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  . 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 . Thirty minutes later, lambda carrageenin,  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  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  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  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  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 , sodium pentobarbital from Ceva  and heparin from B. Braun Medicals . PACs and lambda carrageenin  were dissolved in saline. Indomethacin  was dissolved in Tris-HCl .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:


Anopheles gambiae at selected times during the first two days following blood ingestion, at 5 and 30 min during a 40 minute blood meal and at 0, 1, 3, 5, 12, 16, 24 and 48 hours after completion of the blood meal and compared their expression to transcript levels in mosquitoes with access only to a sugar solution.Blood feeding, or hematophagy, is a behavior exhibited by female mosquitoes required both for reproduction and for transmission of pathogens. We determined the expression patterns of 3,068 ESTs, representing ~2,000 unique gene transcripts using cDNA microarrays in adult female k-means clustering into Early Genes, Middle Genes, and Late Genes, containing 144, 130, and 139 unique transcripts, respectively. Several genes from each group were analyzed by quantitative real-time PCR in order to validate the microarray results.In blood-fed mosquitoes, 413 unique transcripts, approximately 25% of the total, were expressed at least two-fold above or below their levels in the sugar-fed mosquitoes, at one or more time points. These differentially expressed gene products were clustered using The expression patterns and annotation of the genes in these three groups  are discussed in the context of female mosquitoes' physiological responses to blood feeding, including blood digestion, peritrophic matrix formation, egg development, and immunity. Hematophagy, blood-feeding, is a behavior exhibited by most arthropod vectors of human pathogens. In anautogenous mosquitoes, the female generally feeds to repletion on a single blood meal and then proceeds to use this nutrition as the basis for the development of a batch of eggs. The cycle of host seeking, blood feeding, egg development, and oviposition is generally called the gonotrophic cycle, a term coined by Beklemishev in 1940 .Once the adult mosquito acquires a blood meal, she spends approximately 48 hours converting about 20% of it into egg constituents , using aAn. gambiae contains approximately 50 functional egg-production structures, the ovarioles. Each ovariole is comprised of two parts, a distal germarium and a vitellarium proximal to a common oviduct through which eggs will pass as they are laid. In the germarium, mitosis of the primordial germ cells creates a syncitium with an oocyte and seven nurse cells interconnected by intracellular bridges, or ring canals as a result of incomplete cytokinesis. Both the germ cell and the nurse cells are surrounded by a somatically derived follicular epithelium . Thus, iewed in .Ae. aegypti . These Drosophila ovarian development and early embryogenesis opens windows into interpreting our An. gambiae microarray results and understanding mosquito egg development. The majority of An. gambiae genes upregulated at least twofold following a blood meal appear to function in egg production. The only gene possibly involved in oogenesis during the early phases of the ovarian cycle is the Early gene AS 670. It shares sequence similarity with peter pan, a Drosophila gene product required during oogenesis. Oocytes in peter pan mutants often have an incorrect number of associated nurse cells, suggesting that the peter pan protein influences the separation of cells within the germaria [The extensive literature on genes involved in germaria . The ideDrosophila gene CG13388 encoding the protein kinase anchor protein 200, Akap200. Akap200 protein localizes to ring canals during oogenesis, regulates protein kinase C activity, and controls their morphology [Drosophila pendulin gene product, encoded by CG4799. This gene product is also required for assembly of fully functional ring canals. pendulin encodes an importin-α2, a protein necessary for the localization of the kelch gene product, CG7210. kelch encodes an actin organizer without which the ring canals become occluded and nurse cell-oocyte cytoplasmic transport is inhibited [pendulin gene, we did not find kelch. A Late gene product, AS 578, is the homolog of Cdc42, which encodes a small monomeric RHO GTPase involved in signal transduction. Rohatgi et al. [Cdc42 protein most likely links signal transduction to the actin cytoskeleton in Xenopus. In Drosophila ovaries, mutations in Cdc42 caused nurse cells to deflate and coalesce, and inhibited transfer of nurse cell cytoplasm to oocytes in late stage egg chambers [Several differentially expressed gene products found in the present study may be involved in the formation of ring canals, structures necessary for the delivery of maternal factors to oocytes. In particular, bulk transfer of cytoplasmic content from nurse cells to oocytes depends on actin structures . A Middlrphology . Late genhibited ,60. Thoui et al.  suggestechambers .Drosophila nurse cells transcribe the bicoid anterior determinant gene and the resulting mRNA is transported to the anterior region of developing oocytes via polarized microtubules [bicoid does not appear to be an anterior determinant in other insects, but other genes important for its localization are conserved. The Early gene AS 2047 shares similarity with the Drosophila cornichon gene, CG5855. In Drosophila, cornichon is required for formation of a functional microtubular cytoskeletal scaffold used to transport bicoid mRNA and the posterior group oskar gene product to their proper location within the embryo [D. virilis exuperantia, a gene whose product is also required for proper bicoid mRNA localization D. melanogaster [An. gambiae homolog of Drosophila Notch. In Drosophila, Notch signaling regulates a large number of ovarian events beginning with cyst development in the germarium and extending through oogenesis [Notch signaling activates transcription of its target genes are reviewed by Barolo and Posakony [Notch as a Late gene, its activities may be more restricted in An. gambiae, and/or reflect fundamental differences in ovarian biology. AS 1391, a Late gene product, shares sequence similarity with Drosophila Rab-protein 11. This Drosophila small monomeric RAB GTPase is also involved in the polarization of the microtubules for the organization of the posterior pole and for oskar localization there [otubules . bicoid e embryo . The Latnogaster ,66. The ogenesis . The mecPosakony . Since won there .Drosophila gene product Bicaudal C, a RNA binding protein that may play a role in translational silencing of maternal mRNAs in addition to its role in eggshell patterning [Bicaudal C result in premature translation of oskar mRNA before it has reached the posterior region of the oocyte [Drosophila gene product vasa (CG3506), an ATP dependent helicase involved in pole plasm assembly that may also be involved in translational modification of maternal mRNAs [Drosophila cup protein (CG11181). cup protein interacts with nanos, the posterior determinant, and a translational regulator of the gap gene hunchback mRNA during oogenesis, although the exact function of the cup protein still remains unknown [vasa also influences oocyte differentiation and the development of the Drosophila embryo body plan via translation of oskar, nanos, and gurken during oogenesis [An. gambiae Middle gene product, AS 2449, shared sequence similarity with the Xenopus laevis poly(A)-specific ribonuclease and also has mRNA binding motifs, thus it may also repress translation of mRNAs in embryos.To regulate the progress of oogenesis and embryogenesis, stored maternal mRNAs are translationally repressed during early oocyte development. The Middle gene product AS 2031 shares sequence similarity with the tterning . Mutatioe oocyte . The Midal mRNAs . The Lat unknown . A DEAD ogenesis -77. In aogenesis ,79. One nop5, encoding a maternally derived product of the Drosophila CG10206 gene, a component of the small nucleolar ribonucleoprotein (snoRNP) complex involved in rRNA processing [Drosophila minidiscs gene product, an amino acid transporter. In Drosophila ovarian nurse cells, JH induces the expression of minidisks and its transcripts are most likely transferred to the oocyte during nurse cell cytoplasmic streaming [Maternal nurse cells not only provide the biosynthetic machinery and mRNA needed for oocyte axis determination, but also many transcripts and proteins required for zygotic development through the cellular blastoderm stage. The Middle gene product AS 1032 shares sequence similarity with ocessing . The Lattreaming .Drosophila, the localization of the oocyte depends on cadherin-associated adhesion [Drosophila [Drosophila Star protein, a RAS 1 enhancer involved in the EGF receptor signaling pathway, either