Electroconvulsive therapy (ECT) often results in a number of short- and long-time side effects including memory impairment for past and current events, which can last for several months after ECT treatment. It has been suggested that unilateral ECT (uECT) with electrodes placed over the non-dominant  hemisphere significantly reduces side effects, especially memory disturbances. It is important to note that cerebral dominance equates to speech dominance and avoiding this area of the brain also reduces speech dysfunction after ECT. Traditionally, the routine clinical determination of cerebral dominance has been through the assessment of hand, foot and eye dominance, which is an easy and inexpensive approach that, however, does not ensure accuracy. This review of literature on different methods and techniques for determination of cerebral dominance and provides evidence that functional transcranial Doppler sonography (fTCD) represents a valid and safe alternative to invasive techniques for identifying speech lateralisation. It can be concluded that fTCD, notwithstanding its costs, could be used as a standard procedure prior to uECT treatment to determine cerebral dominance, thereby further reducing cognitive side-effects of ECT and possibly making it more acceptable to both patients and clinicians. Electroconvulsive therapy (ECT) is often regarded by the general public as a controversial procedure for the treatment of mental disorders. This is despite evidence of its safety and efficacy , and itsA further possible explanation for the reduction in ECT use may relate to the concern over adverse effects of the procedure. There are a number of short-term side effects including headache, nausea and, sometimes, brief confusion. However, the main side effect of concern is memory impairment for past events (retrograde amnesia) and for current events (anterograde amnesia) that can last for several months after a course of ECT treatment. Some of these side effects are substantially reduced by advances in safety and the introduction of controlled-current ECT machines. The utilisation of muscle relaxants, anaesthetics and resuscitation equipment, and electroencephalographic monitoring during the application of ECT are considered now considered routine. In addition, ECT guidelines issued by the UK National Institute for Clinical Excellence  restrictIt has been suggested that unilateral treatment significantly reduces side effects, especially memory disturbances ,7. DespiThe practice of determining cerebral dominance from handedness appears to mirror Broca's view that a person's handedness is opposite to hemispheric language specialisation. This, however, is incorrect, since there is no "mirror-image" cortical language organisation in left-handers. Several attempts to improve cerebral dominance assessment by introducing additional clues such as handwriting posture (i.e. inverted or hooked style versus non-inverted) and familial sinistrality have not substantially improved the prediction as to determination of cerebral dominance ,13. For A great majority of left-handers have also an ipsilateral functional specialisation for language (i.e. left hemispheric, as in the majority of right-handers). Although right-handers are more clearly lateralised than left-handers in this regard, a certain proportion of right-handers have language localised in the right-hemisphere. This has been confirmed by various techniques, ranging from the old and invasive procedures such as the intracarotid sodium amytal test and ECT, to the new and more sophisticated techniques such as functional magnetic resonance imaging (fMRI) and functional transcranial Doppler sonography (fTCD). The pooling of empirical data from a number of studies -27 whichFrom Table Until recently, an accurate determination of speech dominance prior to a course of ECT treatment was possible only through invasive procedures such as intracarotid sodium amytal test , also knThe advent of sophisticated and non-invasive technologies during the 1980s and 1990s has enabled a non-invasive approach to the assessment of speech dominance. One of the most elegant, mobile, and cost effective methods for determining cerebral dominance for speech is functional transcranial Doppler sonography (fTCD). fTCD is increasingly used in both clinical and research settings and is a new and robust technique based on the same principles as fMRI. Subjects in studies using this method are asked to generate as many possible words within 5-second periods after a letter presented on the computer screen cues for word generation. Basically, fTCD measures cerebral blood flow velocity which corresponds to brain activity. The physical foundation for this technique is quite old and is based on the work of the Austrian mathematician and physicist, Christian Doppler (1803–1853), who discovered that the change in pitch results from a shift in the frequency of the sound waves. This means that the speed of a physical object (i.e. blood) can be estimated by measuring the rate of change of pitch. To complete the fTCD procedure, the additional sound produced through the arteries is required.Recently, it has been argued that fTCD can reliably replace the Wada procedure in patients undergoing brain surgery . The valThis review of the literature on ECT and cerebral dominance provides evidence that fTCD represents a valid and safe alternative to invasive techniques for identifying speech lateralisation. It seems therefore, that fTCD, notwithstanding costs, could be used as a standard procedure prior to uECT treatment to determine cerebral dominance, thereby further reducing cognitive side-effects of ECT and possibly making it more acceptable to both patients and clinicians.none declared.


Bisdioxopiperazine anti-cancer agents are inhibitors of eukaryotic DNA topoisomerase II, sequestering this protein as a non-covalent protein clamp on DNA. It has been suggested that such complexes on DNA represents a novel form of DNA damage to cells. In this report, we characterise the cytotoxicity and DNA damage induced by the bisdioxopiperazine ICRF-187 by a combination of genetic and molecular approaches. In addition, the well-established topoisomerase II poison m-AMSA is used for comparison.Saccharomyces cerevisiae single-gene deletion strains, homologous recombination was identified as the most important DNA repair pathway determining the sensitivity towards ICRF-187. However, sensitivity towards m-AMSA depended much more on this pathway. In contrast, disrupting the post replication repair pathway only affected sensitivity towards m-AMSA. Homologous recombination (HR) defective irs1SF chinese hamster ovary (CHO) cells showed increased sensitivity towards ICRF-187, while their sensitivity towards m-AMSA was increased even more. Furthermore, complementation of the XRCC3 deficiency in irs1SF cells fully abrogated hypersensitivity towards both drugs. DNA-PKcs deficient V3-3 CHO cells having reduced levels of non-homologous end joining (NHEJ) showed slightly increased sensitivity to both drugs. While exposure of human small cell lung cancer (SCLC) OC-NYH cells to m-AMSA strongly induced γH2AX, exposure to ICRF-187 resulted in much less induction, showing that ICRF-187 generates fewer DNA double strand breaks than m-AMSA. Accordingly, when yeast cells were exposed to equitoxic concentrations of ICRF-187 and m-AMSA, the expression of DNA damage-inducible genes showed higher levels of induction after exposure to m-AMSA as compared to ICRF-187. Most importantly, ICRF-187 stimulated homologous recombination in SPD8 hamster lung fibroblast cells to lower levels than m-AMSA at all cytotoxicity levels tested, showing that the mechanism of action of bisdioxopiperazines differs from that of classical topoisomerase II poisons in mammalian cells.By utilizing a panel of Our results point to important differences in the mechanism of cytotoxicity induced by bisdioxopiperazines and topoisomerase II poisons, and suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms. Type II topoisomerases are essential nuclear enzymes found in all living organisms . Their bTopoisomerase II is also a major drug target in human cancer therapy, where a number of clinically active drugs such as the epipodophyllotoxins VP-16 and VM-26, the aminoacridine m-AMSA, and antracyclines such as doxorubicin, daunorubicin and epirubicin are widely used. These drugs have collectively been called topoisomerase II poisons due to their mechanism of action on topoisomerase II. Rather than inhibiting the basic catalytic activity of the enzyme, these drugs perturb the topoisomerase II catalytic cycle resulting in an increase in the level of a transient reaction intermediate, where DNA is cleaved and covalently attached to DNA .Catalytic inhibitors of topoisomerase II have a different mode of action. These drugs exemplified by merbarone, aclarubicin, F11782 and the bisdioxopiperazines work by inhibiting topoisomerase II at other stages in the reaction cycle where DNA is not cleaved as reviewed in ,6. Amongi) Expression of bisdioxopiperazine-sensitive topoisomerase II in cells also expressing bisdioxopiperazine-resistant topoisomerase II confers dominant sensitivity to these drugs [ii) Mouse embryonic stem cells [iii) Killing of yeast cells by exposure to ICRF-193 occurs more rapidly and to a higher level than killing of yeast cells induced by the depletion of endogenous topoisomerase II catalytic activity [iv) The ICRF-193-induced topoisomerase II closed clamp complexes on DNA work as a "road block" signalling selective degradation of topoisomerase II β as well as p53 activation in a transcription dependent fashion [Several studies indicate that the closed clamp complex on DNA represents a novel form of DNA lesion to cells, – and that inhibition of topoisomerase II catalytic activity (DNA strand passage activity) is not responsible for bisdioxopiperazine-induced cell kill: (se drugs ,11 – a mem cells  and chicem cells  having oactivity . (iv) Th fashion .in vitro and in vivo [Some studies have recorded elevated levels of DNA breaks in cells after exposure to the bisdioxopiperazine analog ICRF-193. In one study, ICRF-193 was found to increase the level of topoisomerase II-DNA covalent complexes  in vivo . However in vivo . In this in vivo .Based on these observations, the present study aims to further elucidate the mechanism of cytotoxicity induced by the bisdioxopiperazines. We here characterise the effect of the clinically approved analog ICRF-187 (dexrazoxane) by using a number of different cell-based pharmacological assays, taking advantage of genetically modified yeast and mammalian cells.To pinpoint the mechanism of cytotoxicity of ICRF-187 versus m-AMSA, we employed a panel of human topoisomerase II α-transformed haploid single-gene knockout yeast strains, defective in various aspects of DNA repair, checkpoint control, membrane transport and protein degradation. All yeast strains are depicted in table RAD50, MRE11 and XRS2 together form the Rad50/Mre11/Xrs2 hetero-trimer protein complex that has catalytic and structural functions in many kinds of DNA metabolic processes including HR as reviewed in [rad50, Δmre11, and Δxrs2 single knockout strains were extremely hypersensitive towards m-AMSA, while they displayed considerably less hypersensitivity towards ICRF-187  (rad6 cells are hypersensitive towards m-AMSA due to impaired PRR activity. The involvement of both HR repair and PRR in determining the sensitivity of yeast cells towards the topoisomerase II cleavage complex stabilising drugs mitoxantrone and idarubicin has previously been reported [rev1 and Δrev3 strains (The DNA binding Rad18p forms a hetero-dimer with Rad6p that is involved in post replication repair (PRR) . We founA figure . Interesactivity , and theCRF-187) , suggestreported . The obs strains  suggestsRAD1 and RAD14, the base excision repair (BER) pathway – APN1, and the mismatch repair (MMR) pathway – MLH1, PMS1, MSH2 and MSH3. None of these deletions caused cells to become more sensitive towards ICRF-187 and m-AMSA, indicating that these pathways are not involved in repairing DNA damage induced by these drugs. Interestingly, deleting genes involved in the MMR and NER pathways caused cells to become somewhat resistant to ICRF-187, and to a lesser extent towards m-AMSA  pathway – MEC3, DDC1, RAD17, TEL1, RAD9 and CHK1  as reviewed in . Among ts m-AMSA , suggestt2–4 yeast cells expressing human topoisomerase II α as their sole active topoisomerase II to equitoxic doses of ICRF-187 and m-AMSA for two hours at 34°C. This treatment resulted in a 50 % reduction in clonogenic survival after exposure to both drugs  have wild-type sensitivity towards both drugs .The yeast clonogenic assays presented above point to an important role of HR in the repair of m-AMSA-induced DNA damage, while the importance of this pathway in the repair of ICRF-187-induced DNA damage is less so. Because HR is the major repair pathway in yeast , while bXRCC3 fully reversed the m-AMSA hypersensitivity as CXR3 cells were no more hypersensitive than AA8 wild-type cells, confirming the notion that HR plays a role in the repair of topoisomerase II-induced DNA breaks in mammalian cells. We also observed that irs1SF cells were hypersensitive towards ICRF-187 (figure XRCC3 homolog reversed the hypersensitivity as CXR3 cells displayed near wild-type sensitivity towards ICRF-187.We observed a strong dependence on HR for the sensitivity towards m-AMSA figure . Thus, o7 figure , but thecs deficient, with concomitant reduced level of NHEJ). The results of these experiments are depicted in figure To assess the effect of NHEJ on drug sensitivity we also employed the V3-3 cell line  activity in crude protein extracts isolated from the four cell lines used in clonogenic assays, by applying a radioactive decatenation assay. No significant difference in the level of topoisomerase II DNA strand passage activity was recorded between the four cell lines . This rehprt gene by the activity of HR. From figure The hypersensitivity of the recombination defective irs1SF cells towards both drugs suggests that HR is involved in repairing DNA lesions induced by both drugs. To address this directly we applied a mammalian recombination assay to measure stimulation of HR by ICRF-187 and m-AMSA by using SPD8 hamster cells . This asInduction of γH2AX is a well-established marker for topoisomerase-induced DNA double strand breaks in mammalian cells -42. We trad52-cells had the same sensitivity towards ICRF-187 and ICRF-193 as did RAD52+ cells [We initiated the study by assessing the clonogenic sensitivity of yeast single-gene deletion mutants ectopically expressing human topoisomerase II α towards m-AMSA and ICRF-187. The results presented in table 2+ cells ,44. Our 2+ cells . The selXRCC3 deficient irs1SF cells is general to topoisomerase II poisons, suggesting a role for HR in the repair of topoisomerase II-induced DNA breaks in these cells. The involvement of HR in the repair of DNA lesions induced by topoisomerase II poisons in higher eukaryotes is also supported by a recent work suggesting that RAD51 plays an important role in the repair of etoposide-induced DNA damage in human small cell lung cancer cells [RAD54 in chicken DT40 cells enhances their sensitivity towards the topoisomerase II poison etoposide [We find that the relative sensitivity of AA8, irs1SF, and CXR3 cells towards m-AMSA  Functional ATR, but not ATM, is required for a cell cycle checkpoint arrest induced by ICRF-193 [ii) Exposure of mammalian cells to the topoisomerase II poison etoposide induces degradation of the large subunit of RNA polymerase II indicative of DNA breaks, while this is not the case for ICRF-193 [iii) Our finding that cell survival in the presence of ICRF-187 depends less on HR than cell survival in the presence of m-AMSA suggests that ICRF-187-induced DNA breaks contribute less to overall cytotoxicity than m-AMSA-induced DNA breaks. If ICRF-187-induced DNA breaks were more toxic to cells than m-AMSA-induced DNA breaks, cell survival in the presence of ICRF-187 would be expected to depend at least as much on HR as cell survival in the presence of m-AMSA. This is not the case.The finding that ICRF-187 induces lower levels of HR than m-AMSA in SPD8 cells at equitoxic doses may be explained in at least two ways. Bisdioxopiperazine-induced DNA breaks could be more toxic to cells than breaks induced by topoisomerase II poisons, or the DNA breaks could be only partly responsible for killing the cells. Three lines of evidence support the latter possibility. (ICRF-193 , suggestICRF-193 . (iii) OWhat mechanisms are then responsible for producing the DNA breaks induced by bisdioxopiperazines in cells? In a recent work by Oestergaard and colleagues).), it is sIn a recent study by Lundin and colleagues, it was demonstrated that inhibiting DNA replication by exposing cells to hydroxyurea resulted in the generation of DNA breaks . FurtherTogether our HR, γH2AX, and cytotoxicity data suggest that bisdioxopiperazines kill cells by a combination of DNA break-related and DNA break-unrelated mechanisms. This raises the question as to which mechanism(s) is / are involved in mediating the DNA break-unrelated part of bisdioxopiperazine cytotoxicity. Exposure of mammalian cells to ICRF-193 represses global transcription and mediates selective degradation of topoisomerase II β via a transcription dependent mechanism . InhibitTreatment of mammalian cells with high doses of ICRF-187 for one hour is capable of antagonising DNA breaks and the cytotoxicity of topoisomerase II poisons ,56, and To summarise, our data are consistent with a model where bisdioxopiperazine-induced cytotoxicity results from a combination of DNA break-related and -unrelated mechanisms, where the DNA-break unrelated mechanism is clearly not mediated by the inhibition of catalytic topoisomerase II activity in the cells.Since the discovery by Andoh and colleagues in 1991, that the bisdioxopiperazines target eukaryotic topoisomerase II ,64, theiICRF-187 (Cardioxane, Chiron group) was dissolved in sterile water at 20 mg/ml and kept at – 80°C. To avoid hydrolysis of the drug, fresh aliquots were used for each experiment. m-AMSA (Pfizer) was diluted in DMSO and stored at – 80°C at 1 mg/ml. L-azaserine and thymidine (both from Sigma) were added directly to tissue culture medium. 6-thioguanine and hypoxanthine (both from Sigma) were dissolved in 5 M NaOH and immediately added to the tissue culture medium.Saccharomyces cerevisiae cells (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0) and a panel of single-gene deletion derivatives hereof (table t2–4 cells with the relevant genotype (MATa ura3–52 leu2 trp1 his7 ade1–2 ISE2 top2-4) were kindly provided by Dr. John L. Nitiss, St. Jude Children's Research Hospital, Memphis TN, USA. This strain and the construct for functional expression of human topoisomerase II α in yeast pMJ1 (URA3) have been described previously [rad6, Δrad50, Δrad52, Δsae2 and Δyku70 cells were also transformed with an empty URA3 vector (pYX112). The ICRF-187 and m-AMSA sensitivity of pYX112-transformed cells was assessed to assure that the drug sensitivity of the pMJ1-transformed cells (Table BY4741 haploid eviously . All yea6 cells/ml in pre-warmed YPD medium, and 3 ml cultures were exposed to different concentrations of drug at 34°C for 22.5 hours. After drug exposure the samples were diluted up to 105 times (depending on the combination of strain and drug used) in distilled sterile water. Yeast cells that were not diluted before plating were spun down by brief centrifugation, and re-suspended in the same volume of sterile water. Next, 200 μl of diluted cells were transferred to SC-URA plates, which were incubated for 5 days at 30°C before counting. 200 to 600 colonies were typically counted for each drug concentration in each single experiment. Finally, the relative survival at the different drug concentrations as compared to the no drug sample was calculated to generate dose-response curves. For each combination of yeast strain and drug, at least three dose-response curves were generated using pMJ1-transformed cells from at least two independent clones (mostly from three).The clonogenic sensitivity of the yeast cells towards ICRF-187 and m-AMSA was determined using a clonogenic assay essentially performed as described in [t2–4 transformed with pMJ1 to functionally express human topoisomerase II α. Fresh colonies were inoculated into YPD medium and grown overnight at 34°C, 180 rpm. The cultures were then diluted to obtain an OD600 of 0.2. Cultures of 50 ml in YPD medium were first grown for two hours to assure exponential growth of the cells. 1 mg/ml ICRF-187 or 50 μg/ml m-AMSA (equitoxic concentrations) were then added to the cell cultures , and the cells were grown for an additional two hours. Each treatment was performed in duplicate. The used concentration of both drugs resulted in a reduction in the clonogenecity of the cells of 50 %. After treatment cells were harvested by centrifugation. Total RNA was isolated by the hot acidic phenol method [24 primer containing a T7 RNA polymerase promoter sequence and SuperScript II reverse transcriptase (Invitrogen) for 1 h at 42°C followed by second-strand synthesis using DNA polymerase I and RNase H digestion followed by isolation of cDNA using GeneChip Sample Cleanup Module (Affymetrix). The cDNA was used as template for synthesis of biotin-labeled cRNA by incubation with biotin-labeled ribonucleotides and T7 RNA polymerase for 5 h at 37°C. Biotin-labeled cRNA was purified using GeneChip Sample Cleanup Module. Biotinylated cRNA was fragmented and 15 μg used for hybridization to Affymetrix Yeast Genome S98 arrays at 45°C for 16 h as described in the Affymetrix users' manual. Washing and array staining with streptavidin-phytoerythrin were performed using the GeneChip Fluidics Station 400 and scanning was performed with a Gene Array Scanner G25 (Agilent technology). Data was analyzed using the DNA-Chip Analyzer (dChip) software [Microarray experiments were performed with yeast strain JN362l method . All thesoftware . using the Primer 3 input program . All primers were purchased at DNA Technology A/S, with melting temperatures close to 60°C. Reaction mixtures containing the following components at the indicated end-concentrations were prepared. To make a total of 40 μl in sterile water, 20 μl 1x SYBR® green PCR master mix (Applied Biosystems), 250 nM forward primer, 250 nM reverse primer, and 5 ng template was mixed. Cycling conditions: 95°C for 10 min, followed by 40–45 cycles of 95°C for 15 s and 60°C for 60 s. Relative values of gene expression were calculated with untreated samples as calibrator, and normalized to levels of actin, according to the 2-ΔΔCt method [The RNA preparations used for microarray analyses were also used for real-time PCR. This analysis was performed on an ABIPrism 7900HT (Applied Biosystems). RNA samples were DNase treated using the DNA-free™ DNase treatment and removal kit (Ambion), and RNA concentrations were measured before conversion to cDNA using the TaqMan RT kit (Applied Bioscience). Priming was performed by random hexamers converting 2 μg RNA pr 100 μl reaction volume, to make 20 ng/μl cDNA. Primers were designed for coding sequences from the Saccharomyces genome database t method  and , unprocessed kDNA network and decatenated DNA circles were separated by filtering, and the amount of unprocessed kDNA in each reaction was determined by scintillation counting. The amount of crude extract required to fully decatenate 200 ng of kDNA under these assay conditions  was then determined, and the specific activity of the crude extract was calculated as U/μg protein.Topoisomerase II activity in crude extract was determined by using a decatenation assay previously described . BrieflyFour hours prior to continuous treatment with either ICRF-187 or m-AMSA, 250 cells of each of the hamster cell lines were plated onto 100 mm dishes. After 7 days colonies were fixed and strained in methylene blue in methanol (4 mg/ml), and colonies with more than 50 cells were counted. Finally, the relative survival compared to the no drug treatment was calculated, and plotted against drug concentrations to generate dose-response curves. 150 – 250 colonies were typically counted in the "no drug" dishes.6 SPD8 cells were inoculated into 75 cm2 flasks. When transferred, 6-thioguanine was omitted from the medium. Cells were trypsinised and resuspended in 10 ml medium at 100,000 cells/ml, and exposed to the indicated drug concentrations for 24 hours. To determine clonogenic survival, for each drug-treatment 500 cells were transferred to each of two 100 mm petri dishes containing 10 ml of non-selecting medium and the cells were cultured for 7 days. For selection of recombination events, 300,000 cells were transferred to each of three 100 mm petri dishes containing 10 ml medium supplemented with 50 μM hypoxanthine, 10 μM L-azaserine and 5 μM thymidine and selection was carried out for 10 days. Colonies were fixed by using methylene blue in methanol (4 mg/ml) and counted. Finally, the recombination frequency was determined as the plating efficiency in recombination selective medium divided by the plating efficiency in normal medium, for all concentrations of ICRF-187 or m-AMSA. To enable comparison of recombination frequency at equitoxic levels of m-AMSA and ICRF-187, the recombination frequency was plotted against the relative clonogenic survival of cells receiving only drug.A mammalian recombination assay was performed as described . Briefly2SO4, and incubated for 1 hour at 4°C prior to centrifugation. The supernatant was transferred to a clean tube and 1 ml ice-cold acetone was added followed by incubation overnight for histone precipitation. After centrifugation, the pellet was air-dried and resuspended in 40 μl H2O, and the protein concentration was determined by Bradford protein assay (Bio-Rad Laboratories)Human SCLC OC-NYH cells were grown to sub-confluence and histones were extracted as follows. After the relevant drug treatments, the cells were pelleted and washed in cold PBS, and lysed in lysis buffer (10 mM TRIS-HCl pH = 6.5, 50 mM Sodium Bisulphate, 1% Triton X-100, 10 mM MgCl, 8.6% sucrose) at 4°C by applying 20 strokes in a tight fitting Dounce homogenizer. Released nuclei were pelleted by centrifugation at 2500 g for 10 min at 4°C, and washed in lysis buffer followed by wash buffer (10 mM TRIS-HCl, 13 mM EDTA pH 7.4). The pellet was next resuspended in 100 μl ice-cold 0.4 M HWestern blotting was performed by loading 10 μg of total histones on a 4–12% gradient gel (NuPageTM Bis-Tris Gel, Invitrogen). Separated proteins were transferred to nitrocellulose membranes (Bio-Rad) which were blocked in 10% skimmed milk (Fluka) and incubated overnight with anti γH2AX primary antibody diluted 1:500 (Upstate Technology, cat no 16–193) followed by detection with goat-anti-mouse (Amersham) 1:2000 for one hour. Detection with ECL Plus™(Amersham) was performed by scanning on STORM™ 840 (Molecular Dynamics Inc), on which the image was optimized and bands quantified by Image Quant™ version 5.0 (Molecular Dynamics).BER, Base Excision Repair; CHO, chinese hamster ovary; HR, Homologous Recombination; ICRF-187, (+)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane; m-AMSA, (N-[4-(9-acridinylamino)-3-methoxyphenyl]methanesulphonanilide); MMR, Mismatch Repair; NER, Nucleotide Excision Repair; NHEJ, Non-Homologous End Joining; PCR, Polymerase Chain Reaction; PRR, Post Replication Repair; SCLC, Small Cell Lung Cancer; SC-URA, Synthetic medium lacking uracil; SSA, Single Strand Anealing; YPD, Medium containing Yeast extract, Peptone and Dextrose.Lars H. Jensen: Participated in planning the experiments, performed yeast transformations and clonogenic assays, and prepared the manuscript. Marielle Dejligbjerg: Performed γH2AX western blots, primer design, real-time PCR experiments, and data quantitation. Lasse T. Hansen: Performed mammalian clonogenic assays and recombination assays. Morten Grauslund: Performed RNA purification and microarray analysis. Peter B. Jensen: Participated in planning and monitoring the study. Maxwell Sehested: Participated in the initiation and conduction of the study. All authors read and approved the final manuscript.Clonogenic sensitivity of mutant single-gene deletion yeast strains towards ICRF-187 and m-AMSA. Clonogenic sensitivity of a panel of human topoisomerase II α-transformed haploid yeast deletion strains towards equitoxic (to wt cells) concentrations of ICRF-187 and m-AMSA. Error-bars represent SEM of 3 – 10 independent experiments.Click here for filet2–4 cells were inoculated into YPD medium and grown overnight at 34°C, 150 rpm. The cultures were then diluted into 50 ml YPD medium to obtain an OD600 of 0.2. After growing the cells for 2 hours to assure exponential growth, equitoxic concentrations of ICRF-187 and m-AMSA were applied and the cells were grown for an additional 2 hours before RNA was isolated. The figure depicts the clonogenecity of drug treated and untreated cells. Exposure of the cells to the two drugs resulted in a reduction of their clonogenecity of approx. 50 %. Error bars-represent SEM of three independent experiments.Level of killing of yeast cells used in transcriptional profiling experiments. Fresh colonies of pMJ1-transformed JN362AClick here for fileTranscriptional response towards ICRF-187. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to ICRF-187.Click here for fileTranscriptional response towards m-AMSA. A list of yeast genes whose average expression in two independent experiments is induced or repressed more than 1.5 fold by exposure to m-AMSA.Click here for fileTopoisomerase II activity levels in hamster cell lines. The levels of topoisomerase II catalytic activity in crude extracts from wt and recombination defective hamster cell lines. Error-bars represent SEM of two independent experiments. No difference in the level of topoisomerase II catalytic (DNA strand passage activity) is observed.Click here for file


Buchnera aphidicola. We show that bacteriocytes of Acyrthosiphon pisum express several gene products : Distal-less, Ultrabithorax/Abdominal-A, and Engrailed. Using these markers, we find that a subpopulation of the bacteriocytes is specified prior to the transmission of maternal bacteria to the embryo. In addition, we discovered that a second population of cells is recruited to the bacteriocyte fate later in development. We experimentally demonstrate that bacteriocyte induction and proliferation occur independently of B. aphidicola. Major features of bacteriocyte development, including the two-step recruitment of bacteriocytes, have been conserved in aphids for 80–150 million years. Furthermore, we have investigated two cases of evolutionary loss of bacterial symbionts: in one case, where novel extracellular, eukaryotic symbionts replaced the bacteria, the bacteriocyte is maintained; in another case, where symbionts are absent, the bacteriocytes are initiated but not maintained. The bacteriocyte represents an evolutionarily novel cell fate, which is developmentally determined independently of the bacteria. Three of five transcription factors we examined show novel expression patterns in bacteriocytes, suggesting that bacteriocytes may have evolved to express many additional transcription factors. The evolutionary transition to a symbiosis in which bacteria and an aphid cell form a functional unit, similar to the origin of plastids, has apparently involved extensive molecular adaptations on the part of the host cell.Symbiotic relationships between bacteria and insect hosts are common. Although the bacterial endosymbionts have been subjected to intense investigation, little is known of the host cells in which they reside, the bacteriocytes. We have studied the development and evolution of aphid bacteriocytes, the host cells that contain the endosymbiotic bacteria  Molecular markers show that bacteriocytes, the aphid cells that house the bacterial endosymbionts, are specified in a conserved two-step process that does not depend on the presence of the bacteria Buchnera aphidicola, of several species of aphids has revealed extensive gene loss  (Hemiptera: Aphididae). Using these markers, we aimed to determine the developmental origin of bacteriocytes and to what extent bacteria are required for the formation of the bacteriocytes. We also tested whether the observed patterns of bacteriocyte development are evolutionarily conserved among distantly related aphid species. We show that three transcription factors are expressed in a specific temporal order during early bacteriocyte development of the pea aphid. The final population of bacteriocytes originates from two distinct populations of nuclei recruited at different times of development. Furthermore, we experimentally demonstrate that the specification and proliferation of bacteriocytes occur independently of B. aphidicola. In distant relatives of the pea aphid, we found that the two-step determination of bacteriocytes is conserved. We also investigated two cases involving the loss of B. aphidicola. In the first case, in which the bacterial symbionts have been replaced with extracellular, eukaryotic symbionts, bacteriocyte development appears to proceed normally. In a second case, in which males do not inherit B. aphidicola, the bacteriocytes have been lost.We have identified bacteriocyte-specific markers that allow us to track the proliferation of bacteriocytes throughout the development of the pea aphid A. pisum. We infer that these antibodies are recognizing the homologues, or possibly paralogues, of their respective target proteins. The three proteins are expressed in a specific temporal order. We first observe expression of the Distal-less (Dll) protein (FlyBase ID: FBgn0000157) (We tested five cross-reacting antibodies see  for thei0000157)  in syncy0000157) C. As the0000157)   D–1F. The0000157)  that sta0000157) D′. As th0000157)  is detec0000157) G.. The nuclei of these cells are visibly smaller than those of the original bacteriocytes  approximately 80–150 million years ago (Pemphigus spyrothecae (Eriosomatinae) and Tuberaphis styraci (Hormaphidinae) (discussed below). In both cases, Dll is expressed in a small number of bacteriocyte nuclei of the blastoderm-stage embryo and additional Dll-expressing cells are recruited later. In P. spyrothecae, one or two nuclei are originally determined as bacteriocytes, as suggested by The two-step determination of bacteriocytes described in the previous sections appears to be a conserved feature of the aphids. Using the anti-Dll antibody, we examined development of the bacteriocytes in two species of aphids that diverged from ears ago : PemphigB. aphidicolahas been lost in the lineage leading to T. styraci and has been replaced by a yeast-like symbiont  or alfalfa (Medicago sativa)  . P. spyreviously .) (a gift of C.-C. Chang [A limited number of antibodies recognize the homologues of their target proteins across insects. We tested five of these antibodies in aphids and found that three stained the bacteriocyte nuclei: rabbit anti-Dll , mouse aC. Chang ) and mouBuchnera can be eliminated by treating animals with antibiotics (Medicago arborea in Petri-dish cultures (In the pea aphid, ibiotics . First- ibiotics . Aphids cultures . Controlcultures  were discultures .The FlyBase accession numbers discussed in this paper are FBgn0000014, FBgn0000157, FBgn0000577, FBgn0000606, FBgn0003944, and FBgn0003970.


Wall Street Journal Online poll. The respondents may not know that populations of large predatory fish, such as tuna, swordfish, and marlin, have declined 90% over the past 50 years or that less-prized species are increasingly overfished. Or that ever more fish and seafood species show rising levels of mercury contamination, rendering them unfit for human consumption—and contaminating other organisms in the ocean food chain. Humans are also affecting marine life in unexpected ways, as when large numbers of seals in Antarctica in 1955 and in Siberia in 1987 succumbed to canine distemper virus, presumably contracted from domestic dogs. In 2000, more than 10,000 Caspian seals—which also had contact with domestic dogs—died of the same virus. Such human incursions cause even more damage by exacerbating the effects of naturally occurring parasitic and pathogenic diseases that already wreak havoc as they ripple through the food chain.After the recent mad cow scare in the United States, 61% of Americans said they would start eating more fish, according to a With recent studies suggesting that disease rates have increased over the past 30 years—and are expected to increase even more, thanks to global climate change—prospects for protecting marine ecosystems depend on understanding the causes and nature of these disease outbreaks. While all indicators point to a real increase in disease rates, scientists have no baseline data to measure these increases against and so cannot directly test the hypothesis that marine diseases are increasing. Now Jessica Ward and Kevin Lafferty report a method that uses the recorded incidence of disease as a proxy for baseline data to identify disease trends in major groups of marine organisms.Ward and Lafferty conducted an online search of 5,900 journals published from 1970 to 2001 for reports of disease in nine taxonomic groups: turtles, corals, mammals, urchins, mollusks, seagrasses, decapods (crustaceans), sharks/rays, and fishes. Their approach takes into account three potentially confounding factors in determining trends in this type of search. Fluctuations in publication numbers could skew results, since an increase in the number of scientific reports published in a particular taxonomy might not reflect a true increase in the incidence of disease; a particularly prolific author could bias the search results by turning up more cases of disease in a population than actually occurred; or a single disease event reported multiple times in different papers could create the impression that disease had suddenly increased. To normalize publication rates over time, Ward and Lafferty used a proportion of disease reports from a given population relative to the total number of reports in that group. To determine whether there was an “author effect,” they removed the most prolific author in each taxonomic group and found that an author's abundant contributions did not skew the results. Finally, they confirmed that a single disease didn't bias their results by removing multiple reports of the same disease from the literature before analyzing the trends.When they analyzed the searches without adjusting for the total number of reports published, Ward and Lafferty found that reports of disease increased for all groups. But when they analyzed the normalized results, they found that trends varied. While there was a clear increase in disease among turtles, corals, mammals, urchins, and mollusks, they found no significant trends for seagrasses, decapods, and sharks/rays. And they found that disease reports actually decreased for fishes. (One explanation for this decrease could be that drastic reductions in population density present fewer opportunities for transmitting infection.) Ward and Lafferty tested the soundness of this approach by using a disease (raccoon rabies) for which baseline data exist and showing that normalized reports of raccoon rabies increased since 1970, just as the disease increased from one case reported in Virginia in 1977 to an “epizootic” outbreak, affecting eight mid-Atlantic states and Washington, D.C., by 1992.The pattern of increased reports, the authors propose, confirms scientists' perceptions about the rising distress of threatened populations and thus reflects a real underlying pattern in nature. The fact that disease did not increase in all taxonomic groups suggests that increases in disease are not simply the result of increased study and that certain stressors, such as global climate change, likely impact disease in complex ways. By demonstrating that an actual change in disease over time is accompanied by a corresponding change in published reports by scientists, Ward and Lafferty have created a powerful tool to help evaluate trends in disease in the absence of baseline data. It is only by understanding the dynamics of disease outbreaks that scientists can help develop sound methods to contain them.


Dishevelled (Dsh) is a key component of multiple signaling pathways that are initiated by Wnt secreted ligands and Frizzled receptors during embryonic development. Although Dsh has been detected in a number of cellular compartments, the importance of its subcellular distribution for signaling remains to be determined.Xenopus embryonic explants or mammalian cells are incubated with inhibitors of nuclear export or when a specific nuclear-export signal (NES) in Dsh is disrupted by mutagenesis. Dsh protein with a mutated NES, while predominantly nuclear, remains fully active in its ability to stimulate canonical Wnt signaling. Conversely, point mutations in conserved amino-acid residues that are essential for the nuclear localization of Dsh impair the ability of Dsh to activate downstream targets of Wnt signaling. When these conserved residues of Dsh are replaced with an unrelated SV40 nuclear localization signal, full Dsh activity is restored. Consistent with a signaling function for Dsh in the nucleus, treatment of cultured mammalian cells with medium containing Wnt3a results in nuclear accumulation of endogenous Dsh protein.We report that Dsh protein accumulates in cell nuclei when These findings suggest that nuclear localization of Dsh is required for its function in the canonical Wnt/β-catenin signaling pathway. We discuss the relevance of these findings to existing models of Wnt signal transduction to the nucleus. Drosophila and convergent extension cell movements and tissue separation in Xenopus [The specification of cell fates during embryonic development frequently depends on inductive interactions, which involve transmission of extracellular signals from the cell surface to the nucleus. In the transforming growth factor β (TGFβ) signal transduction pathway, Smad proteins that are initially associated with TGFβ receptors move to the nucleus to regulate target genes . Another Xenopus -12. ThusXenopus ectoderm, and this redistribution requires the DEP domain [Dsh is a common intracellular mediator of several pathways activated by Frizzled receptors and is composed of three conserved regions that are known as the DIX, PDZ and DEP domains . DiffereP domain ,18,19. TP domain ,18,20,21Xenopus ectodermal cells and mammalian cells upon inhibition of nuclear export. Dsh also accumulated in the nuclei after stimulation of mammalian cells with Wnt3a-containing culture medium. By analyzing various mutant Dsh constructs in Xenopus ectoderm, we show that the signals responsible for Dsh nuclear localization reside in a novel domain and that the nuclear translocation of Dsh is essential for its ability to activate Wnt/β-catenin signaling.The current study was based on our initial observation that a Dsh construct lacking the carboxy-terminal DEP domain was found in cell nuclei. We have now identified a nuclear export signal in the deleted region and also discovered that Dsh proteins accumulate in the nuclei of Xenopus ectodermal cells. In contrast to Dsh-GFP, which is localized in punctate structures within the cytoplasm [We studied the subcellular distribution of fusions of Dsh with green fluorescent protein (GFP) in ytoplasm ,18, the ytoplasm ,23. WhenTo examine whether inhibition of nuclear export abrogates Dsh activity, we compared the abilities of DsNESm and wild-type Dsh-GFP to induce secondary axes in frog embryos. Although the molecular mechanism operating during axis induction remains to be elucidated, this assay faithfully reflects the biological activity of Dsh in the canonical Wnt/β-catenin pathway ,16,18,24Xenopus embryonic cells under conditions in which nuclear export is blocked. When ectodermal cells expressing Dsh-GFP were incubated with N-ethylmaleimide (NEM), an inhibitor of the nuclear export receptor CRM1/exportin [Accumulation of DsNESm in the nucleus implies that the wild-type Dsh shuttles between the nucleus and the cytoplasm. We therefore studied the subcellular distribution of Dsh in exportin ,26, Dsh-exportin ,23  293 cells treated with LMB accumulated Dvl2 in the nucleus, contrasting with the cytoplasmic localization of Dvl2 in untreated cells Figure . We alsoTo identify specific amino-acid sequences that direct the transport of Dsh to the nucleus, we studied the subcellular distribution of mutated Dsh-GFP fusion constructs Figure . The remHydra to humans , a positive regulator of the β-catenin pathway ,31, or A protein   pathway, which involves Rho GTPase and JNK and controls morphogenetic movements in early embryos ,9,37-39.Xenopus, the PCP pathway involving Dsh is implicated in the control of convergent extension movements [In ovements ,41,42. Oovements ; Figure ovements ,37-39. BOur findings are consistent with a scenario in which Wnt signaling may cause nuclear translocation of Dsh followed by formation of a stable β-catenin/Tcf3 complex and transcriptional activation of target genes. In support of this hypothesis, Dsh was reported to move to the nucleus in response to Wnt signaling in primary embryonic kidney cells . In Rat-In the current view, Wnt signaling causes inactivation of the β-catenin degradation complex, leading to stabilization and nuclear translocation of β-catenin . Given tXenopus ectoderm cells overexpressing Dsh (data not shown), this process may be cell-context-dependent. On the other hand, we recently showed that Frodo, a nuclear Dsh-interacting protein, associates with Tcf3 and influences Tcf3-dependent transcription [Alternatively, nuclear localization of Dsh may affect β-catenin stability indirectly, by regulating protein interactions that sequester β-catenin in the nucleus, thereby preventing its cytoplasmic degradation . Althougcription . It is tGFP-tagged Dsh constructs were all derived from the DshGFP-RN3 plasmid that encodes the Xdsh protein fused at amino acid 724 to the first amino acid of GFP Figures , 4a. Ds1To generate these constructs, DshGFP-pRN3 was used as a template. The in-frame deletion in Ds3 was made by PCR. Other GFP fusion constructs were synthesized with specific primers and PfuI DNA polymerase followed by DpnI digestion of the template . The folIn vitro fertilization, culture and microinjections of Xenopus eggs were essentially as described previously [eviously . Stages eviously . Axis in2, 0.1 mM MgCl2, 0.5 mM Hepes, 1.2 mM sodium phosphate, (pH 7.5), 0.6 mM NaHCO3 and 0.06 mM EDTA) for one hour prior to fixation. In some experiments, nuclei were stained by addition of 1 μg/ml 4,6-diamidino-2-phenylindole (DAPI) to the final PBS wash. For membrane localization studies, Xfz8 RNA was coinjected with RNAs encoding the Dsh constructs in the animal-pole region; animal-cap explants were dissected at stage 9–9.5 and mounted for observation. Fluorescence was visualized using a Zeiss Axiophot microscope.For subcellular localization of Dsh-GFP constructs, mRNAs were injected into the animal pole region of 2–4-cell embryos. Animal cap explants were dissected at stages 9–10.5, incubated for 60 min in 10 mM N-ethylmaleimide  in 0.8 × MMR (Marc's Modified Ringer's solution, 1 × MMR: 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM HEPES, pH 7.4), or in control (0.8 × MMR), then fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30–45 min, washed three times in PBS, and mounted in 70% glycerol, 30% PBS containing 25 mg/ml of diazabicyclo(2,2,2)-octane (Sigma). Leptomycin B was used at 50 ng/ml in low-calcium medium (76 mM NaCl, 1.4 mM KCl, 0.2 mM CaClFor luciferase assays, pSiaLuc reporter plasmid (20–40 pg) was coinjected with mRNAs encoding Xwnt3a  or Xwnt8Rat-1 fibroblasts, human embryonic kidney (HEK) 293 cells and MCF7 human breast carcinoma cells were cultured in 1 × Dulbecco's Modified Eagle Medium  supplemented with 10% fetal calf serum and 5 μg/ml gentamicin. Conditioned medium was prepared from L cells stably transfected with Wnt3a as described , with thFor immunocytochemistry, HEK293 cells were treated with 50 ng/ml LMB for 14 h while MCF7 cells were treated with Wnt3a or control conditioned medium for 1, 3, 6 or 8 h. Cells were fixed with 4% paraformaldehyde, immersed in methanol, and incubated with anti-Dvl2 antibodies and then Cy3-conjugated anti-rabbit IgG. Nuclei were stained by addition of 1 μg/ml DAPI as described for animal-cap cells. Fluorescence was observed under the Zeiss Axiophot microscope; 10–15 cells from each group were randomly picked up for measurement of the nuclear and cytoplasmic staining intensity using Image-Gauge software (Fuji Film, Tokyo, Japan).2, 10 mM KCl, 1 mM DTT, 10 mM β-glycerophosphate, 1 mM sodium orthovanadate, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 1 μg/ml pepstatin. Cells were swollen for 30 min, and broken open with 25 strokes in a tight fitting Dounce homogenizer. Lysates were layered into tubes containing 1 M sucrose in hypotonic lysis buffer, and spun at 1600 × g for 10 min. Supernatant remaining above the sucrose cushion was used as the cytoplasmic fraction. The pellet, containing nuclei, was resuspended in an equivalent volume of hypotonic lysis buffer.For subcellular fractionation, confluent cultures of Rat-1 cells were harvested by scraping plates and resuspended in hypotonic lysis buffer containing 1 mM EGTA, 1 mM EDTA, 2 mM MgClImmunoprecipitation and western analysis were carried out with cell and embryo lysates as described . To prepin vitro kinase assays were carried out essentially as described [Four-cell embryos were injected with 4 ng Dsh or DsNLSm RNA into four animal blastomeres. Embryo lysates were prepared at stage 10.5 and escribed , except The following is provided as an additional data file with the online version of this article. Additional data file Cloning details of Dsh mutant constructsClick here for additional data file


Ribes nigrum L.) leaves, were analysed using carrageenin-induced paw oedema and carrageenin-induced pleurisy in rats.The anti-inflammatory effects of proanthocyanidins (PACs), isolated from blackcurrant  reduced paw oedema induced by carrageenin in a dose and time-dependent manner. PACs also inhibited dose-dependently carrageenin-induced pleurisy in rats. They reduced (A) lung injury, (B) pleural exudate formation, (C) polymorphonuclear cell infiltration, (D) pleural exudate levels of TNF-α, IL-1β and CINC-1 but did not affect IL-6 and IL-10 levels. They reduced (E) pleural exudate levels of nitrite/nitrate (NOx). In indomethacin treated rats, the volume of pleural exudate was low, its content in leukocytes and its contents in TNF-α, IL-1β, IL-6 and IL-10 but not in NOx were reduced. These data suggest that the anti-inflammatory properties of PACs are achieved through a different pattern from those of indomethacin.in vivo nitric oxide release.These results suggest that the main mechanism of the anti-inflammatory effect of PACs mainly lies in an interference with the migration of the leukocytes. Moreover, PACs inhibited  Proanthocyanidins are compounds, naturally occurring in various plants, with anti-inflammatory ,2 and anRibes nigrum L., Grossulariaceae) leaves which are used in European traditional medicine for the treatment of inflammatory disorders such as rheumatic diseases [We have isolated prodelphinidins and procyanidins, proanthocyanidins (PACs) from blackcurrant (diseases . MajoritPreviously, we have observed that, in vitro, these compounds profoundly affect the metabolism of chondrocytes : they increase the secretion from these cells of type II collagen and proteoglycans while they decrease the secretion of prostaglandin E2 (PGE2) . On the in vivo, on carrageenin-induced paw oedema and pleurisy in rats. This latter inflammatory reaction allowed us to examine the influence of PACs not only on the exudate volume and polymorphonuclear cell accumulation but also on the release of several cytokines, IL-1β, TNF-α, IL-6, IL-10, CINC-1 and of nitric oxide (NO). These cytokines and NO are among the more important mediators involved in inflammatory processes [The present studies were designed to evaluate the potential anti-inflammatory activities of these compounds, rocesses -16.Carrageenin-induced oedema was significantly inhibited by PACs dose-dependently Figure . This in6 per rat . This exudate contained a large number of cells, mostly (> 95%) polymorphonuclear leukocytes (PMNs). The total leukocytes number in the exudate was 119.71 ± 29.29 × 10t Figure . PACs sit Figure  was signHigh levels of TNF-α, IL-1β, IL-6, IL-10 and CINC-1 were found in pleural exudates induced by carrageenin Figure . IndometThe pleural exudate of carrageenin-treated rats contained a large amount of NOx  Figure . The amoHistological examination of lung sections revealed significant tissue injury Figure  when comRibes nigrum leaves reduced the inflammatory reactions induced by carrageenin in rats : the extent of the paw oedema was halved, the volume of the pleural exudates and its content in TNF-α, IL-1β, CINC-1 and NOx were reduced, the infiltration of leukocytes into the lungs and the accumulation of leukocytes into the pleural cavity were largely diminished.Proanthocyanidins (PACs) from et al [PACs have been reported to be able to scavenge free radicals and NO . This pret al , during The development of carrageenin-induced inflammatory reactions in rats results from the activation of the kinin system, the accumulation of leukocytes and the release of several mediators such as prostanoids and cytokines ,20. IndePro-inflammatory cytokines TNF-α, IL-1β and IL-6 are sequentially released in the pleural exudates induced by carrageenin in rat . These cTNF-α plays an important role in promoting and amplifying lung inflammation through the release of chemotactic factors such as CINC-1 (rat IL-8), an important mediator that promotes the migration of neutrophils  and oesiRecently, grape seed proanthocyanidins have been demonstrated to reduce the expression of soluble adhesion molecules, ICAM-1, VCAM-1 and E-selectin in the plasma of systemic sclerosis patients . The samInjection of carrageenin into the pleural cavity induces the accumulation of leukocytes, a release of cytokines, the expression of inducible NO synthase and of cyclo-oxygenase-2, and thus the release of large amounts of nitric oxide and of prostanoids . The inh2 production. The peak of cyclooxygenase-2 activity measured by prostanoid levels in carrageenin-induced pleural exudates spreads from 2 to 6 h after irritant injection [2 [2 production by high doses of indomethacin could result in a downregulation of IL-6 and IL-10 production [et al [2 and IL-6 pathways.Comparatively, some animals have been treated with indomethacin. The inhibitory effect of this well-known non-steroidal anti-inflammatory drug is larger than that obtained with PACs. Indomethacin greatly reduced plasma exudation, nearly suppressed the accumulation of leukocytes and decreased the levels of the cytokines while, it did not modify the pleural content of NOx. Indomethacin is known to inhibit the cyclooxygenase-1 and -2 responsible of the release of PGEnjection ,32. Bothction [2 ,34. An ioduction ,36. Moren [et al , using cRibes nigrum leaves interfere with the accumulation of circulating leukocytes, associated with a reduction of pro-inflammatory factors such as TNF-α, IL-1β and CINC-1, a decrease of NOx level and a decrease in plasma exudation.In conclusion, we have shown that proanthocyanidins isolated from We used male Wistar rats, weighing 250 – 300 gm. The animals were maintained on a standard laboratory diet with free access to water. The experiments were conducted as approved by the Animal Ethics Committee of the University of Liège, Belgium.Rats were pretreated with an intraperitoneal administration of saline or PACs (10, 30, 60 and 100 mg/kg). Thirty minutes later, lambda carrageenin, (0.1 ml, 10 mg/ml) was injected into the plantar region of the right hind paw. Each experimental group contained six animals. Paw volume was measured using a water plethysmometer (Ugo Basile) before and 1 h, 2 h and 4 h after the injection of carrageenin. After 4 h, the animals were anaesthetized with a large dose of sodium pentobarbital (80 mg/kg).Rats were pretreated with an intraperitoneal injection of saline, PACs (10, 30, 60 or 100 mg/kg) or indomethacin (10 mg/kg) 30 min before the intrapleural injection of the irritant. They were then anaesthetized with ketamine HCl (75 mg/kg) and carrageenin (0.2 ml, 10 mg/ml) or saline (0.2 ml) was administered into the right pleural cavity. Each experimental group contained 6 animals. Four hours later, the animals were anaesthetized with sodium pentobarbital (80 mg/kg). The chest was carefully opened and the pleural cavity rinsed with 2.0 ml saline solution containing heparin (5 U/ml). Exudates and washing solutions were removed by aspiration and the total volume measured. Exudates with blood were rejected. Exudates were aliquoted and kept frozen at -32°C.2HPO4 and 0.187 M KH2PO4 (pH 7.2) at room temperature. They were then dehydrated by graded ethanol and embedded in Paraplast. Tissue sections (thickness 7 μm) were deparaffinized with UltraClear, stained with hematoxylin-eosine and examined using light microscopy.After removal of the exudates, lungs were withdrawn and fixed for one week under 30 cm pressure with 10% formaldehyde aqueous solution containing 0.480 M NaThe volume of the exudates was calculated by subtracting the volume of the washing solution (2.0 ml) from the total volume recovered. A sample of each exudate was diluted in phosphate buffer and total leukocyte count was performed using a hemocytometer.The levels of IL-1β, TNF-α, IL-6 and IL-10 in the exudates were measured using a colorimetric commercial ELISA kit (Biosource, Nivelles, Belgium) with a lower detection limit of 4, 3, 8 and 5 pg/ml, respectively. The levels of CINC-1 in the exudates were measured using a colorimetric commercial ELISA kit (Amersham Biosciences, Freiburg, Germany) with a lower detection limit of 0.49 pg/ml.3 - to NO2 - with a lower detection limit of 1 μM.The amount of NOx (nitrite/nitrate) present in the exudates was determined using a microplate assay method  based on Griess reaction after reduction of NORibes nigrum leaves were extracted and isolated according to a previously described method [Proanthocyanidins from d method . A vouchWe used ketamine-HCl from Pfizer (Bruxelles, Belgium), sodium pentobarbital from Ceva (Bruxelles, Belgium) and heparin from B. Braun Medicals (Diegem, Belgium). PACs and lambda carrageenin (Sigma, Bornem, Belgium) were dissolved in saline. Indomethacin (Merck, Sharp and Dohme, Leuven, Belgium) was dissolved in Tris-HCl (0.15 M, pH 7.4).P-value of less than 0.05 was considered significant.Results are given as mean ± standard error of the mean (s.e. mean) of N observations. For the oedema paw studies, a Mixed Procedure SAS  was used to compare difference of least square means. For the pleurisy studies, data sets were examined by one-way analysis of variance (ANOVA) followed by a Scheffe post-hoc test. A NG carried out PACs isolation, animal experimentation, immunoassays, lung sections and statistical analysis. MT coordinated and participated to the PACs isolation. LA coordinated the PACs isolation. JD participated in animal experimentation, conceived of the study and participated in its design and coordination.


The skin contains a system for producing serotonin as well as serotonin receptors. Serotonin can also cause pruritus when injected into the skin. SSRI-drugs increase serotonin concentrations and are known to have pruritus and other dermal side effects.A 46-year-old man consulted his doctor due to symptoms of depression. He did not suffer from any allergy but drinking red wine caused vasomotor rhinitis. Antidepressive treatment with fluoxetine 20 mg daily was initiated which was successful. After three weeks of treatment an itching rash appeared. An adverse drug reaction (ADR) induced by fluoxetine was suspected and fluoxetine treatment was discontinued. The symptoms disappeared with clemastine and betametasone treatment. Since the depressive symptoms returned sertraline medication was initiated. After approximately two weeks of sertraline treatment he noted an intense itching sensation in his scalp after eating a piece of chocolate cake. The itch spread to the arms, abdomen and legs and the patient treated himself with clemastine and the itch disappeared. He now realised that he had eaten a chocolate cake before this episode and remembered that before the first episode he had had a chocolate mousse dessert. He had never had any reaction from eating chocolate before and therefore reported this observation to his doctor.This case report suggests that there may be individuals that are very sensitive to increases in serotonin concentrations. Dermal side reactions to SSRI-drugs in these patients may be due to high activity in the serotonergic system at the dermal and epidermo-dermal junctional area rather than a hypersensitivity to the drug molecule itself. Serotonin can also cause pruritus when injected into the skin. SSRI-drugs increase serotonin concentrations and are known to have pruritus and other dermal side effects pruritus . In contpruritus  and polypruritus . In thisA 46-year-old man consulted his doctor in September 2003 due to depression. He had then experienced symptoms for a few years that had aggravated during the last six to eight months. Using the Montgomery-Åsberg Depression Rate Scale (MADRS) the patient scored 24 points and was diagnosed as having a clinical depression. He did not take any medication and had no regular medical contact. The patient did not have any history of allergy or dermatological diseases. However, he sometimes suffered from vasomotor rhinitis after drinking red wine. The doctor prescribed fluoxetine 20 mg daily as antidepressive treatment. At the revisit three weeks later the patient was very pleased with the fluoxetine treatment and reported that he "had not felt better in 20 years" although he initially had experienced slight nausea and insomnia.A week later, he visited his doctor due to an itching rash that had started the day before. The doctor noted partly confluent urticae on the abdomen, a modest periorbital oedema and red, warm palms and wrists. An ADR induced by fluoxetine was suspected and fluoxetine treatment was discontinued. The symptoms were treated with 2 mg clemastine and 6 mg betametasone orally and disappeared within 48 hours. However, the symptoms of depression returned. Sertraline medication was initiated 10 days after the cessation of fluoxetine treatment since SSRI medication had shown good effect. During the weeks of sertraline treatment no urticarial symptoms appeared. The patient improved in his depression although full recovery was not achieved this time. After approximately two weeks of sertraline treatment he noted an intense itching sensation in his scalp after eating a piece of chocolate cake. The itch spread to the arms, abdomen and legs within a few hours. This time the patient did not seek his doctor but treated himself with clemastine and the itch disappeared during the night. He now remembered that he had had a chocolate mousse dessert before the first episode. Since he had never had any reaction from eating chocolate before, he found this observation so striking that he reported it to his doctor. The patient, himself a scientist, later tried small doses of chocolate and skin rash and itch appeared at an intensity that to him seemed dependent on the "dose" of chocolate ingested.It has been known for 30 years that serotonin can stimulate cutaneous C-fibres , the typIn the present case the patient experienced skin symptoms from two different SSRIs. However, these symptoms occurred only when he had eaten chocolate. Chocolate contains serotonin, at concentrations which depend on the type of chocolate . A conceApart from the SSRI – chocolate interaction this patient had another possible sign of sensitivity to serotonin. The present case thus suggests that there may be individuals that are very sensitive to increases in serotonin concentrations. Skin side reactions to SSRI-drugs in these patients may be due to high activity in the serotonergic system system at the dermal and epidermo-dermal junctional area rather than a hypersensitivity to the drug molecule itself. However, the reaction of skin to serotonin from food is poorly studied and further studies are necessary to determine how much alimentary serotonin can increase serum serotonin concentrations and to what extent SSRI-medication affects this process. More knowledge in this field could be of help for physicians who encounter patients with dermal reactions to SSRI-drugs and there might be food and beverages containing serotonin that these patients should avoid. Moreover, possible individual differences in the serotonergic system at the dermal-epidermal junction remain to be studied.What happened to the patient and his depression? Due to poor anti-depressive effect of sertraline, the treatment was altered back to fluoxetine. He is now free from his depression and experiences no rash or oedema-like adverse reactions as long as he is avoiding chocolate.5-HT: 5-hydroxytryptamine, ADR: Adverse Drug Reaction, SSRI: serotonin selective reuptake inhibitorsThe author(s) declare that they have no competing interests.SS first described the case, JC and HM performed literature searches and JC first drafted the manuscript. HM and SK took part in the scientific discussion and in finalising the manuscript.The pre-publication history for this paper can be accessed here: