epithelial cells during the mid to late secretory phase . SeveralIn conclusion, the spatial and temporal expression of 17βHSD2 mRNA and protein in the placenta during pregnancy and the comparison of 17βHSD2 expression and steroid levels between term placenta and mid-late secretory phase endometrium provide clear evidences that 17βHSD2 can act as a barrier to protect the foetus against excessive E2 levels.


Development of lung alveolar sacs of normal structure and size at late gestation is necessary for the gas exchange process that sustains respiration at birth. Mice lacking the lung differentiation gene T1α [T1α(-/-)] fail to form expanded alveolar sacs, resulting in respiratory failure at birth. Since little is known about the molecular pathways driving alveolar sacculation, we used expression microarrays to identify genes altered in the abnormal lungs and, by inference, may play roles in normal lung morphogenesis.Altered expression of genes related to cell-cell interaction, such as ephrinA3, are observed in T1α(-/-) at E18.5. At term, FosB, Egr1, MPK-1 and Nur77, which can function as negative regulators of the cell-cycle, are down-regulated. This is consistent with the hyperproliferation of peripheral lung cells in term T1α (-/-) lungs reported earlier. Biochemical assays show that neither PCNA nor p21 are altered at E18.5. At term in contrast, PCNA is increased, and p21 is decreased.This global analysis has identified a number of candidate genes that are significantly altered in lungs in which sacculation is abnormal. Many genes identified were not previously associated with lung development and may participate in formation of alveolar sacs prenatally. Lung development starts in mice at embryonic day 9.5 (E9.5). By E16.5, airways have extensively grown and branched to form the bronchial tree. Between E16.5 and term (E20.5) lung cell proliferation is gradually reduced, and the distal lung undergoes significant morphogenetic changes to form the alveolar sacs. While a population of distal epithelial cells flattens, thins, and spreads to form type I cells, other distal epithelial cells remain cuboidal, acquire surfactant filled lamellar bodies and differentiate into type II cells. Differentiation of epithelial cells is accompanied by vascular remodeling and thinning of the mesenchyme, and results in enlargement of the diameter and surface area of the alveolar sacs. Overall this process is known as sacculation, and it is critical to increase the efficiency of fluid absorption and gas exchange processes at birth .Very little is known about the molecular regulation of sacculation in normal animals. Abnormal sacculation has been reported in many genetically altered animals carrying null mutations, or transgenes that mis- or over-express growth factors, transcription factors, and other regulatory molecules. These molecular abnormalities result in formation of alveolar spaces that are either too small, as in glucocorticoid receptor (GR) , corticoWe have previously shown that mice carrying a null mutation of the T1α gene fail to form expanded alveolar sacs near term and die at birth due to an inability to inflate their lungs with the first few breaths . In normSome insights into the process of alveolar sacculation in normal animals come from gene expression microarray data using lungs of normal mice at different developmental time points from embryonic day 9 through postnatal week 4 . This stAs an alternative approach for understanding the molecular regulation of sacculation, it is reasonable to compare lungs with normal alveolar sac formation to those with altered sac formation and infer from differences in gene expression a set of candidate genes involved in the process. Preliminary expression microarray analysis of the GR knockout lung  has explHere, we use a microarray approach to begin to understand the molecular mechanisms by which formation of alveolar sacs is altered in T1α null mice, animals that serve as a highly reproducible model of altered lung sacculation. We have identified genes that are preferentially altered in the T1α null lung prior to recognizable structural abnormalities, such as ephrinA3, and identified that a number of regulators of the cell cycle are altered at term. Our study also shows that p21, a negative cell cycle regulator, is decreased at term but not at E18.5 at both the mRNA and protein levels, a finding that is likely associated with the failure of sacculation. These genes are now candidates for future studies designed to understand the molecular mechanisms that regulate sacculation in the normal lung.T1α protein is expressed in the apical surface of lung epithelial cells throughout mouse development Figure . Gross mTo determine whether morphological alterations observed in the absence of T1α at E18.5 are also accompanied by changes in cell proliferation, we compared expression of PCNA and p21 genes by northern and western blot analyses in (+/+), (+/-) and (-/-) lungs at E18.5 and term. Neither PCNA nor p21 protein level is altered at E18.5 in T1α(-/-) lungs compared to T1α(+/+) Fig. . HoweverTo identify genes potentially involved in the abnormal alveolar sac formation of the T1α null lungs we analyzed global gene expression profiles of T1α(-/-) vs. T1α(+/+) lungs, at E18.5 and at term, using oligonucleotide microarrays. The data were analyzed using Student's t-test and the more stringent Mann-Whitney analysis  mice was described previously . T1α hetE11.5 whole embryos or E15.5, 18.5, 19.5 and P1 dissected lungs were fixed in 4% paraformaldehyde/PBS pH 7.4 solution and embedded in paraffin as described previously . SectionTotal RNA from E18.5 and term T1α(+/+) and T1α(-/-) lungs was isolated , re-purified using RNeasy Mini Kit (QIAGEN), and DNase treated on column with DNA-free . First and second strand syntheses were performed as recommended by Affymetrix using 8 μg total RNA as template. Biotin-labeled cRNA was generated using the ENZO BioArray High Yield RNA transcript labeling kit . Each biotin-labeled cRNA sample (15 μg) was hybridized (16 hours at 45°C and 60 rpm) in an Affymetrix GeneChip System to the Murine Genome U74 Av2 GeneChip (Affymetrix) that contains ~12,000 sequences of the UniGene database or ESTs . ConfocaFive E18.5 T1α(+/+) lungs, five E18.5 T1α(-/-) lungs, three term T1α(+/+) lungs and three term T1α(-/-) lungs were analyzed on microarrays. A single weighted mean expression level for each gene was derived using Microarray Suite 5.0 software . Using a one-sided Wilcoxon signed-rank test, the MAS 5.0 software generated a detection p-value for each gene indicating whether or not the transcript was reliably detected. Data from each array was scaled in order to normalize the results for inter-array comparisons.2 = 0.953 +/- 0.023, E18.5 T1α(+/+) (5 arrays) R2 = 0.939 +/- 0.026, term T1α(-/-) (3 arrays) R2 = 0.945 +/- 0.016, and term T1α(+/+) (3 arrays) R2 = 0.931 +/- 0.026.To filter out arrays of poor quality, several quality control parameters on each array were assessed. We reviewed the scanned image for significant artifacts, and the presence on the array of bacterial genes spiked into the hybridization mix (bioB and bioC). Background and noise measurements were below 100 in all arrays. The average 3' to 5' ratio of the signal for GAPDH was 1.01 ± 0.15 and for β-actin was 1.63 ± 0.09 in E18.5 arrays. In term arrays, the 3' to 5' ratio for GAPDH was 1.63 ± 0.82 and for β-actin was 1.72 ± 0.54. A ratio less than 3 is considered acceptable. An average of 45.4 ± 3.8% probe sets exceeded the probe pair threshold in all arrays . Correlation values were calculated among arrays in the same genotype and developmental group. The average correlation values were: E18.5 T1α(-/-) (5 arrays) RPrior to performing statistical analyses, we selected genes whose median detection p value was ≤ 0.05  in at least one of the 2 comparative groups (+/+ or -/-). To compare T1α(-/-) lung gene expression vs. T1α(+/+) at E18.5 (n = 5 each) or at term (n = 3 each), a Student's t-test was performed with a p-value less than 0.05 considered significant . E18.5 and term total RNA, treated with DNA-free DNase (Ambion), was reversed transcribed (RT) (1 μg in 25 μl) using AMV reverse transcriptase . RT reactions diluted 1:32 were analyzed by QRT-PCR in an ABI 7000 instrument . Primers were designed using PrimerExpress version 2.0 software (Applied Biosystems) or were obtained from Assays-on-Demand (Applied Biosystems). Reactions were performed in 50 μl using either TaqMan PCR universal master mix or SybrGreen PCR master mix (Applied Biosystems). For all primers and probes, optimal concentrations were found and dissociation curves were performed for primers used in SybrGreen reactions to confirm a single product. The relative concentration of RNA for each gene tested was obtained using calibration curves performed with wild type E18.5 or term total lung RNA (n = 3), respectively. Samples and calibration curves were reverse transcribed and amplified under equal conditions. E18.5 T1α (+/+), E18.5 T1α (-/-), term T1α (+/+), and term T1α (-/-) lungs were analyzed in duplicate (n = 3-6). Data were normalized to β-actin mRNA at E18.5 and at term; the level of expression of β-actin mRNA does not change between T1α(+/+) and T1α(-/-) samples at the respective time point. Oligonucleotides used in SybrGreen reactions were: Additional sex combs like (F) TCTTCCGCCATTTCATCTGA, (R) CACAACATGGACTTGGCCAA; Complement C1qa (F) GTGAAAGGCAATCCAGGCAA, (R) TGGTTGGTGAGGACCTTGTCA; Cyr61 Applied Biosystems Assays-on-Demand; Dual specificity phosphatase 1 (MKP-1) (F) ATGCTTGACACACCCACCAGT, (R) TCATACGCGCATGTCATCG; Dynein cytoplasmic (F) AAATCCACCCCAGCGTTTCTAC, (R) CCCCATTTGTTTTTGAGACGG; Early growth response 1 (F) TTTCAGCCTGAGTCCTTACCCA, (R) TGCATGGTCAGTTCCCAGAAC; Ephrin A3 (F) TGCCCCCTTCCTATGACAGAGAG, (R) GCCCAGAGAGATGTACAGTGCA; FosB (F) CGGCCTCTCCCTTTATCCTTT, (R) TGTCCCCGACAAATCCAGAA; Gas5 (F) TTCATTTGGCTGGCTTGCTT, (R) GGAATTCAAGGCCCCCAAT; Nuclear receptor subfamily 4A1/Nur77 (F) CCAGCCTGGTTTTGAGCTAAG, (R) CCATCTTCTGGAGTGAGGAGGT; OTS8/T1α (F) TGGCGTCTTGTTAGCCATTG, (R) GGCGAGACCTTCCAGAAATC; Prostaglandin-endoperoxide synthase 1 (F) CAAACCTACGTCTACGCCAA, (R) AGTCCATCTGTTCCCTCCAC; Rb1 (F) GAATTCTCAGAGATCTTGCTTCG, (R) AGTGGAGTGTCTGCAGCTTTAAG; Sp1 (F) CACCCAGTGCCAGAGACATATG, (R) CCAATTCTTCCTACCACCAAGC.Total lung RNA (10 μg) was electrophoresed, blotted and hybridized as described previously . p21 proE18.5 and term lung proteins were extracted using RIPA buffer . ProteinGM, has previously participated in the generation of the T1α null mutant mice. She obtained all the developmental lung samples for the studies and prepared samples for microarray hybridizations at the microarray core facility at Boston University. She also validated gene expression patterns by real time PCR; AH, performed histology and immunostaining analyses; JW, analyzed patterns of expression of cell cycle regulators by northern and western blot methods; AS participated in the design of the study, analyzed the microarray data, performed the statistical analyses, and provided his expertise to interpret the microarray data; MCW participated in the design of the study, in the interpretation of the data, and in the drafting of the manuscript; MIR participated in the design and coordination of the study, in the interpretation of the data, and in the drafting of the manuscript. All authors read and approved the final manuscript.Supplementary Tables. Table S1. Genes dysregulated in T1α (-/-) vs. (+/+) lungs at E18.5 (p ≤ 0.05). Table S2. Genes dysregulated in T1α (-/-) vs. (+/+) lungs at term (p ≤ 0.05). Table S3. Mann-Whitney p values for genes dysregulated in E18.5 T1α (-/-) lungs compared to wild type, with Student's t test p ≤ 0.05. Table S4. Mann-Whitney p values for genes dysregulated in term T1α (-/-) lungs compared to wild type, with Student's t test p ≤ 0.05.Click here for fileFigure S1. Ratios of epithelial vs. mesenchymal marker genes in T1α (+/+) and (-/-) lungs at E18.5 and term.Click here for fileReferences in Table Click here for file


Hibiscus rosa sinensis in myocardial ischemic reperfusion injury, particularly in terms of its antioxidant effects.The present study investigates the cardioprotective effects of Hibiscus rosa sinensis (Chinese rose) have been mentioned in ancient literature as useful in disorders of the heart. Dried pulverized flower of Hibiscus rosa sinensis was administered orally to Wistar albino rats (150–200 gms) in three different doses , 6 days per week for 4 weeks. Thereafter, rats were sacrificed; either for the determination of baseline changes in cardiac endogenous antioxidants  or the hearts were subjected to isoproterenol induced myocardial necrosis.The medicinal values of the flowers of Hibiscus Rosa sinensis. In the 250 mg/kg treated group, there was significant increase in superoxide dismutase, reduced glutathione, and catalase levels but not in the 125 and 500 mg/kg treated groups. Significant rise in myocardial thiobarbituric acid reactive substances and loss of superoxide dismutase, catalase and reduced glutathione (suggestive of increased oxidative stress) occurred in the vehicle treated hearts subjected to in vivo myocardial ischemic reperfusion injury.There was significant increase in the baseline contents of thiobarbituric acid reactive substances (TBARS) [a measure of lipid per oxidation] with both doses of Hibiscus rosa sinensis (250 mg/kg) augments endogenous antioxidant compounds of rat heart and also prevents the myocardium from isoproterenol induced myocardial injury.It may be concluded that flower of  Morphological observations of the ischemic myocardial tissue undergoing reperfusion suggest that it is a true pathological phenomenon and a distinct entity from the preceding ischemic injury ,2. ReperHibiscus rosa sinensis  [HRS], has been reported in the ancient Indian medicinal literature with beneficial effects in heart diseases .The flowers of The flower was air dried under shade and pulverized. A suspension of the flower in 2% carboxymethyl cellulose (Vehicle) was made daily.Hibiscus rosa sinensis flower powder of three doses (125 mg/kg (H1), 250 mg/kg (H2) and 500 mg/kg (H3)] or with vehicle by oral gavage once a day for 4 weeks (6 days/week), along with standard rat chow  and water, ad libitum. There was no significant difference in body weight of the treated rats, when compared with control, either at the beginning or at end of the study period. The treated rats did not offer any abnormal resistance to drug administration. The treatment schedule did not cause any change in food and water intake pattern. After 48 hours of the last dose, rats were heparinised [375 units/200 g i.p] [Male Wistar albino rats weighing 150–200 g were obtained from the Tamil Nadu Veterinary and Animal Science University and were housed at 25° ± 5°C under 12 hour light and dark cycle. Experiments were carried out according to the guidelines of the animal ethics committee of the Institute. The rats were divided into 4 groups (20 in each group) and fed either with the suspension of dried pulverized 0 g i.p]  and halfHearts from ten rats of each group were harvested and stored in liquid nitrogen for estimation of basal endogenous antioxidants and in 10% buffered formalin for histological studies.The groups studied wereGroup control: Vehicle treated ratsGroup H1 BL: 125 mg/kg of Hibiscus rosa sinensis treated ratsGroup H2 BL: 250 mg/kg of Hibiscus rosa sinensis treated ratsGroup H3 BL: 500 mg/kg of Hibiscus rosa sinensis treated ratsAt the end of the treatment period 6 rats from each group were administered isoproterenol (ISO) 85 mg/kg s.c., for two consecutive days to induce myocardial injury ,12. AfteGroup control: Vehicle + saline injected ratsGroup IR : Vehicle + ISO treated ratsGroup H1 IR: 125 mg/kg of Hibiscus rosa sinensis + ISO treated ratsGroup H2 IR: 250 mg/kg of Hibiscus rosa sinensis + ISO treated ratsGroup H3 IR: 500 mg/kg of Hibiscus rosa sinensis + ISO treated ratsTBARS levels in the myocardium were determined by the method described by Ohkawa et al., .Myocardial GSH was estimated by the method of Ellman, .SOD levels in the hearts were determined by McCord and Firdovich method (1969) and modified by Kakkar et al .Catalase level was estimated by the method described by Aebi, .Protein estimation for the tissue samples were done by the method of Bradford .The hearts were removed, washed immediately with saline and then fixed in 10% buffered formalin. The hearts stored in 10% buffered formalin, were embedded in paraffin, sections cut at 5 μm and stained with hematoxylin and eosin. These sections were then examined under a light microscope for histological changes.All values were expressed as Mean ± SE. (n = 10 in each groups). One way ANOVA was applied to test for significance of biochemical data of the different groups. Significance is set at p < 0.001.Hibiscus rosa sinensis treated groups and the Hibiscus rosa sinensis treated groups subjected to ISO administration. Three rats died in the vehicle + ISO injected group (IR).There was no mortality in the There were no significant changes in the levels of TBARS and antioxidants between the vehicle treated and vehicle + saline injected groups of rats. Hence, the results given in the result portions represent the values of vehicle treated rats as control (C).Baseline changes brought about by pretreatment for 4 weeks (6 days/week) with three different doses of 125 mg/kg (H1BL), 250 mg/kg (H2BL) and 500 mg/kg (H3BL) or vehicle treated groups are given below:Myocardial baseline TBARS were significantly high (p < 0.001) in H2BL and H3BL groups (58.3 ± 2.6 and 63.6 ± 2.6 nmole/g wet wt), but not in H1BL group (48.8 ± 2.4 nmole/g wet wt) in comparison to control group (46.2 ± 1.5 nmole/g wet wt).Myocardial baseline GSH was significantly increased (p < 0.001) in both H2BL and H3BL groups (441.1 ± 2.9 and 375.3 ± 1.5 μg/g wet wt), whereas in H1BL group the GSH level was not significantly altered (312.8 ± 4.6 μg/g wet wt) in comparison to that of control group (320.3 ± 6.9 μg/g wet wt).Myocardial baseline SOD was significantly (p < 0.001) increased in H2BL and H3BL groups (5.9 ± 0.6 and 4.4 ± 0.8 I.U/mg protein) in comparison to control group (2.8 ± 0.3 I.U/mg protein). But in H1BL group, there was no significant change (2.7 ± 0.6 I.U/mg protein) in comparison to control group.Myocardial baseline catalase was significantly (p < 0.001) increased in H2BL groups (59.4 ± 1.6 I.U/mg protein) whereas in H1BL and H3BL groups the catalase showed no significant change (40.7 ± 5.8 and 50.1 ± 1.4 I.U/mg protein) in comparison to the control group (43.1 ± 5.3 I.U/mg protein).The results obtained in the different groups subjected to in-vivo ischemic reperfusion injury are presented below. Table Myocardial TBARS in IR group (67.8 ± 3.4 nmole/g wet wt) was significantly (p < 0.001) higher than that in control group (46.2 ± 1.5 nmole/g wet wt). In H2 IR and H3 IR treated groups there was a significantly (p < 0.001) lower TBARS (49.3 ± 1.9 and 57.8 ± 1.7 nmole/g wet wt) respectively, whereas in the H1 IR group the TBARS shows no significant change (74.1 ± 5.3 nmole/g wet wt) in comparison to IR groupMyocardial GSH level was significantly lower (p < 0.001) in IR group (289.7 ± 2.4 μg/g wet wt) in comparison to that of the control group (320.3 ± 6.9 μg/g wet wt). There was a significant increase (p < 0.001) in the levels of GSH in the H2 IR (372.1 ± 2.9 μg/g wet wt), whereas in there was no change in the levels of GSH levels H1 IR (279.3 ± 9.8 μg/g wet wt) and H3 IR (271.2 ± 7.4 μg/g wet wt); in comparison to the IR group.Myocardial SOD activity was significantly lower (p < 0.001) in IR group (1.5 ± 0.5 I.U/mg protein) than that in control group (2.8 ± 0.3 I.U/mg protein). Myocardial SOD levels showed no significant change in the H1 IR and H3 IR groups (2.5 ± 0.8 I.U/mg protein and 2.5 ± 1.1 I.U/mg protein respectively) in comparison to IR group. However, the myocardial SOD level was significantly higher (p < 0.001) in the H2 IR group (4.2 ± 0.7 I.U/mg protein) in comparison to IR group.Myocardial catalase was significantly lower (p < 0.001) in the IR group (30.6 ± 2.7 I.U/mg protein) in comparison to that of the control group (43.1 ± 5.3 I.U/mg protein). There was no change in myocardial catalase levels in the H1 IR (37.8 ± 4.5 I.U/mg protein) and the H3 IR (35.6 ± 1.8 I.U/mg protein) groups, whereas in H2 IR group myocardial catalase was significantly (p < 0.001) higher (46.4 ± 4.3 I.U/mg protein) in comparison to the IR group.Light microscopy of the tissue sections of group control showed normal myofibrillar structure with striations, branched appearance and continuity with adjacent myofibrils Fig . Group IThe group H2 BL showed normal myofibrillar structure with striations, branched appearance and continuity with adjacent myofibrils Fig. . The H2 Hibiscus rosa sinensis flower powder caused significant rise in myocardial TBARS as well as with endogenous antioxidants  in the 250 and 500 mg/kg treated groups but not with other baseline treated groups. The increase in TBARS is indicative of an enhanced oxidative stress, which in the absence of any evidence of cellular injury , may be considered as non-lethal. It is, therefore possible that the increase in oxidative stress was non-lethal and might be responsible for cellular adaptive mechanisms. Recent studies show that various plants and plant extracts can also stimulate the synthesis of cellular antioxidants [Hibiscus rosa sinensis flowers [Hibiscus rosa sinensis prevents the oxidative stress and the structural changes associated with IRI. The mechanism of such protection of chronic oral administration of Hibiscus rosa sinensis may be due to myocardial adaptation, oxidative stress is mediated through augmentation of cellular antioxidants such as glutathione, SOD, catalase [In the present study chronic oral administration of oxidants -20. Hibi flowers  and Grap flowers  contain  flowers . The pri flowers ,23-25. Ccatalase . In ischcatalase -29. Althcatalase .in-vivo ischemic reperfusion injury of the 250 mg/kg treated rat, there was a significant decrease in the TBARS but not in the other treatment groups. Significant rise in the levels of GSH, SOD and catalase were observed in all the treatment groups, and it shows better recovery profile along with histological improvement which, was seen only in the 250 mg/kg treated group subjected to in-vivo ischemic reperfusion injury. This indicates that this dosage withstands the oxidative stress associated with in-vivo ischemic reperfusion injury. It is particularly noteworthy that both superoxide dismutase and catalase were increased, since it has been shown that an increase in cellular SOD without a concomitant increase in catalase is more harmful by favoring the generation of H2O2 [In  of H2O2 . The exaHibiscus rosa sinensis are particularly useful agents, as they could enhance myocardial endogenous antioxidants without producing any cytotoxic effects. Histopathological evidence of myocardial injury following ischemic reperfusion injury was also preserved. Therefore, the protection against myocardial ischemic reperfusion injury in the treated rats is attributed to enhanced endogenous antioxidant activity.In this respect, the present study showed for the first time that the flowers of Ischemic heart disease (IHD), Isoproterenol (ISO), ischemia and reperfusion (I-R), ischemic reperfusion injury (IRI), Thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD).The author(s) declare that they no competing interests.MS, TP, VP, KK SJ carried out the experimental work, participated in the sequence alignment, drafted the manuscript and performed the statistical analysis. ND conceived the study, and participated in its design and coordination. KG participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Amyloid is a generally insoluble, fibrous cross-β sheet protein aggregate. The process of amyloidogenesis is associated with a variety of neurodegenerative diseases including Alzheimer, Parkinson, and Huntington disease. We report the discovery of an unprecedented functional mammalian amyloid structure generated by the protein Pmel17. This discovery demonstrates that amyloid is a fundamental nonpathological protein fold utilized by organisms from bacteria to humans. We have found that Pmel17 amyloid templates and accelerates the covalent polymerization of reactive small molecules into melanin—a critically important biopolymer that protects against a broad range of cytotoxic insults including UV and oxidative damage. Pmel17 amyloid also appears to play a role in mitigating the toxicity associated with melanin formation by sequestering and minimizing diffusion of highly reactive, toxic melanin precursors out of the melanosome. Intracellular Pmel17 amyloidogenesis is carefully orchestrated by the secretory pathway, utilizing membrane sequestration and proteolytic steps to protect the cell from amyloid and amyloidogenic intermediates that can be toxic. While functional and pathological amyloid share similar structural features, critical differences in packaging and kinetics of assembly enable the usage of Pmel17 amyloid for normal function. The discovery of native Pmel17 amyloid in mammals provides key insight into the molecular basis of both melanin formation and amyloid pathology, and demonstrates that native amyloid (amyloidin) may be an ancient, evolutionarily conserved protein quaternary structure underpinning diverse pathways contributing to normal cell and tissue physiology. The authors show that native Pmel17 amyloid found in mammalian melanosomes accelerates melanin synthesis. Proteins typically adopt a well-defined three-dimensional structure, but can misfold and form aggregates with a specific cross-β sheet fold called amyloid –4. The msilver locus results in a progressive loss of pigmentation, apparently through loss of melanocyte viability . X. X27]. XrMα amyloid was used to further investigate the native function of Mα amyloid fibers in melanogenesis. The monomeric precursor for melanin polymerization, indole-5,6-quinone (DHQ), is one of the terminal products of a series of oxidation steps initiated by the action of the type I transmembrane enzyme tyrosinase on the substrate tyrosine . MelaninThe discovery of amyloid as a prominent structure in eukaryotic cells now adds the amyloid fold to the repertoire of structures used in normal mammalian cell physiology. We provide a number of lines of in vitro and ex vivo evidence to support this conclusion. Ex vivo melanosomes exhibit selective environment-sensitive thioflavin T and Congo red fluorescence and have detergent-resistant properties expected of amyloid. rMα assembles faster than any known polypeptide into amyloid fibers, at least four orders of magnitude faster than the Aβ and α-synuclein peptides associated with Alzheimer and Parkinson disease, consistent with an evolved sequence that adopts an amyloid structure. The structure produced spontaneously by Mα in vitro exhibits the characteristic X-ray fiber diffraction, CD and FT-IR spectra, and fibrillar morphology expected of amyloid. Finally, rMα amyloid fibers can hasten melanin formation in vitro by serving as a template for DHQ polymerization, thereby recapitulating the fibers' putative native function .E. coli and Salmonella extracellular curli fibers ) and disrupted by Dounce homogenization. The homogenate was centrifuged at 2,000 g at 4 °C for 10 min to obtain a postnuclear supernatant. The postnuclear supernatant was layered onto a sucrose step gradient (0.75 M/1.5 M/2 M sucrose) and centrifuged at 85,000 g for 1 h. The melanosome-rich fraction was collected from the 2 M layer of the gradient and washed in 0.25 M sucrose buffer. To prepare the detergent-insoluble fraction, isolated melanosomes were resuspended in extraction buffer . Triton-X 100 was added from a 10% stock solution to a final concentration of 1%. The suspension was shaken at 4 °C for 2 h. The insoluble fraction was collected by centrifugation at 10,000 g for 1 min and washed three times with extraction buffer.Melanosomes were isolated from the RPE and choroid layers of bovine eyes by sucrose density ultracentrifugation as previously described  with minE. coli. Shaken cultures (1 l) were grown at 37 °C to OD600 = 0.5 in the presence of 270 μM ampicillin and then induced with 1 μM IPTG for 4 h. Cells were collected via centrifugation at 4 °C, resuspended in TBS, and frozen at −80 °C. The resuspended pellet was thawed and the cells were lysed by probe sonication. rMα formed inclusion bodies that were collected by centrifugation, and washed by resuspension followed by centrifugation twice in washing buffer  and then in TBS. The inclusion body pellet was dissolved in extraction buffer  by magnetic stirring at 4 °C for 48 h. The resulting solution was centrifuged, filtered through a 0.22 μM cellulose acetate filter, and then frozen at −80 °C. After thawing, the solution was gel filtered using extraction buffer with a Superdex 200 26/60 column. Purified rMα fractions were assayed via SDS-PAGE and Western blot using the GP100 anti-Pmel17 antibody. rMα was concentrated using a 3-kDa MWCO Centricon filter  and stored at room temperature.The lumenal fragment of Pmel17, rMα, consisting of amino acids 25–467 was subcloned into a pET3c vector and expressed in BL21-DE3 2PO4/K2HPO4, 100 mM KCl, 5 mM EDTA, 20 μM thioflavin T) at the appropriate pH was placed in a stirred cuvette. The solution was excited at 440 nm and data were collected at 485 nm. After data collection had been initiated, an aliquot of concentrated rMα in extraction buffer was rapidly added to a final concentration of 10 μM. Data shown represent several normalized traces that have been averaged. Stagnant thioflavin T binding kinetics were assayed using an Aviv  ATF105 fluorimeter. Stock solutions of Aβ, α-synuclein, and rMα in 8 M GdmCl, 50 mM KH2PO4/K2HPO4 (pH 7.4), and 100 mM KCl were diluted to a final concentration of 10 μM in assay buffer at pH 7.4. The solutions were allowed to aggregate for various amounts of time, whereupon thioflavin T was added from a stock solution to a final concentration of 20 μM. The samples were excited at 440 nm and data were collected at 485 nm. Control experiments were performed to ensure that guanidine-resistant seeds were not affecting Mα amyloidogenesis rates using stock rMα solutions in 8 M GdmCl that were centrifuged for 1 h at 500,000 g. These controls gave identical time courses to experiments in which the centrifugation step was not performed.Thioflavin T binding kinetics were assayed using a Cary Eclipse fluorimeter . Assay buffer . The solution was vortexed and allowed to incubate for 5 min, whereupon Congo red was added from a concentrated stock solution to a final concentration of 20 μM. Absorbance spectra were recorded using a Hewlett-Packard  8453 spectrometer. Units of Congo red bound were calculated using the following equation: OD540/25,295 − OD477/46,306 , 100 mM KCl stock) rMα into 125 mM CH3COOH/CH3COOK buffer (pH 5.0) at a final concentration of 10 μM and allowing it to stand at room temperature for 24 h. rMα aggregates were adsorbed onto 200-mesh carbon-coated copper grids , stained with 1% aqueous uranyl acetate (Electron Microscopy Sciences), and visualized with a Philips  CM100 transmission electron microscope.rMα fibers were generated by diluting  from a concentrated stock solution . Aβ was purchased  and rendered seed-free by dissolving in water, sonicating for 15 min, adjusting the pH to 10.5 using 100 mM NaOH, sonicating for 15 min, filtering through a 0.22-μm syringe filter, and finally filtering through a 10-kDa MWCO concentrator (Centricon). The resulting solution (400 μM Aβ) was mixed in equal volumes with a solution of 600 mM NaCl, 300 mM NaPO4 (pH 7.5), and 0.04% NaN3 and allowed to form amyloid with rocking at 37 °C for 4 d. α-Synuclein was expressed and purified from E. coli using a procedure adapted from Lashuel et al. ) was added to the supernatant, which was then stirred at 4 °C for 60 min. The precipitated proten was removed by centrifugation, NH4SO4 (0.129 g/ml) was added, and the solution was stirred at 4 °C for 60 min. The supernatant was decanted and the pellet resuspended in one-tenth of the culture volume of 10 mM Tris buffer (pH 7.4). The protein was then purified on a source Q column . α-Synuclein-containing fractions were concentrated to one-tenth of their original volume and further purified using a Superdex 200 26/60 column with 100 mM (NH4)2CO3. The purified α-synuclein was then lyophilized and stored at −80 °C until used. Lyophilized α-synuclein was dissolved in 25 mM MES buffer (pH 6.0) and induced to form amyloid by rocking at 37 °C for 48 h. The amyloid nature of the α-synuclein and Aβ aggregates was tested by far-UV CD (which showed β-sheet) and TEM or AFM (which showed fibers). α-Synuclein or Aβ amyloid was collected by centrifugation  and resuspended in assay buffer at a final concentration of 0.5 mg/ml. Collagen IV  was rendered insoluble by lyophilization and resuspended in assay buffer at a final concentration of 0.5 mg/ml. These solutions were vortexed, and 10 μg of tyrosinase was added. The solutions were allowed to react at room temperature for varying periods of time, after which they were centrifuged at 16,000 g for 15 min. The pellets  were resuspended in 125 mM CH3COOH/CH3COOK buffer (pH 5.0) and centrifuged again. The pellets were then resuspended in 1 M NaOH and then heated to 60 °C for 5 min and vortexed to effect dissolution. Absorbance spectra were recorded at 350 nm.The ability of rMα to enhance melanogenesis was evaluated using D,L-DOPA (Sigma) and tyrosinase . rMα (0.5 mg) was added to 1.0 ml of fresh assay buffer .Click here for additional data file.http://www.ebi.ac.uk/swissprot/) accession numbers for the gene products discussed in this paper are Pmel17 (P40967) and type I transmembrane enzyme tyrosinase (P14679).The Swiss-Prot (


Inhibiting ERK and JNK pathways significantly reduced PAI and uPA induction and inhibited the invasive cell phenotype. These results suggest that TGFβ Smad-dependent signalling is perturbed in Barrett's carcinogenesis, resulting in failure of growth-arrest. However, TGFβ can promote PAI and uPA expression and invasion through MAPK pathways. These data would support a dual role for TGFβ in oesophageal adenocarcinoma.In cancer, Transforming Growth Factor β (TGFβ) increases proliferation and promotes invasion via selective loss of signalling pathways. Oesophageal adenocarcinoma arises from Barrett's oesophagus, progresses rapidly and is usually fatal. The contribution of perturbed TGFβ signalling in the promotion of metastasis in this disease has not been elucidated. We therefore investigated the role of TGFβ in Barrett's associated oesophageal adenocarcinoma using a panel of cell lines . 4/5 adenocarcinoma cell lines failed to cell cycle arrest, down-regulate c-Myc or induce p21 in response to TGFβ, and modulation of a Smad3/4 specific promoter was inhibited. These hyperproliferative adenocarcinoma cell lines displayed a TGFβ induced increase in the expression of the extracellular matrix degrading proteinases, urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1), which correlated with an invasive cell phenotype as measured by  Transforming Growth Factor β (TGFβ), and the components of its signal transduction pathway, are known to demonstrate tumour suppressor activity. In many human cancers, there are inactivating mutations in components of the TGFβ pathway resulting in uncontrolled proliferation. In addition, through its actions on both the tumour cells and the surrounding stromal cells, TGFβ can enhance invasion, motility and metastasis. TGFβ can thus play a dual role in the initiation and malignant progression of human cancer INK4B, and the cdk2 inhibitor p21CIP1/WAF1. Another key event in TGFβ mediated cell cycle arrest involves the repression of c-Myc. This transcription factor binds the promoters of p15INK4B and p21CIP1/WAF1 and suppresses their expression 1, and thus potently inhibit proliferation TGFβ signalling occurs through activation of type I and II trans-membrane serine/threonine kinase receptors, which leads to the phosphorylation of Smad 2 and 3. This complex, in association with Smad 4 translocates to the nucleus resulting in transcriptional activation of downstream targets TGFβ is frequently overexpressed by epithelial cancer cells which become unresponsive to its anti-proliferative effects and this leads to paracrine stimulation of stromal cells within the tumour microenvironment in vivo and in vitro evidence suggests that inactivation of SMAD4, lack of RUNX3 and inability to degrade SnoN may be some of the causes for the unresponsiveness of oesophageal cancer to TGFβ anti-proliferative effect In contrast to the plethora of information about mutations in the TGFβ signal cascade in colon, gastric and pancreatic cancers c-jun N-terminal kinase (JNK) The signalling pathways by which TGFβ exerts its effects on migration and invasion are gradually being elucidated. The activated TGFβ receptor complex is now known to activate kinase pathways independently of Smad signalling and these have been shown to stimulate the expression of both uPA and PAI The specific aims of this study were therefore: 1). To determine whether TGFβ can simultaneously affect proliferation  and invasion indices  in oesophageal adenocarcinoma cell lines; 2). To elucidate the cell signalling pathways involved in these responses.CIP/WAF1 and a decreased expression of c-Myc, the effect of TGFβ on these mRNA expression levels was examined. Real time PCR demonstrated that only OE33 cells exhibited a significant increase in p21 and decrease in c-Myc expression , which is known to induce cell cycle arrest, and cells treated with SF media followed by release into the cell cycle by complete media (C), . All celpression , , and an increase in the ability of PAI to retard the action of uPA in all cells lines analysed (increase in the dark band intensity), . Inhibitin vitro assays: aggregation, invasion and wounding. An aggregation assay measures the ability of cells to grow in an anchorage-independent manner; invasion assays through matrigel measures the ability of cells to degrade the basement membrane and to migrate through it while the wounding assay only measures the ability of cells to migrate to occupy an empty space. Therefore a highly invasive cell in vitro should not form aggregates, should migrate through matrigel and to close up a wound. TGFβ had significant effects on at least one assay for each of the adenocarcinoma cells lines, compared to the control (no TGFβ), (We determined the ability of TGFβ to influence the invasiveness of cells using three o TGFβ), . In ordeTherefore, aggregation was performed for TE7, SEG, FLO and OE33, invasion for TE7, SEG and OE33 and wounding for OE33. With regards to aggregation, the presence of all three kinase inhibitors diminished the cell scattering ability (a higher score denotes less scattering) of TGFβ in all the cell lines to a greater or lesser extent . The incin vitro. 4/5 BE cell lines are unable to mount an anti-proliferative response to TGFβ. However, the loss of TGFβ responsiveness is selective, such that the same cell lines that fail to exhibit an anti-proliferative response demonstrate a TGFβ dependent increase in the expression of PAI and uPA via PI3K, ERK and JNK  in BE adenocarcinoma cells . This isin vitroThe combination of uPA, uPA receptor and PAI is crucial in the modulation of ECM homeostasis and tumour invasion as demonstrated in breast and prostate cancer cells in vivo. For example, in prostate, breast and pancreatic carcinoma cells, JNK, or the upstream activator of both kinases, MKK4, has been demonstrated to mediate growth of cells in vitro, and as xenografts in vivouPA and PAI expression through the activity of ERK and JNK may also be relevant for tumour progression In conclusion, we have shown that there is a divergent response to TGFβ in cells from BE adenocarcinoma. The Smad dependent anti-proliferative response is lost; however, the ability of TGFβ to induce ECM genes PAI and uPA prevails. This occurs predominantly through the ERK and JNK MAPK pathways, and thus contributes to the invasive properties of the oesophageal tumour cell. Such a differential response utilising different cell signalling pathways may allow TGFβ to play a role in both the initiation and progression of oesophageal adenocarcinoma.For this study cell lines derived from Barrett's adenocarcinoma were used. Barrett's associated adenocarcinoma cell lines OE33 , TE7 , were maintained in RPMI-1640, and SEG, BIC and FLO  were maintained in DMEM. All cells were supplemented with 10% (FBS), 100 U/mL penicillin, 100 mg/mL streptomycin and 2 mM glutamine.Cells were grown to 70% confluence in complete medium, which was then replaced with serum free medium for 3 h. Cells were pre-treated for 1 h with inhibitors at the minimal concentration leading to kinase inhibition : PI3K inhibitors - Wortmannin (5 nM) and LY294002 (20 µM) ; ERK inhibitors - U0126 (inhibits MEK 1 and 2), (10 µM) and PD098059 (inhibits MEK 1), (20 µM) ; and JNK inhibitors - SP600125 (20 µM) (Sigma-Aldrich) and curcumin (10 µM) . Cells were then treated with 10 ng/mL TGFβ1 for 24 h in the presence of inhibitors. This concentration was used as we have previously shown in dose response experiments that it is the optimum concentration to induce a decrease in proliferation 4 cells were seeded in 6 well plates in complete growth medium, and all cells were synchronised overnight in serum-free medium. Cells were then either incubated in complete medium with or without 10ng/mL recombinant TGFβ1 , or kept in serum free medium, for 24 h. Cells were harvested, fixed in 70% ethanol at 4°C and then incubated in PBS, 0.1% Tween 20 containing 2 µg/mL propidium iodide (Sigma-Aldrich) and 200 µg/mL RNAse A for 30 min at 37°C. DNA content was analysed by fluorescence associated cell sorting (FACS) using the BD LSR1 flow cytometer , and data captured using Cell quest software .10Cells were seeded overnight onto sterilised coverslips in a 24 well plate. Media was removed and replaced with fresh media with or without 10 ng/ml TGFβ1. Following a 6 h incubation, media was removed and the coverslips washed twice in 0.5 ml PBS. The coverslips were then stained with Smad2/3 antibody  following a procedure described previously 4 cells/well and left overnight. Using Fugene6 transfection reagent  cells were transfected with the reporter plasmid (CAGA)12-Luc, containing 12 repeats of the CAGA Smad3/4 binding sequence. Cells were also transfected with Renilla luciferase reporter plasmid to normalise transfection efficiency. After 24 h medium was replaced with serum free medium with or without TGFβ1 (10 ng/mL). After a further 24 h luciferase activity was determined using the Dual Luciferase Reporter System  in a TD 20/20 Luminometer (Promega).Cells were seeded in 24 well plates at a density of 5×10T) was obtained and relative quantities determined compared to β actin.Two micrograms of total RNA isolated using Trizol Reagent  were reversed transcribed, and 1 µL of cDNA was amplified in a 50 µl volume containing 25 µL of 2× Quantitect SYBR Green PCR Master Mix  and 0.2 µM of each primer . TriplicμL of immunoprecipitated sample or 50 µg of proteins were then separated by gel electrophoresis on 8% polyacrylamide gels and transferred to a PVDF membrane . Membranes were incubated overnight at 4°C with the following antibodies: phospho-ERK1/2 (0.1 µg/mL) , phospho-JNK (1∶1000) , phospho-Akt (New England Biolabs) or pSmad2/3 . Membranes were incubated with a 1∶10,000 dilution of peroxidase conjugate secondary IgG antibody , and signals detected by chemiluminescence .Immunoprecipitation was required due to the low sensitivity of western blotting alone for the phosphorylated forms of AKT, ERK and JNK. Protein lysates were prepared with ice-cold lysis buffer  containing protease inhibitors . 200 µg of protein was immunoprecipitated with 1 µg ERK1/2 antibody , JNK antibody (1∶100)  and Akt antibody (1∶1000) . 20To assess uPA activity 50 µg of protein lysate were incubated with non-reducing buffer for 20 min at 37°C, then electrophoresed in a polyacrylamide gel containing 2 mg/mL α-casein (Sigma-Aldrich) and 5 µg/mL plasminogen (Sigma-Aldrich). Gels were washed 2 times for 30 min each in 50 mM Tris-HCl (pH 7.6), 2.5% Triton X-100, incubated for 16 h at 37°C in 50 mM Tris-HCl (pH 7.6) and then stained for 1 h in a solution of 30% methanol, 10% acetic acid and 0.5% Coomassie brilliant blue G250 and de-stained. Urokinase activity was detected as digested clear bands on a blue background. To determine PAI activity lysates were subjected to SDS-PAGE in gels containing 2 mg/mL α-casein, 5 µg/mL plasminogen and 0.8 U/mL human urokinase . Gels were then washed and stained as above. PAI activity was detected as dark undigested bands against a light background.Cells were grown in a 60 mm dish to confluency, and then wounded using a hand held engraving drill (Minicraft MB168) fitted with a cut down 10 µL pipette tip. Wound area was measured using an eyepiece graticule and calibrated using a haemocytometer grid, at 10× magnification. The wounded monolayer was washed with PBS, and then cultured in serum free medium, containing 10 ng/mL TGFβ1 alone or with LY294002 (20 µM), PD98590 (20 µM) or SP600125 (20 µM) for 24 h. The wound area was again measured, the absolute change in wound size deduced and the % healing at 24 h then calculated. In each experiment 8 wounds were made and each experiment was repeated at least three times.5 cells were resuspended in 200 µl of serum free RPMI containing 10 ng/mL TGFβ1 alone or with LY294002 (20 µM), PD98590 (20 µM) or SP600125. Cells were then applied to the upper compartment of the invasion chamber. In the lower compartment 500 µl of serum containing medium was added and the cells were then incubated at 37°C for 24 h. Medium was then removed and the cells were then fixed for 20 min in methanol at −20°C. Chambers were stained in haematoxylin for 1 min and a cotton swab used to remove non-invasive cells in the upper compartment. Membranes were excised from the chamber and fixed on a microscope slide. Membranes were quantified by counting the number of cells that had invaded the lower chamber in 5 high-power fields.Fifty µL of a 10 mg/mL solution of matrigel (BD Biosciences), containing 25 µg/mL plasminogen was applied to the upper compartment of a Transwell 24 well insert (BD Biosciences) of 8 µm pore size, and incubated at 37°C. Cells were washed three times in serum free RPMI, and then 1×104 cells (100 µl) were seeded into each well and left for 1 h. 100 µL of serum free medium containing TGFβ1 alone or with LY294002 (20 µM), PD98590 (20 µM) or SP600125 (20 µM). 2 using an inverted microscope. Replicates were scored 0 if no aggregates were present, 1 for small aggregates and 2 for large aggregates.100 µL of agar was solidified in 96 well plate formats. Cells were washed with Moscona's solution, trypsinised and then resuspended in serum free media. 2×10The Kruskal-Wallis test was used to compare values between different data sets, and the Dunn's Multiple Comparison Test was used to identify specific differences, using Graphpad Prism software. A p<0.05 was necessary for statistical significance.Table S1(0.04 MB DOC)Click here for additional data file.Figure S1Densitometry analysis of phosphorylated MAPK. The western blots (as presented in (10.47 MB TIF)Click here for additional data file.Figure S2Densitometry analysis of uPA and PAI activity. The zymographies (as presented in (9.82 MB TIF)Click here for additional data file.