upstream or in parallel to EGFR, during formation of the embryonic ventral midline. Star encodes a single pass transmembrane protein that may be involved in the processing of gurken protein. The DNA damage checkpoint 14-3-3epsilon protein also participates in RAS 1 signaling, normally functioning downstream or in parallel to RAF, but upstream of transcription factors. The Middle gene AS 106 shares sequence similarity with Drosophila 14-3-3epsilon. The 14-3-3epsilon protein is also capable of binding to a large number of other proteins in a phosphorylation-dependent manner. One of its functions may be to alter the cell cycle by binding Cyclin B and appears to have homologs in most if not all eukaryotes [An. gambiae Cyclin B homolog, AS 1357, grouped as a Late gene.Similar to the oocyte, the eggshell undergoes dorsal-ventral patterning. Crucial to this process is the correct placement of the oocyte relative to the maternal somatic follicle cells. In adhesion . The Latosophila . The RASosophila ,42. The karyotes ,83. The Drosophila have led to the identification of a number of other gene products that may be involved in RAS 1 signaling. TppII (tripeptidyl-peptidase II), and smt3 (SUMO) were discovered in a search for lethal mutations that could enhance a weak RAS 1 eggshell phenotype [Drosophila tripeptidyl-peptidase II, the CG3991 gene, encoding a serine protease that degrades neuropeptide signals [Drosophila smt3, CG4494, whose product is ubiquitin-like protein that may tag proteins for nuclear localization or retention in the cytoplasm [smt3 protein may modulate activity of transcription factors in the follicle cells downstream of EGFR activation. However, we feel that smt3 is likely to be a minor player in RAS 1 signaling in the events following blood ingestion in the mosquito, because its mRNA reaches maximal expression after 12–24 hours PBM, unlike the other gene products we identified as potentially influencing RAS 1 signaling.Mutation screens in henotype . The Mid signals . The Latytoplasm ,87. smt3smt3 protein may also play a role in Toll signaling. This signal transduction pathway is known to be necessary for dorsal/ventral patterning of the Drosophila embryo. smt3 protein binds the NFκB homolog dorsal protein and targets this Rel transcription factor for migration to the nucleus [et al. [smt3 conjugation to the dorsal protein enhanced its transcriptional activity. smt3 protein may play other roles in the cell by altering the interactions of septins, cytoskeletal proteins involved in cytokinesis [Drosophila, septins have been found in the cytoplasm of nurse cells and at the baso-lateral surfaces of follicle cells [smt3 gene product in oogenesis. We also found that the Late gene AS 2034, a homolog of Drosophila Aos1, the CG12276 gene, the smt3 (SUMO) activating enzyme, was also expressed at least twofold more abundantly during the height of smt3 expression. This result reinforces the importance of smt3 in the events occurring between 24–48 hours PBM. nucleus ,89. Bhas [et al.  demonstrokinesis ,91. In Dle cells . These rAe. aegypti ornithine decarboxylase antizyme, a protein that modulates polyamine synthesis. The homologous Drosophila ornithine decarboxylase antizyme gene, formerly known as gut feeling, has been shown to be important in developing oocytes. It is one target of Sex lethal which encodes an RNA binding protein that regulates mRNA splicing and the mitotic events in early germ cells via regulating Cyclin B [Drosophila polo CG12306 gene product, a protein kinase required for cytokinesis and another regulator of Cyclin B [Drosophila black pearl CG5268 gene product. This protein contains DnaJ domains implying that it is necessary for cellular growth [black pearl RNA from various developmental stages showed two transcripts with greatest expression in Drosophila embryos 0–6 hours old [Drosophila Imaginal disc growth factor4 (Idgf4), a mitogen with a non-functional chitinase domain. Transcripts of Idgf4 are detected in Drosophila nurse cells, oocytes, and in the yolk cytoplasm of early embryos [Drosophila β Integrin. An. gambiae β Integrin may interact with the Middle gene AS 985 product to promote somatic cell adhesion and cell migration during oogenesis and embryogenesis. This is due to the similarity of the AS 985 gene to Drosophila Receptor of activated protein kinase C, RACK1. RACK1 can bind a number of different signaling and cell adhesion molecules including the activated form of protein kinase C (PKC), Src family kinases, and β Integrins [et al. [et al. [An. gambiae β Integrin gene was expressed at highest levels 48 hours PBM, whereas we found that the β integrin homolog reached at least twofold increased expression by 24 hours PBM.In addition to genes regulating the polarity of the embryo and eggshell, genes involved in cellular growth and differentiation were differentially expressed during the 48 hours PBM ingestion. AS 337 and 495 shared sequence similarity with the Cyclin B . The LatCyclin B . The Ear embryos . The Midntegrins -99. Cox  [et al.  demonstr [et al.  found thAe. aegypti [Dopa decarboxylase (Ddc) gene, Ddc is an enzyme involved in the tyrosine metabolic pathway necessary for eventual chorion melanization in Ae. aegypti, and other melanization events. The gene is up-regulated in response to blood meal with transcripts initially detectable by 12 hours PBM, and maximal levels achieved between 24 and 48 hours PBM [Ae. aegypti Dopachrome conversion enzyme [Ae. aegypti females, but becomes upregulated when they are infected with Dirofilaria [An. gambiae differs from Ae. aegypti in the substrate metabolized to produce chorionic melanin.A number of genes have been implicated in the development of the egg shell structures. Our screen does not appear to have identified any homologs of the several endochorionic structural proteins characterized in  aegypti ,103. Thi aegypti ,103. Ourours PBM . Howevern enzyme . This enofilaria . Since iDrosophila thioredoxin and Ae. aegypti 2-Cys thioredoxin peroxidase, may have roles in regulating reactive oxygen species that can be produced from the highly reactive quinones which are normally cross-linked into melanin immediately after they are formed.Several Middle and Late genes encoding antioxidants were upregulated at least twofold 12–48 hours PBM. The Middle gene, AS 2033, a glutathione S-transferase D3, and the three Late genes, ASs 1684, 35, and 2156, encoding glutathione S-transferase 1–6 class theta, and homologs of An. gambiae there is a small, polymorphic tandem array of vitellogenin genes and a single dispersed vitellogenin gene, all located on Chromosome 2R in division 18B . Vitellogenin mRNA becomes detectable by Northern blot analysis by 8 hrs PBM, though it is detectable earlier by RT-PCR, increases dramatically by 12 hours, reaches maximal levels by 24 hours, and declines to undetectable levels by 48 hours PBM [Paramount to the development of the embryo is the massive accumulation of vitellogenin by the oocyte. In ours PBM . Our micours PBM .Drosophila Adaptin subunit, AP-1σ, CG5864, and the Late gene, AS 2086, homolog of another Drosophila clathrin-associated protein, AP-50, CG7057.Following synthesis in the fat body, vitellogenins are released into the hemolymph. Eventually, they diffuse through channels between the cells of the follicular epithelium and are accumulated by the oocyte by receptor-mediated endocytosis in clathrin-coated pits . The incAe. aegypti, the lysosomal cathepsin D-like aspartic protease (AeLAP) exhibited a similar transcription profile to vitellogenin [et al. [Ae. aegypti protein, exhibits the same expression profile, and may be its homolog. At approximately 30 hours PBM, 6 hours after peak production of vitellogenin, the activity of four other lysosomal enzymes, arylsulfatase A, acid phosphatase-1, β-galactosidase, and Cathepsin D, has dramatically increased to reach maximal levels by 36–42 hours PBM [When vitellogenesis has ceased, the biosynthetic machinery in the fat body is degraded in lysosomes . In Ae. llogenin . Cho et  [et al.  also ideours PBM ,109. Theours PBM . It willet al. [An. gambiae by identifying 168 ESTs that differed in statistical abundance between cDNA libraries made from adult female mosquitoes fed on sugar and 24 hours PBM. Ribeiro [in silico data can complement each other. However, our study is unique in that we have determined the temporal patterns of expression of the genes we identified. The observed similarities between the gene expression patterns and production of the two principal insect hormones suggest that gene transcription may be influenced by changes in JH titers as well as by 20-E levels, a phenomenon that has been well-studied in the context of Drosophila metamorphosis and in Ae. aegypti vitellogenesis. Future analysis may reveal genes co-regulated via the same promoters. Indeed, this now appears possible for organisms whose genomes have been sequenced [Holt et al.  performe Ribeiro  extended Ribeiro  studies.equenced -114. As An. gambiae genome, culminating in the public announcement of the genome sequence in 2002 and its subsequent updates. Yet, although we have identified 413 differentially expressed gene products, we could not assign almost half of them to a biological process. Of the 200 "Unknowns," 43 unique transcripts shared no significant identity with sequences in the Nr and dbEST databases. The genes corresponding to these transcripts may be identified following the second gene build of the An. gambiae genome. Functional studies using microarray analysis verified by qRT-PCR must confirm in silico predicted annotations and provide biological information about gene products. Many of the gene products identified in this study share sequence similarity with Drosophila proteins. Much of the information generated by studies of fruit fly cell biology and development may also apply to mosquitoes, although it will be more difficult to test in An. gambiae, since it is not easily manipulated genetically. This study underscores the importance of ongoing functional studies including tissue-specific expression profiling using microarray analysis and qRT-PCR. Understanding how the events following blood feeding are related to each other on a molecular level will provide a more comprehensive picture of this unique behavior and may also delineate new vector-control strategies.Great progress has been made in the annotation of the An. gambiae which had been sugar-fed (harvested 30 hours post-eclosion), rat blood-fed (harvested 30 hours PBM), and P. berghei-infected rat blood-fed (harvested 30 hours PBM), all at 19°C . Clones from all three libraries were subjected to PCR-based insert amplification using λTriplEx2 vector specific primers . Amplification reactions contained 1.0 μL eluted phage, 0.03 pmol of each primer, 1 × Taq Polymerase Buffer (Invitrogen), 3 mM MgCl2, 1 mM of each dNTP, and 0.2 U Taq Polymerase (Invitrogen), in a total volume of 100 μL. Reactions were conducted in 96-well plates on a Perkin-Elmer 9700 Thermocycler using the following cycling conditions: initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing/elongation at 70°C for 2 min, and a final elongation step at 68°C for 3 min. Samples of all PCR products were electrophoresed on 1% agarose, 1 × TBE gels and visualized by ethidium bromide staining. PCR products were purified on a Beckman Biomek FX using Montage PCR 96 Cleanup kits (Millipore), eluted in 100 μL of water, evaporated overnight and the pellets resuspended in 30 μL of 3 × SSC microarray spotting buffer. A total of 3060 resuspended cDNA inserts and 108 controls were spotted in triplicate on CMT-Gaps II slides  using the Affymetrix Arrayer 417 at 19 – 20°C and relative humidity between 50 – 60%. Slides were post-processed by baking at 80°C for three hours, incubation in 1% SDS for 2 min, in 95°C purified water for a further 2 min, and then plunged 20 times into 100% ethanol kept at -20°C and air-dried via centrifugation at 500 RPM for 5 min.Three cDNA libraries were constructed from abdomens of adult female Total RNA was extracted from blood-fed and sugar-fed whole adult female mosquitoes of the malaria susceptible 4Arr strain, 5–7 days post eclosion, using Trizol  according to the manufacturer's directions. Mosquitoes were blood-fed on anesthetized white rats and maintained under conditions similar to those for sugar-fed mosquitoes, 25°C with 80% humidity and a 12-h light/dark cycle with available 20% sucrose solution, until collection. Fully-engorged mosquitoes were identified visually and harvested at the following 10 time-points: 1) 5 min after initiation of blood feeding during the acquisition of the blood meal (DBM), 2) 30 min DBM, 3) 0 hr post-blood meal (PBM), immediately after they ceased feeding on the rat, 4) 1 hr PBM, 5) 3 hr PBM, 6) 5 hr PBM, 7) 12 hr PBM, 8) 16 hr PBM, 9) 24 hr PBM, and l0) 48 hr PBM. For each blood-fed sample, total RNA was extracted from batches of approximately 10–15 females. Total RNA was also extracted from batches of 100 sugar-fed females for reference samples. RNA samples were then treated with 1.0 μL DNase I (Life Science Technology) according to manufacturer's instructions. Following DNase I treatment, total RNA was re-extracted with Trizol. First strand cDNA synthesis and labeling with Cyanine 3 (Cy3) or Cyanine 5 (Cy5), were performed on 15 μg of total RNA from each sample using the Genisphere 3DNA Array 50 kit according to the manufacturer's protocol. Hybridizations were conducted following the two step protocol recommended by the manufacturer: 1) cDNA hybridization to the amplified cDNA probes spotted on the slides, 2) hybridization of 3-DNA fluorescent dendrimers (Genisphere) to cDNAs via the capture sequences incorporated into them during first strand synthesis. All cDNA and fluorescent dye hybridizations were performed in a volume of 50 μL using the formamide-based hybridization buffer provided by the manufacturer. The cDNA hybridizations were performed at 45°C overnight. The slides were then washed according to the 3DNA Array 50 kit protocol and air dried by centrifugation for 3 min at 800 RPM. The 3-DNA hybridizations were performed at 53°C for 2 hours as described above, except that 0.5 mM DTT was added to the first two wash solutions to protect the fluorochromes from oxidation. Five replicate slides were generated for each of the ten time points for a total of 50 hybridized and labeled slides. These included two dye-swap experiments performed to eliminate dye fluorescence bias. Pilot experiments conducted with total RNA from the same sample labeled with both Cy3 and Cy5, self-self hybridization, indicated that there was no dye labeling bias following data normalization (data not shown).Following hybridization and washing, microarray slides were scanned successively at 532 and 635 nm using the Affymetrix 428 Array Scanner. Raw signal intensities were acquired using the adaptive circle algorithm and spot intensities quantified using the Jaguar 2.0 segmentation and data analysis software . Average signal intensities were normalized using the Loess curve for intensity dependent normalization followed by a per gene median normalization using the Genespring 5.1 software . Signal intensities were filtered such that only gene products exhibiting a raw signal intensity value greater than 300 pixels in one channel and greater than two-fold expression difference between the sugar-fed and blood-fed samples from at least one time point hybridized to the same array were utilized in further analysis. Gene expression level measurements falling outside one standard deviation from the mean signal intensity of each gene product calculated from the five replicates were excluded from further analysis. As an additional quality control, only genes whose PCR amplified products migrated as a single band in agarose gel electrophoresis and that generated high quality sequences for use in EST assembly were analyzed.k-means clustering (data not shown). From this preliminary analysis it was determined that three major clades existed and the genes were clustered using Genespring software using a k-means clustering algorithm with a centroid number of 3 and the Pearson Correlation distance metric. Finally, an independent analysis using principal components analysis (PCA) was conducted on the genes induced or repressed at least twofold, using the Genespring software.Gene products that were induced or repressed at least twofold during blood feeding were initially clustered hierarchically using the Genespring software to determine the user-defined number of centroids (clusters) to be used in Transcript levels of several selected genes were measured using SYBR dye technology  and quantitative real-time PCR (qRT-PCR) analysis in order to validate microarray data,. The Primer Express Software v. 1.5  was used to design the following primers to nine genes: the two Early Genes agCP4871  and CG8690 , the four Middle Genes agCP8064 , agCP12846 , CG7758 , and agCP14019 , and the two Late Genes agCP14623  and agCP2112 , and the ribosomal protein S7 (RP S7) gene control . RP S7 was used as an internal control since its expression is constitutive during blood-feeding . All ampAND carried out the cDNA library construction, microarray fabrication, data analysis, and drafted the manuscript. YSH performed the microarray experiment, data analysis, and helped draft the manuscript. MKK performed the qRT-PCR experiments and MEH annotated the ESTs. BWH constructed the microarray genechips and carried out microarray data acquisition. NFL sequenced the cDNA library and JRH maintained and provided mosquito samples throughout the project. PR assisted AND and YSH to draft the manuscript and reviewed it. FHC (P.I.) initiated and supervised the project. All authors read and agreed on the final version of this manuscript.Supplementary Table S1 in a Microsoft Excel format where gene annotations for all 413 at least twofold differentially expressed gene products are given.Click here for file


Chlamydiales are familiar causes of acute and chronic infections in humans and animals. Human pulmonary emphysema is a component of chronic obstructive pulmonary disease (COPD) and a condition in which chronic inflammation manifested as bronchiolitis and intra-alveolar accumulation of macrophages is common. It is generally presumed to be of infectious origin. Previous investigations based on serology and immunohistochemistry indicated Chlamydophila pneumoniae infection in cases of COPD. Furthermore, immunofluorescence with genus-specific antibodies and electron microscopy suggested involvement of chlamydial infection in most cases of pulmonary emphysema, but these findings could not be verified by PCR. Therefore, we examined the possibility of other chlamydial species being present in these patients.Tissue samples from patients having undergone lung volume reduction surgery for advanced alpha-1 antitrypsin deficiency  or non-alpha-1 antitrypsin deficiency emphysema (n = 34) or wedge resection for hamartochondroma (n = 14) were examined by transmission electron microscopy and PCR.Chlamydia psittaci was demonstrated by PCR in lung tissue of 66.7% AATD vs. 29.0% non-AATD emphysema patients. Partial DNA sequencing of four positive samples confirmed the identity of the agent as Chlamydophila psittaci. In contrast, Chlamydophila pneumoniae was detected only in one AATD patient. Lung tissue of the control group of non-smokers with hamartochondroma was completely negative for chlamydial bodies by TEM or chlamydial DNA by PCR.In all cases of AATD and 79.4% of non-AATD, persistent chlamydial infection was detected by ultrastructural examination. Intra-alveolar accumulation of macrophages and acute as well as chronic bronchiolitis were seen in all positive cases. The presence of Chlamydophila psittaci in pulmonary emphysema by linking this chronic inflammatory process to a chronic infectious condition. This raises interesting questions on pathogenesis and source of infection.These data indicate a role of  Chlamydiaceae are well-known etiological agents of acute and chronic infections in humans and animals [Chlamydia (C.) psittaci, the agent responsible for this disease, has had several different names and, according to a recent proposal, should now be called Chlamydophila (Cp.) psittaci [C. pneumoniae [Chlamydophila pneumoniae [Cp. pneumoniae infection based on serology was reported in severe cases of chronic obstructive pulmonary disease (COPD), in which emphysema is dominant [Cp. pneumoniae by immunohistochemical staining was elevated in lung tissue from subjects with COPD, but controls were not completely negative [Cp. pneumoniae was thought to be virulent for humans only, but recent descriptions of isolates from horse, koala, frog and reptiles suggest a wider host spectrum and even the possibility of zoonotic transmission [Several species of the family  animals ,2. The f animals . Chlamydpsittaci . A centueumoniae ,6 curreneumoniae . Meanwhidominant ,8, as wedominant  and in pdominant ,11. The negative . Initialsmission -15.Chlamydia spp. in patients having undergone lung volume reduction surgery for advanced pulmonary emphysema [Cp. pneumoniae was detected in two out of ten cases [Chlamydia spp. in over 80% [Chlamydiaceae. Here we report the results of a more detailed study involving a larger number of cases and samples including controls.Our previous investigations by means of immunofluorescence using a genus-specific antiserum against chlamydial LPS and scanning as well as transmission electron microscopy showed infection of the alveolar parenchyma and the bronchioles by mphysema ,17. Accumphysema . In prelmphysema , DNA speen cases . But thiLung tissue of adequate quality from patients with advanced emphysema undergoing lung volume reduction surgery was used for the present study ,21. SampFormalin-fixed lung tissue was embedded in paraffin wax , slides of 3–7 μm thickness were cut using a rotatory microtome  and stained by hematoxylin and eosin. For TEM, tissue was fixed in 2.5% buffered glutaraldehyde or 3.5% formaldehyde and embedded in epon after postfixation with osmium tetroxide and block contrastation with uranyl acetate. In the cases of the hamartochrondroma control group, cores with diameters of 0.60 cm and 0.24 cm were obtained from paraffin blocks using a prototypical self-made manual tissue puncher (a device for tissue microarray construction developed by M.W.). These cores were used to select an area well defined by light microscopy for PCR (0.60 cm cores) and TEM (0.24 cm cores). For TEM, tissue was dewaxed with xylene and processed as described above. Semithin sections  were stained with basic fuchsin and methylene blue to define blocks of adequate quality. Ultrathin sections from two to five blocks were stained with lead citrate and examined using a Zeiss EM 900 transmission electron microscope .Tissue from two different resources was used. Firstly, frozen tissue was collected immediately after resection and stored at -80°C (n = 31). Secondly, paraffin-embedded tissue (PET) containing histologically discernible inflammation sites was used, and sections or 0.60 cm cores were dewaxed using xylene (n = 12 and 14 controls). In five cases, material was available as both frozen and PET. DNA was isolated from lung tissue using the High Pure PCR Template Preparation Kit  according to the instructions of the manufacturer. Five μl of the DNA extract were used as template in PCR.C. psittaci and Cp. pneumoniae by a modified version of the nested PCR procedure described by Kaltenböck et al. [ompA gene. The first step was genus-specific amplification using primers 191CHOMP (5'-GCI YTI TGG GAR TGY GGI TGY GCI AC-3') and CHOMP371 (5'-TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC-3'). For the second amplification, we used 1 μl of the genus-specific product and primer combination 218PSITT (5'-GTA ATT TCI AGC CCA GCA CAA TTY GTG-3') / CHOMP336s (5'-CCR CAA GMT TTT CTR GAY TTC AWY TTG TTR AT-3') for C. psittaci, or 201CHOMP (5'-GGI GCW GMI TTC CAA TAY GCI CAR TC-3') / PNEUM268 (5'-GTA CTC CAA TGT ATG GCA CTA AAG A-3'), for Cp. pneumoniae, respectively. The sizes of specific amplicons are: 576–597 bp (genus-specific product), and 389–404 bp for C. psittaci or 244 bp for Cp. pneumoniae after nested PCR. A detailed protocol of the procedure is contained in [Samples were tested fork et al. , which tained in .C. psittaci-group [In order to discriminate the different members of the ci-group , five μlci-group . NucleotThe median value of age was 46.5 in AATD, 58.0 in non-AATD emphysema and 64.5 in hamartochondroma. The rate of females varied between 33.3% in AATD, 37.5% in non-AATD and 42.9% in hamartochondroma. Histology revealed destruction of the alveoli, intra-alveolar accumulation of macrophages, and acute alongside chronic bronchiolitis in all cases of AATD Fig.  and non-TEM images illustrate that the cell and tissue morphology appeared severely destroyed in emphysema samples. Chlamydial elementary and reticular bodies of 0.2 to 0.8 μm diameter were found on the surface of alveolar or bronchiolar epithelium and showed adherence to microvilli as previously described ,17. RetiC. psittaci-specific DNA in four (66.7%) specimens with AATD and nine (29%) with non-AATD emphysema . In five cases, where both PET and frozen tissue were examined, one patient was positive in TEM and another one in PCR.Examination by PCR revealed the presence of  in four 6.7% specema Fig. . In one Cp. psittaci.To confirm the identity of the chlamydial species, DNA from four of the positive samples, i.e. N16, N25, N26, and N33, was sequenced in the 16S rRNA signature region  indicate an association with regional activity of infection and inflammation. Besides psittacosis, Cp. psittaci has been recently associated with chronic inflammation in patients with ocular adnexal lymphomas [Cp. psittaci was found at comparable and statistically significant rates in AATD and non AATD emphysema.Higher detection rates of ymphomas . In COPDymphomas . The relymphomas  and by cymphomas . These fCp. psittaci provides an important indication on the source of infection. It is conceivable that the patients were infected through contact with birds, although this could not be verified for lack of relevant data on history. The detection rate of chlamydiae in cases of AATD emphysema vs. non-AATD emphysema was clearly higher, thus indicating a relevant role of Cp. psittaci infection in this disorder, or at least a higher susceptibility of AATD patients for an infection of their lungs with Cp. psittaci. Further investigations concerning smokers and non-smokers, pathogenetic relevance and zoonotic implications are required. In any circumstances, Cp. psittaci has to be considered an underestimated pathogen with considerable importance in public health, although the various facets of its specific impact have yet to be evaluated.The fact that the chlamydial agent present in the emphysema tissue was identified by DNA sequencing as an avian serovar of None declared.Dirk Theegarten has designed and organized this study, done light microscopy, written most of the manuscript, participitated in its statistical analysis and reviewed results of transmission electron microscopy. Olaf Anhenn participitated in designing the study, collected the data, performed statistical analysis and reviewed the manuscript. Helmut Hotzel carried out PCR analysis and DNA sequencing. Konrad Sachse has done the sequence alignments and written the parts of the manuscript concerning molecular biology and veterinary aspects, and also participated in PCR analysis. Mathias Wagner developed and used the tissue puncher for this study. Georgios Stamatis has done lung volume reduction surgery. Alessandro Marra and Georgios Stamatis have done the clinical parts. Alessandro Marra has reviewed the history of the patients. Grigori Mogilevski performed transmission electron microscopy. All authors have discussed and approved the final manuscript.The pre-publication history for this paper can be accessed here:


There is great interest in appropriate phenotypes that serve as indicator of genetically transmitted frontal (dys)function, such as ADHD. Here we investigate the ability to deal with response conflict, and we ask to what extent performance variation on response interference tasks is caused by genetic variation. We tested a large sample of 12-year old monozygotic and dizygotic twins on two well-known and closely related response interference tasks; the color Stroop task and the Eriksen flanker task. Using structural equation modelling we assessed the heritability of several performance indices derived from those tasks.h2 = nearly 50 %). In contrast, we found little evidence of heritability on flanker performance. For both tasks no effects of sex on performance variation were found.In the Stroop task we found high heritabilities of overall reaction time and – more important – Stroop interference (These results suggest that normal variation in Stroop performance is influenced by underlying genetic variation. Given that Stroop performance is often hampered not only in people suffering from frontal dysfunction, but also in their unaffected relatives, we conclude that this variable may constitute a suitable endophenotype for future genetic studies. We discuss several reasons for the absence of genetic effects on the flanker task. The Stroop test  is arguaThe Stroop test has been used both to tap fundamentals of human information processing (e.g. ), and asThere now exist numerous versions of the Stroop test. For example, instead of using color words, researchers have adopted more ecologically relevant items, such as emotion words, pictures of food items or of threatening objects, etc. In addition, it is now also common to use computerized versions of the Stroop task, permitting a trial-by-trial analysis of performance. But what all these different Stroop versions have in common is that the subject is always presented with a stimulus that simultaneously activates two conflicting response channels; one response is activated by the instructions, whereas the other response is activated by elements in the array that strongly invite an alternative – yet incorrect – response. In order to resolve this response conflict the subject has to direct attention to task relevant information and ignore information from the task irrelevant channel. The time needed to resolve this conflict is derived using subtractive logic, and can be used as an index of the efficiency of the attentional system under investigation.A task that is less widely used in clinical circles, but that also indexes the efficiency of the frontal network is the Eriksen flanker task. In the arrow version of this task, subjects have to respond to the direction of a left or right pointing arrow, and ignore flanking arrows that point in the opposite direction as the target arrow . SimilarThe Stroop task  has thus acquired a strong neuropsychological validation, and is nowadays widely used in clinical settings. However, studies adopting an individual differences paradigm have revealed that the time needed to resolve the response conflict in the Stroop task does not predict the time needed to resolve the response conflict in the flanker task. In an earlier study we  found thFurther insight into the nature of these interference tasks might be gained by adopting a genetic perspective on normal and abnormal frontal functioning. A wealth of studies has now shown that many frontal psychopathologies are influenced by genes. For example, the heritability of ADHD is estimated to be around 80% (e.g.). In a sdrd4 and dat1, and the efficiency of the frontal executive attention network ). The pattern of twin correlations is hardly indicative of genetic effects on performance. Even though the MZ twin correlations on response speed were higher than the DZ correlations, the highest twin correlation was obtained with the DOS zygosity group. Furthermore, the highest twin correlation for the flanker effect was obtained with the DZF group. In addition, the twin correlations within the monozygotic groups were low. We therefore conclude that there were no genetic effects of flanker performance.Table The twin correlations obtained with the Stroop task thus appeared strongly indicative of genetic effects. Using structural equation modelling, these effects were formally tested. But prior to testing we had to establish whether there were significant differences in variances across sex and zygosity, since one of the assumptions underlying structural equation modelling is the assumption of homogeneity of variances. We conducted the Levene test on all variables, separately for the first-born and the second born twins. For none of the variables the Levene test yielded a significant effect, with the possible exception of the completion time of Card 1 for the second born twins, F = 2.664, p = 0.05. So, we felt it was legitimate to use structural equation modelling to test for genetic effects.2 ranged from 7.732 to 8.883, df = 9, p's ranged from .448 to .561), with the possible exception of Card 1 , although the most parsimonious model for Card 1 fitted slightly better . Sex differences in parameter estimates could be discarded from the models, although they were almost significant for Card 3 . Common environmental influences were not necessary to describe the data, but additive genetic influences explained a significant part of the variance in all 4 variables. Heritabilities for the 3 cards were 75%, 70% and 74% respectively, with confidence intervals indicating that these were well above half the variance. Heritability of the interference effect was 49%, with a 95% confidence interval between 29 and 64%.In this study we assessed the heritability of performance on two well-known response interference tasks: the color word Stroop task and the Eriksen flanker task, using a large sample of 12-year old twins. The aim was to test whether Stroop performance and/or flanker performance could qualify as a suitable endophenotype for genetic frontal abnormalities, such as ADHD. First, we found that the time to complete each of the three cards was highly heritable. This may represent a general factor related to processing speed and/or rapid naming speed. Of greater importance was the finding that the interference score (the difference between completion times of Card 2 and 3) was also heritable: nearly 50% of the variation in performance was due to genetic variation. Thus, the efficiency of the network that deals with response conflict is -in part- under genetic influence.For the theoretically similar flanker task, however, there was little evidence of genetic influences on performance. Even though the MZ twin correlations on response speed were higher than the DZ correlations (as in ), the hiThe question now is whether Stroop and flanker performance can qualify as a suitable endophenotype of frontal pathologies. Recently a list of 5 criteria was compiled that are ideally possessed by endophenotypes . CriteriCriteria 1 and 2 of  state thWe have found evidence for the existence of strong genetic effects on conflict resolution, although the effects are task dependent. We conclude that performance on the Stroop test yields a better endophenotype for frontal (dys)function than performance on the flanker task. So, despite the overlapping regions of brain activation in the Stroop and flanker tasks, and despite their face-value similarity, we believe that these interference tasks differ in important, yet unknown ways.The subject group consisted of a group of 290 12-year old twins. There were 33 monozygotic male pairs (MZM), 24 dizygotic male pairs (DZM), 45 monozygotic female pairs (MZF), 16 dizygotic female pairs (DZF), and 27 opposite sex pairs (DOS). The twins participate in a longitudinal study of attention and attention problems. The twins are registered in the Netherlands Twin Registry (NTR), which is hosted by the Vrije Universiteit of Amsterdam . The twiTwin pairs were first asked in writing whether they were willing to participate in the study. Permission was also asked of the parents or caretakers. If permission was granted, the families received further information on the study, and were invited to come to the campus site to do the tests. The study was approved by the local Ethics Committee, and on the day of testing the children and their parents / legal representatives signed an informed consent form.Twins were tested on the same day. The children performed a range of neuropsychological tests that lasted approximately 4 hours per child. All tests were performed in the same order. Here we focus on the Stroop Color and Word Test  and the In the Eriksen flanker task subjects were presented with a horizontal array of 5 arrows. Subjects were instructed to attend to the direction of the center arrow, and ignore the 4 flanking ones. Subjects had to press the left key to a left facing center arrow, and the right key to a right facing center arrow. The flanking arrows could either all point in the same direction as the target arrow , or they all pointed in the opposite direction . Subjects received 40 congruent and 40 incongruent trials in a random order. For each trial, the computer stored the RT and whether the correct key was pressed. The number of valid cases in the flanker task is lower than in the Stroop task. This was due either to practical problems (some data were not collected or not stored on the computer), or because some subjects made an extremely high number of errors (less than 75 % correct). Further details of the Stroop task and the flanker task can be found in .In order to test for the effects of card type and sex on Stroop performance we performed an analysis of variance on the completion times with card type  as within-subjects factor, and sex  as between subjects factor. For the flanker task, we performed an ANOVA on the mean correct response times and on the mean percentages correct, with stimulus type (congruent vs. incongruent) as within-subjects factor, and sex  as between subjects factor. The same analysis was done for the error rates. These analyses were done separately for the first-born twins and the second born twins, because twins within a family do not yield independent data. We adopted an alpha-level of .05.minus Completion time Card 2. There exists another method to determine the size of Stroop effect, which also takes into account the completion times of Card 1  [Pearson correlations were calculated for the different measures between first born and second born twins for all zygosity groups. A first indication of the heritability can be derived by doubling the difference between correlations for MZ twins and those for DZ twins [MZ-rDZ)] .a, c and e represent the influence of genes, common environment and unique environment on the phenotypes (P) of twin 1 and twin 2. The total variance of the phenotype (P) = a2 + c2 + e2. The heritability (h2) is calculated as a2/VP.A structural equation modeling approach as implemented in Mx  was useda, c and e to be equal across sexes was compared to one in which they were allowed to vary. After this, the significance of c and a was investigated by dropping them one by one from the model and comparing the fit of a full model to that of a reduced model. The chi-squared statistic is computed as twice the difference between the likelihood for the full model (-LL0) and that for a reduced or constrained model (-LL1) (χ2 = 2 × (LL0-LL1)) and is tested against the difference in degrees of freedom between the two models.To test if parameter estimates are equal for boys and girls the fit of a model with constrained parameter estimates for JFS drafted the manuscript, and performed the descriptive analyses. GCMvB performed the genetic analyses. TJCP collected the data. FCV and DIB conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.h2), common environmentability (c2), and unique environmentability (e2), for completion times of the three cards, and of the Stroop interference score. Best fitting models are shown in bold. Parameter estimates are given in percentages .Model fitting and parameter estimates Model fitting and parameter estimates of heritability (Click here for file


Intravenous morphine use is associated with elevated histamine release leading to bronchoconstriction, edema and hemodynamic instability in some patients. This study evaluated the possibility that sulfite, which is present as a preservative in many morphine preparations, might contribute to histamine release in vitro.The human mast cell line, HMC-1, was exposed to various morphine concentrations, in the absence of sulfite, under cell culture conditions. Clinically attained concentrations of morphine (0.018μg/ml and 0.45μg/ml) did not cause increased histamine release from mast cells. There was a significant increase in histamine release when the morphine concentration was increased by 1184-fold (668μg/ml morphine). Histamine release from mast cells exposed to morphine and/or sulfite required the presence of prostaglandin H synthetase. Histamine release in experiments using sulfite-containing morphine solutions was not statistically different from that observed in morphine-only solutions.in vitro experiments of the human mast cell line, HMC-1. This does not preclude the fact that sulfite may lead to elevation of histamine levels in vivo.Sulfite in sulfite-containing morphine solutions, at concentrations seen clinically, is not responsible for histamine release in  Morphine is among the most common analgesic agents used in the intensive care setting. It is usually administered parenterally as an intravenous infusion. Despite its efficacy, its use is associated with a number of hazards including respiratory depression , toleranA number of clinical studies have demonstrated an increase in plasma histamine concentration following intravenous morphine administration -7, but tIn the body, sulfites are generally oxidized by the mitochondrial enzyme sulfite oxidase and non-toxic metabolites are excreted in the urine . A smallSulfite radicals are chemically reactive and have been implicated in lipid , proteinThe potential generation of free radicals from sulfite preservatives may confound our understanding the association of morphine with histamine release in man. A number of studies have demonstrated that, when exposed to free radicals, mast cells may release histamine -26. It iTotal histamine content of mast cells was calculated by freezing and thawing suspensions for 3 cycles. Cell suspensions containing 500,000 cells/ml released 1.27μg/ml (0.21) histamine, corresponding to 2.53 pg (0.41) histamine per mast cell.Mast cells stimulated with 0.25μg/ml calcium ionophore released 63% (7.2) of total histamine.The effect of histamine release from mast cells treated with both clinically attained concentrations of morphine and concentrations above those seen clinically was determined.To chemically defined reaction mixtures, morphine was added at varying concentrations . Enzymes causing free radical activation were not present. The blank sample contained no morphine. Reaction mixtures were incubated in a water bath at 37°C for 60 minutes. Experiments were performed in triplicate. Histamine release from samples was determined following derivitization with o-phthaldialdehyde and mercaptoethanol using high-performance liquid chromatography as detailed below. Histamine release from reaction mixtures was expressed as a % of total histamine present in the cell pellet.Histamine release from reaction mixtures containing morphine at all concentrations was not statistically different p > 0.1) from histamine release from blank samples  was added to the reaction mixtures described previously. Blank samples contained neither morphine nor sulfite. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as above.There was no statistical difference in histamine release at any concentration of morphine with sulfite compared with blank samples Table .Reaction mixtures containing 668μg/ml morphine with 0.1% sodium bisulfite were incubated for 5, 10, 20, 40 and 60 minutes yielded results identical to those observed in the absence of sodium bisulfite (results not shown).Prostaglandin H synthestase may catalyze free radical formation . The firProstaglandin H synthetase was added to reaction mixtures at a concentration of 25 mU. In reaction mixtures containing no drug, the enzyme was added to mast cell samples containing neither morphine nor sulfite. Controls contained no enzyme.Reaction mixtures containing the enzyme alone (no sulfite or morphine) produced similar histamine release compared to blank samples (3.2% (0.7) of total and 2.9% (2.6) of total respectively). The presence of prostaglandin H synthetase had no effect.In vivo studies show histamine release at these concentrations of morphine [in vivo concentration following an intravenous morphine bolus) and 18 ng/ml  [The effect of clinically attained concentrations of morphine in the presence of prostaglandin H synthetase on mast cell histamine release was investigated. morphine -7. Morphnfusion) . Mast ceBlank samples contained water instead of morphine. All samples contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Histamine release values are shown in Table The effect of sodium bisulfite 0.1% in varying concentrations of morphine in the presence of prostaglandin H synthetase was investigated. This preservative is present in many morphine preparations and its effect on histamine release from mast cells is determined here. Morphine was added to the mast cell samples at concentrations of 668μg/ml, 6.68μg/ml, 450 ng/ml and 18 ng/ml. Sodium bisulfite was added to samples at a concentration of 0.1% of morphine concentration. Controls contained morphine only. Each sample also contained 25 mU of prostaglandin H synthetase. Specimens were incubated, dried, re-dissolved, derivitized and analyzed as described below. Reaction mixtures containing morphine 668μg/ml with 0.1% sodium bisulfite showed a statistically significant increase in histamine release compared with blank samples (p = 0.03). There was no statistical difference in histamine release between morphine only samples at all concentrations and samples containing morphine and 0.1% sodium bisulfite  showed a significant increase in histamine release in the presence of prostaglandin H synthetase compared with no enzyme. This was true for both sulfite-free (p = 0.003) and sulfite-containing (p = 0.009) morphine solutions. This suggests that even at clinically-attained morphine concentrations, it is not only possible to cause the metabolic activation of sulfite, but also that of morphine.The effect of sulfite alone (without morphine) on histamine release from mast cells was investigated. To 4 mast cell reaction mixtures, varying concentrations of sulfite  were added. These concentrations of sulfite correspond to the concentration of sulfite present in morphine solutions of 18 ng/ml, 450 ng/ml, 6.6μg/ml and 668μg/ml. To each sample 25 mU prostaglandin H synthetase was added. The highest concentration of sulfite (0.67μg/ml) was incubated for 10, 20, 40 and 60 minutes to assess time dependence. The reaction mixtures were incubated, centrifuged and analyzed as described below.In the presence of prostaglandin H synthetase, sulfite resulted in histamine release from mast cells. There was a significant increase in histamine release (9.7% (0.65) of total) in the reaction mixture containing 0.67μg/ml sulfite (p = 0.01). There was no statistically significant difference in histamine release compared with blank samples at all other concentrations of sulfite (0.018 ng/ml sulfite (2.8% 0.3) of total, p = 0.91), 0.45 ng/ml sulfite (1.93% (0.35) of total, p = 0.53) and 0.007μg/ml sulfite (4.5% (0.7) of total, p = 0.36)  at 10 minutes incubation, 7.7% of total (2.2) at 20 minutes, 10.5% of total (6.1) at 40 minutes and 9.7% of total (0.65) at 60 minutes.Our experiments showed similar histamine release from the mast cell line HMC-1 stimulated with morphine with or without 0.1% sodium bisulfite. Maximum histamine release was 16.1% of total in morphine only samples and 17.6% of total in morphine with 0.1% sodium bisulfite samples. This percentage is similar to other studies of histamine release in the human mast cell line HMC-1, where maximum release was 20–30% -30. In oin vivo situation, where maximum release usually occurs within 5 minutes of morphine administration, and suggests that we were not able to simulate exactly the human environment where histamine release occurs. It is likely that although the sulfite radical is formed rapidly in vitro, it may take some time for its effects to become evident [HMC-1 cells contain histamine and release their histamine content in response to various exogenous stimuli -30. Our  evident .in vivo.Metabolic activation of sodium bisulfite may occur through a variety of mechanisms. We investigated a number of enzyme systems and our final model used prostaglandin H synthetase. Other enzyme systems (horseradish peroxidase/ hydrogen peroxide and hypoxanthine/ xanthine oxidase) did release histamine in our model (results not shown), but they produced excess background interference in the chromatograms and made identification of peaks difficult. Activation of sulfite following morphine administration may not occur through this mechanism, however, and is another possible flaw in our aim to construct a model similar to the one that exists in vivo, but it has not been investigated whether sulfite, in the form of the sulfite radical, is the cause of this release. Our study finds that sulfite in sulfite-containing morphine solutions, at clinically attained concentrations, is not responsible for histamine release in in vitro experiments of the mast cell line HMC-1, but this does not preclude the fact that sulfite may lead to marked elevation in histamine levels in vivo. The release of a significant amount of histamine with either sulfite or morphine at concentrations higher than those seen clinically suggests that both substances may be responsible for histamine release from mast cells. Further clinical research studies need to be performed to examine this possibility.Morphine has been shown to cause significant histamine release in vitro experiments of the mast cell line, HMC-1, but this does not preclude the fact that sulfite may lead to marked elevation in histamine levels in vivo. Our experiments suggest that at high concentrations, both morphine and sulfite are responsible for histamine release from mast cells. Further in vivo research needs to be performed to examine this possibility.Our study finds that the sulfite in sulfite-containing morphine solutions, at concentrations seen clinically is not responsible for histamine release in 2 vented tissue culture flasks  containing Iscoves modified Dulbecco's medium  with 10% iron-supplemented calf serum  and monothioglycerol 1.2 mM . Incubations were conducted in a humidified atmosphere at 5% CO2 and 37°C. Cells were passed weekly and fed only when necessary (evidence of color change in media).Human leukemic mast cells, HMC-1  were kinTo pass cells, the cell suspension was placed in a 50 ml polypropylene centrifuge tube  and centrifuged at 250 × g at 4°C for 10 minutes. The culture medium was discarded and the cells were re-suspended in fresh medium. Cell density was maintained at 500,000 cells/ml.Reagent grade O-phthaldialdehyde (OPA), mercaptoethanol, dihydrogen sodium phosphate, disodium EDTA, sodium bisulfite, morphine sulfate, hydrogen peroxide, xanthine oxidase, boric acid and phosphate buffered saline (PBS) were purchased from Sigma . Methanol (HPLC grade), acetonitrile (HPLC grade) and sodium hydroxide were from Fisher Scientific . Phosphate buffered saline (PBS) pH 7.4, contained 120 mmol/L NaCl, 2.7 mmol/L KCl and 10 mmol/L phosphate. Horseradish peroxidase was purchased from Cooper Biomedical. Prostaglandin H synthetase was purchased from Calbiochem .Mast cell suspensions, containing approximately 500,000 mast cells were, aliquoted into 1 ml samples. These were rinsed 4 times with 4 ml of PBS, pH 7.4. Cells were then re-suspended in 1 ml PBS. Reaction mixtures were supplemented with morphine, sulfite and prostaglandin H synthase at this stage according to experimental design, unless otherwise specified. Reaction mixtures were incubated for 60 minutes in a water bath at 37°C. Reactions were stopped by centrifugation at 250 × g for 4 minutes at 4°C and placed on ice. Then 200μl of the supernatant was collected and dried under nitrogen at 40°C.Histamine in the supernatant was measured using derivitization with OPA and mercaptoethanol (ME) as described . Dried sThe response of HMC-1 cells to the stimulant calcium ionophore was investigated to assess the releasability of HMC-1 cells. 0.25μg/ml calcium ionophore was added to mast cell suspensions. Reaction mixtures were incubated and analyzed as above.To extract the remaining histamine from the original sample, the mast cell pellet in suspension was frozen and thawed 3 times. It was then centrifuged at 250 × g for 4 minutes and 200μl of the supernatant was dried under nitrogen as described previously. Samples were re-dissolved, derivitized and analyzed as above. Histamine release during morphine incubations was expressed as a percentage of total histamine release.Histamine was measured by high-performance liquid chromatography using a Varian model 5500 chromatograph. The mobile phase was 0.1 M dihydrogen sodium phosphate and 1 mM disodium EDTA with 16% methanol and 14% acetonitrile adjusted to pH 6.4. The flow rate was 0.8 ml/minute.Separations were performed using a 15 cm × 4.6 mm, Ace 5 C18 reversed-phase cartridge column . A Zorbax C8 guard column with a 5 micron, 4.6 × 12.5 mm analytical guard-column cartridge  was used. Fluorescence detection was used with the fluorimeter (Varian 9070) set at an excitation of 348 nm and an emission of 444 nm .Chromatographic conditions involved the use of a mobile phase gradient to optimize peak separation. A linear gradient starting at 90% mobile phase, 10% acetonitrile and ending at 80% mobile phase, 20% acetonitrile took place over 20 minutes. The column was washed by increasing the acetonitrile to 30% by 22 minutes and by 23 minutes the proportions were returned to the original values. The length of each run was 30 minutes.Values are expressed as a mean +/- SD of 3 experiments. One-way analysis of variance was used to determine the significance of the difference between groups. Results were considered significantly different when p < 0.05.EG participated in experiments and drafted the manuscript. JB conceived of the study and participated in its design. CM suggested the methodology of experiments. All authors read and approved the final manuscript.


Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING  method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project. Rapid progress being made in genome sequencing projects provides raw material for the potential understanding of gene function, so effective reverse genetic strategies are increasingly in demand . Sequencin silico procedure for Arabidopsis researchers, as searchable databases of flanking sequences from T-DNA and transposon insertions are available on-line  [17 mutations] [1.5-kb/1-kb]÷([11-kb] [750 plants]) = ~12 mutations). Since this study was completed, we have expanded the size of our mutagenized population to ~2500 M1 individuals in the B73 genetic background and ~2000 M1 plants in the W22 genetic background that are ready for screening, and populations of ~10,000 M1 plants are being prepared.For effective pooling, each individual in a pool must be represented at a concentration that is equivalent to the other members in a pool. Failure to accomplish this could result in a mutation represented in the pool below the level of detection. The goal is to maximize throughput by increasing pooling while still detecting all possible mutations. We have shown that in Arabidopsis, heterozygous mutations can be as efficiently discovered as homozygous mutations in 8-fold pools, thus providing a minimum estimate of robust discovery in a production setting of 1 in 16 [Sorting Intolerant From Tolerant [Project Aligned Related Sequences and Evaluate SNPs[Position-Specific Scoring Matrix) Difference score based on alignment blocks (Figure in vivo targets of CMT3-dependent methylation [At least one of the maize genes that we screened yielded an excellent allelic series. We discovered three missense mutations in DMT102, all of which are predicted to damage the protein based on sequence conservation. This analysis used two different programs: SIFT (Tolerant , which uuate SNPs, which phylation . Togethehylation , our TILTILLING has several advantages as a general reverse-genetic tool, especially for organisms for which other options are limited. The high density of mutations resulting from chemical mutagenesis means that, relative to insertional or deletional mutagenesis, far fewer plants are required for screening and much smaller genes can be effectively targeted. EMS is a stable and reliable mutagen, whereas the stability, penetrance and accuracy of RNAi-based silencing is uncertain ,31, and B73 pollen was mutagenized with EMS and applied to the silks of B73 ears . Ears weSamples were prepared from lyophilized leaf tissue essentially as described , except The same procedure used for TILLING Arabidopsis  was adapScreening of mutations using LI-COR gel analyzers was performed as previously described . SequencSHR, KY and EB prepared the samples, CB, CAC, LCE and ARO performed the high-throughput screening and data processing, CW and NS provided the mutagenized plant material, EAG performed the data analysis and BJT, LC and SH participated in the design and execution of the study and wrote the paper.


The unique ability of nucleic acids to replicate has recently been combined with the power of combinatorial chemistry, providing a new approach to the science of drug design At some time almost 4 billion years ago, nature likely was faced with a chemical dilemma. Nucleic acids had emerged as replicable information carriers and primitive catalysts , yet theA small but growing number of researchers have begun to tackle a modern version of this dilemma. While proteins and nucleic acids can be manipulated using powerful molecular biology techniques that enable their directed evolution, the size, fragility, and relatively limited functional group diversity of biological macromolecules make them poorly suited for solving many problems in the chemical sciences. Ideally, researchers would like to apply evolution-based approaches to the discovery of functional synthetic, rather than biological, molecules. A solution analogous to nature's translation of mRNA into protein could, in principle, address this contemporary problem . If a laThe translation of DNA into synthetic molecules is conceptually distinct from the use of DNA simply as a tag during the solidphase synthesis of a molecule that is part of a combinatorial library . The latDNA-templated organic synthesis (DTS) has emerged as one way to translate DNA sequences into a variety of complex synthetic small molecules . In thisPLoS Biology, Harbury and co-workers describe an elegant new approach to translating DNA into synthetic peptides called “DNA display.” Their approach uses DNA hybridization to separate mixtures of DNA sequences into spatially distinct locations. The first paper , and can use reaction conditions such as high temperatures or high pH that may not be compatible with DNA hybridization. These features suggest that DNA display may be able to access structures that cannot be created by DTS. Likewise, because DTS approaches use effective molarity rather than intermolecular reactivity to direct organic synthesis, they enable modes of controlling reactivity (such as using otherwise incompatible reactions in a single solution ) and claevolving libraries of diverse synthetic molecules, rather than simply enriching libraries that already contain at the outset the “most fit” molecule, researchers must develop sophisticated library syntheses that generate remarkable complexity (vast numbers of different compounds) in a relatively modest number of DNA-compatible synthetic steps. True evolution takes place when the theoretical complexity of a population exceeds the number of different molecules that can be created in a single library translation step, and when diversification is required to access compounds in later generations that are more fit than any member of the starting pool.In order for either approach to fully realize its potential of truly 8 compounds). However, because so few copies of a DNA-linked synthetic molecule are required for in vitro selection (8 different structures would represent 10 kg of material, not including the mass of beads or plates associated with the library, while a chemically translated library containing 10,000 copies of 108 different species represents less than 1 µg of total material.To my knowledge, no synthetic library to date contains this degree of complexity (indeed, the total size of the Chemical Abstracts Service database of known chemical substances is presently less than 10election —comparedWhile significant remaining challenges face efforts to develop and apply chemical translation, the promise of marrying evolution and organic synthesis is an irresistible combination for some researchers. The work of Harbury and co-workers described in this issue represents the latest approach to the very ancient problem of translating replicable information into functional structures.


Splenic metastases occur in rare cases with a few case reports of patients in the literature. Generally, splenic metastases mean late dissemination of a disease. Solitary splenic metastases from solid tumors are extremely unusual.We report a case of a patient with ovarian mucinous cystadenocarcinoma who underwent splenectomy for isolated parenchymal metastasis.Ovarian epithelial tumors comprised most of isolated splenic metastases from gynecologic tumor. When isolated splenic recurrence is suspected on image studies and serum tumor markers, intraabdominal gross findings should be examined to exclude peritoneal carcinomatosis. If only spleen was under suspicion of recurrence of ovarian cancer, splenectomy may play a therapeutic role. Splenic metastases from solid tumors occur in late stage of a disease, so those can hardly be an indication for a surgery. Cancers of the ovaries, lung, breast, stomach, skin and colon are known to metastasize to the spleen . Even thWe present a case of mucinous cystadenocarcinoma that recurred in the splenic parenchyma.A 29-year-old woman was admitted our hospital on August 1999, had been diagnosed as mucinous tumor of borderline malignancy a year ago. She was followed up at a local clinic. On the follow-up study, the patient's CEA level was raised to 43.32 U/L and CT scan showed splenomegaly with cystic lesion. Her past medical history was not significant. She already underwent two surgeries. The first surgery, right salpingo-oophorectomy, was performed at the age of 22 after being diagnosed as dermoid cyst. She was healthy thereafter. Seven years later, left ovarian mass was found on the routine check. At the second surgery, left ovarian mass excision, mucinous tumor of borderline malignancy was diagnosed.During follow-up after the second surgery, she was referred to our hospital on the suspicion of carcinomatosis peritonei. On the preoperative evaluation Figure , splenicTo exclude peritoneal carcinomatosis, open laparotomy was perfomed. On opening the abdomen, no abnormal gross findings were found except the splenic lesion, which reported as probable metastatic adenocarcinoma on frozen sections. After splenectomy was carried out, peritoneal washings and multiple biopsies on the omentum, peritoneum, mesentery, and left ovary were performed to rule out possible microscopic peritoneal dissemination. Suspecting the transabdominal metastasis, 100 mg of cisplatin was infused into the peritoneal cavity at the end of the operation; however no intraperitoneal recurrence was confirmed after tissue diagnosis.Final pathologic examination Figure  showed mTwo years after the surgery, 3 × 3-sized mass on the left ovary, which assumed to be recurrence, was detected. She has been followed up at outpatient department receiving symptomatic treatment and chemotherapy.The frequency of splenic metastasis has been reported 2.3 to 7.1 per cent from autopsy series of cancer patients -11. SpleUntil now, in the literature, splenic capsular metastasis has been reported to occur in the case of far advanced stage of a disease or in the case of more than one organ was already involved in terminal stage and especially in the case of peritoneal metastasis. When one or more organ in the thoracic cavity and abdominal cavity was involved, the rate of splenic metastasis was 43% . But isoAmong cases of the isolated splenic metastasis, the ovarian cancers make up the most. After Minazawa et al  firstly CT scanning and measurement of serum tumor markers, especially CA 125, are helpful for detecting the recurrence and the infrequent splenic metastasis.We proposed that splenectomy be a proper therapeutic modality for an isolated splenic metastasis, especially parenchymal metastasis, from an ovarian cancer. When isolated splenic recurrence is suspected on the CT scanning and serum tumor markers, intraabdominal gross findings should be examined meticulously. If only spleen was under suspicion of recurrence, splenectomy would be a proper therapeutic procedure.The author(s) declare that they have no competing interests.YS carried out the literature search and prepared the manuscript. JC Kim, the corresponding author, was the main operator in charge of the case. CK was involved in the operation and the patient active management. Three authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: