Plasmodium berghei. Targeted disruption of the spect gene greatly reduced sporozoite infectivity to the liver. In vitro cell invasion assays revealed that these disruptants can infect hepatocytes normally but completely lack their cell passage ability. Their apparent liver infectivity was, however, restored by depletion of Kupffer cells, hepatic macrophages included in the sinusoidal cell layer. These results show that malarial sporozoites access hepatocytes through the liver sinusoidal cell layer by cell traversal motility mediated by SPECT and strongly suggest that Kupffer cells are main routes for this passage. Our findings may open the way for novel malaria transmission-blocking strategies that target molecules involved in sporozoite migration to the hepatocyte.Liver infection is an obligatory step in malarial transmission, but it remains unclear how the sporozoites gain access to the hepatocytes, which are separated from the circulatory system by the liver sinusoidal cell layer. We found that a novel microneme protein, named sporozoite microneme protein essential for cell traversal (SPECT), is produced by the liver-infective sporozoite of the rodent malaria parasite,  Plasmodium protein called SPECTTo infect hepatocytes, malarial sporozoites must traverse Kupffer cells of the liver. This active process is shown to be mediated by a newly identified  Malaria is one of the most devastating infectious diseases in the world, killing more than 1 million people per year. Malaria is transmitted by bites of infected mosquitoes that inject sporozoites under the skin. The first obligatory step for these parasites to establish infection in humans is migration to hepatocytes, where they proliferate and develop into the erythrocyte-invasive form . This liMalarial parasites develop into sporozoites in the mosquito midgut and then invade the salivary gland, where they wait to be transferred to the mammalian host . Once inThe malarial parasite has no locomotory organelles such as flagella or cilia. Motility of the host-invasive stages of the malarial parasite, including the sporozoite, is dependent on secretion of micronemes that are organelles occupying the cytoplasm of the parasite . MicroneSalivary gland sporozoites display three modes of motility in vitro dependent on secretion of micronemes . One modPlasmodium berghei. In this paper, we report a novel microneme protein, named SPECT , which is specifically produced by the liver-infective sporozoite and is essential for the sporozoite's cell-passage ability. By using spect-disrupted parasites, we show that cell-passage ability of the sporozoite plays a critical role in malarial transmission to the vertebrate host and is required for sporozoites to access hepatocytes by traversal of the liver sinusoidal cell layer. In addition, we provide a model of sporozoite liver infection, which suggests an answer to the question of how sporozoites reach the hepatocytes.Aiming at identification of molecules involved in sporozoite infection, we screened an expressed sequence tag (EST) database of the salivary gland sporozoite of a rodent malarial parasite, P. berghei salivary gland sporozoites. We assembled 3,825 ESTs, obtained 502 contigs, and screened them for genes encoding secretory proteins or membrane-associated proteins, which may participate in host–parasite interactions. This screening was started from the contigs containing a high number of ESTs, since the number of ESTs may correlate with the expression levels of the respective genes. In this process, we identified a contig composed of ten ESTs, encoding a putative secretory protein of 241 amino acids . Sequence analysis of the spect gene identified four introns (data not shown). A computer search of Plasmodium genome databases 1 and spect(−)2) were used in the following experiments.To investigate the function of SPECT protein, we generated bination A. The spanalysis B. To excspect-disrupted and wild-type populations (data not shown). Furthermore, disruption of the gene did not affect parasite development in the mosquito vector, as numbers of sporozoites residing in the midgut and in the salivary glands were similar in the spect-disrupted and wild-type populations . It is thought that the parasitemias reflect the liver infectivity of the respective parasite populations, since the growth rates of their intraerythrocytic stages are similar , showing that their gliding motility is not affected by SPECT gene disruption. Second, we examined the ability of the sporozoites to infect hepatocytes. This was assayed by formation of EEFs in a human hepatoma cell line, HepG2    and testarasites B. These Plasmodium sporozoite has the ability to traverse cultured cells rapidly . This suggests that the cell-traversal ability of the sporozoite is realized by cooperation of several microneme proteins. We suggest that these molecules could be targets for antimalarial strategies, since success in crossing this layer is critical for the malarial parasite to establish infection in humans. Elucidation of the molecular mechanisms of passage may lead to novel malaria transmission-blocking strategies that prevent sporozoites from gaining access to the hepatocyte.Our recent work has identified two other genes that are involved in the cell passage activity of the sporozoite. Like SPECT, the products of these genes have a secretory protein-like structure and are localized in the micronemes. Furthermore, sporozoites disrupted for these genes have similar phenotypic character to P. berghei ANKA strain were prepared by peritoneal injection of infected blood that was stored at −70°C. For the purification of sporozoites, infected mosquitoes were dissected 24–28 d after the infective blood meal. The salivary glands and midgut were separately collected in medium 199 on ice and then gently ground to release the sporozoites. Ookinetes and erythrocytic-stage parasites were prepared as described previously  infected with the eviously .P. berghei parasites (2 μg) was digested with ClaI, EcoRI, EcoT22I, HindIII, or XbaI, separated on 1.2% agarose gel and then transferred to a nylon membrane. DNA fragments were amplified by PCR using genomic DNA as template with the following primers: 5′-TGGGCAATTTTGCCTTTAAAAACG-3′ and 5′-TTTCATTGTGTTAAACGATAAGTG-3′. They were labeled with [32P]dCTP and used as probes.Genomic DNA of Recombinant SPECT without signal sequence was expressed as a glutathione S-transferase (GST)–fusion protein using the pGEX 6p-1 system . The recombinant protein was purified with a GST column and used for immunization of rabbits. Specific antibodies were affinity purified using a N-hydroxysuccinimide-activated column (Amersham Bioscience) coupled with recombinant SPECT protein. For CS antiserum production, the peptide DPPPPNANDPAPPNAN, corresponding to the repeat region, was conjugated to keyhole limpet hemocyanin as a carrier and used for the immunization of rabbits. Western blot analysis was performed as described previously .Immunofluorescence microscopy was performed as described previously . Purifiespect gene were amplified by PCR using genomic DNA as template with the primer pairs 5′-CGCGAGCTCGCAATATGGTATTAAATTTTGGGCTAGCCA-3′ and 5′-CGCGGATCCGGTATTTTCATTGTGTTAAACGATATGTGA-3′ and 5′-CCGCTCGAGGTCCTATTTATCATTTTAAAATGTGTTTTATC-3′ and 5′-CGGGGTACCAATCGTCATAAATAGGAGTTATGAAGT-3′. These fragments were cloned into either side of the selectable marker gene in pBluescript . The gene targeting experiment was performed as described previously (For construction of the targeting vector, two fragments of the eviously .n = 5). Before each inoculation, sporozoites were checked for their ability to glide in vitro to confirm that they contained over 60% motile sporozoites. Parasitemia was checked every 12 h by a Giemsa-stained blood smear.Sporozoites collected from mosquito salivary glands were suspended in medium 199 and then injected intravenously into 3-wk-old female Wistar rats (Japan SLC, Inc., Hamamatsu, Japan) (5) were intravenously inoculated into a 3-wk-old female Wistar rat. After 24 h, the liver was perfused with PBS followed by 4% paraformaldehyde. The liver was further fixed in 4% paraformaldehyde for 6 h and frozen in liquid nitrogen. Cryostat sections (20 μm) were prepared from the left lobe and fixed in acetone for 2 min on a glass slide. The EEFs were detected by immunofluorescence staining using rabbit anti-CS antiserum and FITC-conjugated secondary antibody. At least 12 sections were examined under an Olympus (Tokyo, Japan) BX60 fluorescence microscope (200×) and the number of EEFs per square millimeter was calculated.Sporozoites (5.0 × 105) were plated in 8-well chamber slides. Sporozoites (5.0 × 103 or 5.0 × 104) were suspended in 100 μl of complete medium and added to this culture. After 2 h, the media were replaced with 400 μl of fresh complete medium supplemented with 3 μg/ml glucose. The slides were incubated for 2 d with medium changed twice a day and were fixed in acetone for 2 min. The EEFs were detected by immunofluorescence staining as described above. The number of EEFs in one-fifth of the area of each well was counted under an Olympus BX60 fluorescence microscope (200×).The EEF formation assay was performed as described previously  with min5) or HeLa cells (5.0 × 104) were inoculated into 8-well chamber slides (Nunc Inc., Napierville, Illinois, United States). Sporozoites were added 2 d later to cells for 1 h in the presence of 1 mg/ml FITC-labeled dextran . The cells were incubated for an additional 3 h in complete culture medium and fixed with 4% paraformaldehyde in PBS. The number of FITC-positive cells was counted under a fluorescence microscope.The traversing activity of the sporozoite was examined using a standard cell-wounding and membrane repair assay . HepG2 c2MDP or an equal volume of PBS as control. After 48 h, sporozoites were injected into a tail vein and the parasitemia was checked by Giemsa-stained blood smears. Cl2MDP liposomes were prepared as described elsewhere (2MDP was a gift from Roche Diagnostics (Mannheim, Germany).For depletion of Kupffer cells, 3-wk-old female Wistar rats were injected intravenously with 120 μl of liposome-encapsulated Cllsewhere . Elimina


CHD7 on chromosome 8 was the underlying etiology in most of the affected patients.CHARGE syndrome is a complex of birth defects including coloboma, choanal atresia, ear malformations and deafness, cardiac defects, and growth delay. We have previously hypothesized that CHARGE syndrome could be caused by unidentified genomic microdeletion, but no such deletion was detected using short tandem repeat (STR) markers spaced an average of 5 cM apart. Recently, microdeletion at 8q12 locus was reported in two patients with CHARGE, although point mutation in We have extended our previous study by employing a much higher density of SNP markers (3258) with an average spacing of approximately 800 kb. These SNP markers are diallelic and, therefore, have much different properties for detection of deletions than STRs.A global error rate estimate was produced based on Mendelian inconsistency. One marker, rs431722 exceeded the expected frequency of inconsistencies, but no deletion could be demonstrated after retesting the 4 inconsistent pedigrees with local flanking markers or by FISH with the corresponding BAC clone. Expected deletion detection (EDD) was used to assess the coverage of specific intervals over the genome by deriving the probability of detecting a common loss of heterozygosity event over each genomic interval. This analysis estimated the fraction of unobserved deletions, taking into account the allele frequencies at the SNPs, the known marker spacing and sample size.The results of our genotyping indicate that more than 35% of the genome is included in regions with very low probability of a deletion of at least 2 Mb. Although no deletion was detected in the CHARGE study sample, we conclude that this method could be generally useful in other studies in which small deletions occur as part of the allelic spectrum of disease.CHARGE Association is characterized by ocular coloboma, cranial nerve abnormalities, common outflow tract heart defects, choanal atresia, cupped-shaped pinnae, Mondini dysplasia of the inner ear ,2 and grpatients  and may The diagnosis of CHARGE syndrome was established by examination by a participating clinical genetics specialist (CB, JWB, SRL). A medical history questionnaire was completed by the parents or by direct interview. For the core genotyping, 8 Caucasian and 1 Hispanic case/parent trios were selected based on the presence of 4 major criteria or three major and three minor criteria for the syndrome . There wGenotyping for this study was carried out on an Orchid BioSciences SNPstream UHT platform (Princeton, NJ) as previously described . For thiThis genotyping method utilized Orchid's single base primer extension chemistry (SBE) to identify which bases were present at the site of interrogation. Multiplexed reactions (12-plex) were performed in a single tube that incorporated labeled chain terminating nucleotides onto the ends of the SBE oligonucleotides. These reactions were then hybridized onto a microarray format, that facilitates the solid-phase sorting of the labeled extension-primers to a set of universal tagged primers arrayed on the surface of the plate. The universal tags were arranged on the surface of the microarray plate in a 384-well microwell layout. This microarray format created a generic design consisting of 384 4 × 4 arrays that contained 12 oligonucleotides that corresponded to 12 unique universal capture tags. The four additional oligonucleotides, plotted in each array, were used for positive and negative controls. Genotyping calls were determined by the presence or absence of incorporated dyes that appeared at each spot on the printed arrays.Two Assays-on-demand SNPs, rs422951 and ss1309424, flanking rs431722 were obtained from Applied Biosystems and genotyping was performed in 384 well-plates, using the TaqMan polymerase chain reaction-based method. The final volume reaction was 5 μl using 12 ng of genomic DNA, 2.5 μl of Taqman Master mix and 0.25 μl of 20X Assays-on-Demand SNP Genotyping Assay Mix. The plate was heated at 95° for 10 minutes, followed by forty cycles of denaturation at 92° for 15 seconds and annealing/extension at 60° for 1 minute. PCR plates were read on ABI PRISM 7900HT instrument with SDS v2.0 software. Individual genotypes that were ambiguous were excluded.in situ hybridization was performed as described elsewhere [Bacterial Artificial chromosomes (BACs) were selected from the public database  and obtalsewhere . Detectiet al. [The genotype error rate was estimated using the method of Gordon et al.  and as iet al. . ExpecteNOTCH4 gene on chromosome 6p21.32. Human DNA sequence from clone XXbac-300A18 (GenBank accession number AL662884) on chromosome 6p21 was used for FISH. This BAC clone encompasses the NOTCH4 gene, and was confirmed by PCR amplification of the clone sequence using NOTCH4 specific primers (data not shown). The analysis of the metaphase chromosomes after staining with DAPI showed two bright hybridization signals, indicating the presence of both alleles  and RP11-414L17 (GenBank accession number AC023102). Using these BAC clones, microdeletion of this region was excluded in all the affected patients in this study sample.SNP genotyping data were subjected to analysis for Hardy-Weinberg equilibrium. As an additional test of marker integrity, a transmission disequilibrium test was also performed for each of the SNP markers to examine for distortions in allele transmission in the trios. Unequal transmission of alleles from the heterozygote parents to the affected offspring was not determined by this analysis. Non-paternity was excluded in core pedigrees. Using the method of Gordon , we usedBecause none of the pedigrees were consanguineous, we used parental data to estimate the allele frequencies for each of the 3258 SNP markers . The EDDet al. have previously shown that SNP genotyping of child-parent trios provides valuable information about the presence of de novo microdeletion when sufficient families are studied [Despite various efforts to understand the molecular basis of CHARGE syndrome, with candidate genes sequencing ,15, comp studied . HoweverNOTCH4 gene on chromosome 6p21.32. The Notch gene family encodes highly conserved transmembrane receptors that are involved in intercellular signaling. The Notch signaling pathway plays an essential role in regulating embryonic vascular morphogenesis and remodeling [JAG1 is known to result in Alagille syndrome [NOTCH4 was further studied with FISH as well as flanking SNP markers for microdeletion. The results, however conclusively excluded a discernible microdeletion at this locus.Assuming the genotyping error rate of 0.5%, the probability of observing >2 inconsistencies per marker is very low. The SNP marker rs431722 showed Mendelian inconsistency in four of nine pedigrees, with apparent loss of a parental allele in each case i.e. a frequency much higher than expected for the genotyping error rate. Interestingly, this SNP is located within the modeling . Moreovesyndrome , nonsyndsyndrome  and possThis screen is expected to detect deletions of about 1–2 Mb depending on the overlap of the SNP markers with the deletion interval. Variable coverage for each chromosome was determined for approximately 2 Mb microdeletion in this study. Almost 50% of chromosomes 7, 19 and 20 were excluded for any microdeletion greater than 2 Mb. The least coverage was observed for chromosome 15 and 18, with exclusion of 20% of the chromosome for the presence of a similar genetic aberration. Overall, we can estimate that approximately 36% of the genome had >80% chance for detecting a common 2 Mb deletion in at least 2 patients with CHARGE Syndrome.There are several limitations to this approach in studying the genetics of CHARGE syndrome. Although the marker density is high, the reduced amount of information per marker means that only some of the trios give the possibility of a conclusive result. Denser marker sets would be predicted to fill most of the gaps, but the regions around the centromeres are likely to be difficult with any currently available technique.The strategy out lined in this paper would work equally well for conventional Mendelian traits in which the mutant alleles included at least some deletions. Using a much denser SNP map of 1 marker every 5–10 kb, as is anticipated for whole genome association analyses, it would be possible to detect most deletions given a sufficient representation of deletions within the spectrum of gene mutations.In this report we show that a SNP genotyping screen has excluded moderate length submicroscopic deletions in a subset of patients with CHARGE syndrome. Further analysis by microarray comparative genome hybridization methods or denser SNPs will allow a comprehensive assessment of the role, if any, of microdeletions in CHARGE syndrome.The author(s) declare that they have no competing interests.SRL carried out the data analysis, FISH and TaqMan assays and drafted the manuscript. AMS assisted in the data analysis. MP carried out the SNP genotyping, JWB conceived of the study, and participated in its design and coordination. He performed the statistical analysis and drafted the manuscript. SDF, CAB, and NLG participated in patient enrollment and LMM assisted in DNA extraction. WJC and JAT assisted in manuscript preparation. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Corynebacterium diphtheriae has been shown to be an iron-activated transcription regulator that controls not only the expression of diphtheria toxin but also of iron uptake genes. This study aims to identify putative binding sites and operons controlled by DtxR to understand the role of DtxR in patho-physiology of Corynebacterium diphtheriae.The diphtheria toxin repressor, DtxR, of C. diphtheriae genome. In addition, DtxR-regulated operons were also identified taking into account the predicted DtxR regulatory sites and genome annotation. Few of the predicted motifs were experimentally validated by electrophoretic mobility shift assay. The analysis identifies motifs upstream to the novel iron-regulated genes that code for Formamidopyrimidine-DNA glycosylase (FpG), an enzyme involved in DNA-repair and starvation inducible DNA-binding protein (Dps) which is involved in iron storage and oxidative stress defense. In addition, we have found the DtxR motifs upstream to the genes that code for sortase which catalyzes anchoring of host-interacting proteins to the cell wall of pathogenic bacteria and the proteins of secretory system which could be involved in translocation of various iron-regulated virulence factors including diphtheria toxin.Positional Shannon relative entropy method was used to build the DtxR-binding site recognition profile and the later was used to identify putative regulatory sites of DtxR within in silico approach to identify the putative binding sites and genes controlled by DtxR in Corynebacterium diphtheriae. Our analysis shows that DtxR could provide a molecular link between Fe+2-induced Fenton's reaction and protection of DNA from oxidative damage. DtxR-regulated Dps prevents lethal combination of Fe+2 and H2O2 and also protects DNA by nonspecific DNA-binding. In addition DtxR could play an important role in host interaction and virulence by regulating the levels of sortase, a potential vaccine candidate and proteins of secretory system.We have used an  Iron is an important inorganic component of a cell. Iron is required as co-factor for various essential enzymes and proteins some of which are involved in electron transport (Cytochromes), redox reactions (oxidoreductases) and regulation of gene expression (fumarate-nitrate reduction regulatory protein, iron-binding protein) . HoweverCorynebacterium diphtheriae, Escherichia coli, and Bordetella bronchiseptica [C. diphtheriae [tox, hmuO, irp1, irp2, irp3, irp4, irp5 and irp6 have been identified by DNA footprinting methods [tox gene is diphtheria toxin which catalyzes the NAD-dependent ADP ribosylation of eukaryotic aminoacyl-transferase-II, thereby causing inhibition of protein synthesis and subsequent death of the host. The hmuO gene, which encodes a haem oxygenase, oxidizes the haem to release free iron. The operons irp1 and irp6 encode the products with homology to ABC-type ferric-siderophore transport systems. The gene irp3 encodes a homologue of AraC-type transcriptional activators. The products of irp2, irp4 and irp5 do not show any homology to the other known proteins. In addition, C. diphtheriae with inactive DtxR has been shown to be sensitive to killing by exposure to high iron conditions or hydrogen peroxide than the wild type [Inorganic iron is also known to influence virulence in many pathogenic bacteria such as iseptica -5. The dhtheriae ,7. Eightild type .in silico method to identify additional DtxR-binding sites and target genes to understand the role of DtxR in virulence and patho-physiology of C. diphtheriae.This work uses an diphtheriae NCTC13129 genome . Table C. diphtheriae NCTC13129 genome. We could not find irp1 and irp2 motifs as the corresponding genes (irp1, irp2) are not present in the sequenced strain NCTC13129 [irp3, irp4 and irp6 genes in the strain NCTC13129 shows one base change from the binding sites reported in strain C7 [irp3 gene (TTAGGTGAGACGCACCCAT) although exists in strain NCTC13129, but not there in the predicted sites, because it is located within the coding region of irp3 ORF. The predicted ORF of irp3 in the sequenced strain NCTC13129 has different start position and is larger than what was previously reported in strain C7 [Experimentally characterized DtxR-binding motifs were collected from the literature Table . These btrain C7 .recA (DIP1450) and ywjA (DIP1735) are not under a direct DtxR regulation as we could not detect any motif upstream to these gene with scores above the cutoff value [In addition, we have identified binding sites in upstream sequences of eight genes recently reported to be regulated by DtxR . HoweverywjA DIP135 are noM. tuberculosis. DtxR and IdeR are iron-dependent regulators. A pair wise sequence comparison of the two proteins shows a high (58%) overall sequence identity (similarity 72%) which increases further to 92% identity and 100% similarity in DNA recognition domain. In addition, the structural comparison of two regulators also shows a very similar 3D organization, suggesting that the IdeR regulator would be able to recognize the DtxR motif [Since our approach to identify DtxR-regulated genes is purely computational in nature, we decided to test the validity of our predictions. A sample of predicted regulator binding motifs Table   downstream to the predicted DtxR-binding site Table . FunctioC. diphtheriae physiology are discussed here.Our analysis identified putative DtxR motifs upstream to various operons/genes which could be involved in siderophore biosynthesis, ABC-type transport systems, iron storage, oxidative stress defense and iron-sulfur cluster biosynthesis. In addition, we have also identified the motifs upstream of operons that could be involved in anchoring of host-interacting proteins to the cell wall and secretion of various virulence factors. Important functions of some of these DtxR-regulated genes and their role in C. diphtheriae [Predicted member of the DtxR regulon, the gene DIP0586, codes for the IucA/IucC family of enzymes that catalyze discrete step in the biosynthesis of the aerobactin . In addihtheriae .Escherichia coli is induced in response to oxidative or nutritional stress and protects DNA from oxidative stress damage by nonspecific binding [2+ + H2O2 + 2H2O → 2Fe+3OOH(core) + 4H+) which in turn prevents hydroxyl radical formation by Fenton's reaction (Fe2+ + H2O2 → Fe+3 + HO- + HO.) and thereby prevents subsequent DNA damage [xerD gene in E. coli belongs to the oxidative stress regulon [We predict that DtxR could regulate divergently transcribed genes DIP2303 and DIP2304 whose products are similar to starvation inducible DNA-binding protein (Dps) and Formamidopyrimidine-DNA glycosylase (Fpg), respectively. Dps in  binding . Dps alsA damage . The enzA damage . The gen regulon .suf operon of E. coli, could be regulated by DtxR. The suf operon in bacteria encodes the genes for Fe-S cluster assembly machinery [C. diphtheriae also show DtxR motif in their upstream sequences.We predict that the operon DIP1288-DIP1296, which is similar to the achinery . In addiWe predict that DtxR could regulate the recently acquired pathogenic island DIP2271-DIP2272, encoding the sortase srtA and hypothetical protein, respectively . SortasesecA) and DIP0540 (secY) that code for the protein translocation system. The secY-containing operon, which is similar to the streptomycine operon spc from B. subtilis and other bacteria, involves the genes required for protein translation and translocation [Corynebacterium diphtheriae by unmasking receptors on mammalian cells [DtxR could regulate two operons that contain genes DIP0699 , fb,i is observed frequency of each base at position i and qb is the frequency of base b in the genome sequence. The contribution of each base to the positional Shannon's relative entropy is calculated by multiplying positional frequency of each base with positional relative entropy. The binding site profile thus generated was used to scan upstream sequences of all the genes of the Corynebacterium diphtheriae genome. The score of each site is calculated as the sum of the respective positional Shannon relative entropy of each of the four possible bases. A maximally scoring site is selected from the upstream sequence of each gene. The lowest score among the input binding sites is considered as cut-off score. The sites scoring higher than the cut-off value are reported as potential binding sites conforming to the consensus sequence.where Co-directionally transcribed genes, downstream to the predicted binding site were selected as potential co-regulated genes (operons) according to one of the following criteria (a) Co-directionally transcribed orthologous gene pairs, conserved in at least 4 genomes; (b) genes belong to the same cluster of orthologous gene function category and the intergenic distance is less than 200 base pairs; (c) the first three letters in gene names are identical (gene names for putative genes were assigned from COG database); (d) intergenic distance is less than 90 base pairs .The function of predicted genes was inferred using the RPS-BLAST search against conserved domain database . These gtuberculosis was expressed from a recombinant pRSET vector containing the IdeR gene fused to a six His affinity tag (P. Chakhiyar unpublished). The expressed protein was first purified using Ni-NTA Metal Chelate Affinity chromatography; later it was desalted and concentrated using Centricon Ultra filtration device. The concentration of the recombinant protein was estimated using Bradford method.The iron-dependent regulator IdeR from M. 32P]-ATP and were incubated with the recombinant purified IdeR protein in a binding reaction mixture. The binding reaction mixture  contain the DNA-binding buffer , 10 μg of poly(dI-dC) per ml (for nonspecific binding) and 200 μM MnCl2. The reaction mixture was incubated at room temperature for 30 min. Approximately 2 μl of the tracking dye (50% sucrose, 0.6% bromophenol blue) was added to the reaction mixture at the end of incubation and was loaded onto 7% polyacrylamide gel containing 150 μM MnCl2 in 1 × Tris-borate-EDTA buffer. The gel was electrophoresed at 200 V for 2 hours. Subsequently the gel was dried and exposed to Fuji Storage Phosphor Image Plates for 16 hours. The image plates were subsequently scanned in Fuji Storage Phosphor Imaging workstation.Double-stranded oligonucleotides containing the predicted binding motif (19 bp long) were end labeled with T4 polynucleotide kinase and [γDtxR – Diphtheria toxin repressor; IdeR – Iron-dependent regulator; Dps – DNA-binding protein from starved cells; RPS-BLAST – Reversed Position Specific – Basic Local Alignment Search Tool; EMSA – Electrophoretic Mobility Shift AssaySY: carried out the computation, data analysis, and manuscript preparation. SR: Carried out the EMSA and drafted the manuscript. PC: provided the cloned IdeR construct, drafted the manuscript. SH: Manuscript preparation and coordination. AR: Design of the study and coordination. All authors read and approved the final manuscript.


Mitral annular calcification (MAC) and aortic annular calcification (AVC) may represent a manifestation of generalized atherosclerosis in the elederly. Alterations in vascular structure, as indexed by the intima media thickness (IMT), are also recognized as independent predictors of adverse cardiovascular outcomes.To examine the relationship between the degree of calcification at mitral and/or aortic valve annulus and large artery structure (thickness).We evaluated 102 consecutive patients who underwent transthoracic echocardiography and carotid artery echoDoppler for various indications; variables measured were: systemic blood pressure (BP), pulse pressure (PP=SBP-DBP), body mass index (BMI), fasting glucose, total, HDL, LDL chlolesterol, triglycerides, cIMT. The patients were divided according to a grading of valvular/annular lesions independent scores based on acoustic densitometry: 1 = annular/valvular sclerosis/calcification absence; 2 = annular/valvular sclerosis; 3 = annular calcification; 4 = annular-valvular calcification; 5 = valvular calcification with no recognition of the leaflets.Patient score was the highest observed for either valvular/annulus. Mean cIMT increased linearly with increasing valvular calcification score, ranging from 3.9 ± 0.48 mm in controls to 12.9 ± 1.8 mm in those subjects scored 5 (p < 0.0001). In the first to fourth quartile of cIMT values the respective maximal percentual of score were: score 1: 76.1%, score 2: 70.1%, score 4: 54.3% and score 5: 69.5% (p > 0.0001).MAC and AVC score can identify subgroups of patients with different cIMT values which indicate different incidence and prevalence of systemic artery diseases. This data may confirm MAC-AVC as a useful important diagnostic parameter of systemic atherosclerotic disease. Mitral annular calcification (MAC) and aortic annular calcification (AVC) are observed in populations that develop significant atherosclerosis and more frequently in the elderly . PreviouCurrently, there are no accurate and standardized methods to quantify the degree of MAC an AVC; many studies have been performed with the aim to detect categorical scoring systems derived from echocardiographic annular-valvular morphology -12.Alterations in vascular structure such as increased arterial wall thickness, as indexed by the intima media thickness (IMT), are also increasingly recognized as significant independent predictors of adverse cardiovascular outcomes -17.We therefore undertook a cross-sectional study to examine the relationship between the degree of calcification at mitral and/or aortic valve annulus and large artery structure (thickness).We evaluated 128 consecutive patients who underwent transthoracic echocardiography and carotid artery echoDoppler for various indications. Patients with significant common carotid artery stenosis, rheumatic valvular disease, cardiomyopathy, prosthetic valves, ischemic heart disease and carotid artery surgery were excluded. Thus, 102 subjects were enrolled for the present study. All participating patients gave informed consent; the study protocol was approved by the institutional ethics committee.Blood pressure determinations were performed with subjects in the supine position, and following a ten minute quiet resting period. Blood pressure was measured in the nondominant arm with a mercury sphygmomanometer using an appropriately sized cuff. The blood pressure values used in this study are the average of the second and third measurements. Values for systolic blood pressure (SBP) and diastolic blood pressure (DBP) were defined by Korotkoff phase I and V, respectively. Hypertension was defined as either systolic or diastolic elevation of blood pressure (>140/90 mmHg) or ongoing antihypertensive pharmacological therapy. Pulse pressure was computed as PP = (SBP-DBP); mean BP was computed as MBP = DBP +(PP/3).2. Smoking status was ascertained by a questionnaire that classified each subject as a non smoker, former, or current smoker. For the purpose of the present study, current smoker status was used.Height and weight were determined for all participants. Body mass index (BMI) was determined as body weight (kg) / height (m)2 [Blood samples were drawn from the antecubital vein between 7 and 8 AM after an overnight fast. Subjects were not allowed to smoke, engage in significant physical activity or take medications prior to the collection of the sample. The concentrations of plasma triglycerides and total cholesterol were determined by an enzymatic method ,19. HDL-2 . Serum L2 . Fasting® SONOS 5500 with a S3 probe. All patients had an adequate 2D echocardiogram. Evaluation of mitroaortic sclerosis/calcification was made, off line, with the acoustic quantification-densitometry package  wich restitute values based on echogray scale (0 db = black, 64 db = white, fig. All the echocardiographic examinations were performed using the PHILIPSHigh-resolution B-mode carotid ultrasonography was performed with a linear-array 5- to 10-MHz transducer. The subject lay in the supine position in a dark, quiet room. The right common carotid artery (CCA) was examined with the head tilted slightly upward in the midline position. The transducer was manipulated so that the near and far walls of the CCA were parallel to the transducer footprint and the lumen diameter was maximized in the longitudinal plane. A region 1.5 cm proximal to the carotid bifurcation was identified, and the carotid intima media thickness (cIMT) of the far wall was evaluated as the distance between the luminal-intimal interface and the medial-adventitial interface. cIMT was measured on the frozen frame of a suitable longitudinal image with the image magnified to achieve a higher resolution of detail. The cIMT measurement was obtained from 5 contiguous sites at 1-mm intervals, and the average of the 5 measurements was used for analyses. All the measurements were performed by a single sonographer.All analyses were performed using the SPSS 8.0 package. Data are presented as mean ± SD unless otherwise specified.Comparison of groups based on different calcification score was made by ANOVA, followed by Bonferroni's test for all two-way comparisons, or by chi-square analysis – as appropriate. Geometric mean values of vascular end points, adjusting for traditional cardiovascular risk factors, were calculated across categorized features by means of General Linear Model.Of the 102 patients evaluated, 24 were scored 1, 19 were score 2, 20 were score 3, 18 were score 4 and 21 were score 5. There were no statistically significant intergroup differences in age, sex distribution, total cholesterol, HDL and LDL cholesterol, smoking habits, diabetes mellitus, and positive family history of coronary artery disease (tab.1). Similarly, clinical indications for ultrasound examinations were not significantly different in the 5 score groups  had higher values of cIMT.Previous pathologic studies demonstrated that foam cells which represent early atherosclerotic lesions can be found in subjects already during adolescence on the endothelium of the epicardial coronary arteries, the ventricular surface of the posterior mitral leaflet and the aortic aspects of each aortic leaflet ,22. ExpeMany recent studies showed a clear association between mitral annulus calcification and the presence of aortic atheromas, atheroma thickness and carotid artery disease ,5 these In our study we investigated the possible association between AVC, MAC and cIMT in elderly patients; we found a clear and strong significant linear correlation between cIMT and AVC/MAC values; there were no significant influence of the considered variables on this correlation. In addition, we found a strong association of incremental values of score with first to fourth quartile of cIMT Another exclusive characteristic of our study is the creation of a semi quantitative way of AVC-MAC evaluation; in fact, scoring the evolution of AVC and MAC, we were able to correlate this parameter with the continuous variable cIMT.Furthermore, a recent study  showed aOur data indicate that AVC-MAC could be considered a form of polisegmental atherosclerosis; the semi quantitative evaluation of AVC-MAC is strongly associated cIMT; this semi quantitative way of grading mitral-aortic valvular-annular sclerosis and calcification was also able to identify the quartile of cIMT. These results indicate the score evaluations as an important echocardiographic tool for atherosclerotic disease evaluation.This study evaluated only elderly patients, therefore the eventual correlation of MAC-AVC and cIMT can be only supposed in younger patients; future studies are needed to demonstrate this hypothesis. We viewed cIMT as a marker for sub clinical atherosclerosis. Although the significance of carotid thickening, particularly in the distal common carotid artery, continues to be debated, its association with prevalent and future cardiovascular events has been supported by a number of studies -17,24,25Additionally, whether our findings are sufficient to indicate a widespread use of carotid ultrasound in those with presence of MAC-AVC requires further studies.MAC-AVC presence and their scoring can be detected by transthoracic echocardiography, a simple noninvasive imaging method. Using MAC and AVC values we can identify subgroups of patients with different cIMT values, a well-established precursor of systemic atherosclerosis, which indicate different incidence and prevalence of carotid, coronary and aortic artery diseases. Therefore, mitral or aortic valve calcification should not be regarded as a natural correlate of aging, rather as markers of generalized atherosclerosis.MAC: Mitral annular calcificationAVC aortic annular calcificationIMT intima media thicknesscIM carotid intima media thicknessCCA right common carotid artery


Previous studies demonstrated that the organ selector gene pha-4/FoxA is critical to establish the onset of transcription of Caenorhabditis elegans foregut (pharynx) genes. Here, we discover additional cis-regulatory elements that function in combination with PHA-4. We use a computational approach to identify candidate cis-regulatory sites for genes activated either early or late during pharyngeal development. Analysis of natural or synthetic promoters reveals that six of these sites function in vivo. The newly discovered temporal elements, together with predicted PHA-4 sites, account for the onset of expression of roughly half of the pharyngeal genes examined. Moreover, combinations of temporal elements and PHA-4 sites can be used in genome-wide searches to predict pharyngeal genes, with more than 85% accuracy for their onset of expression. These findings suggest a regulatory code for temporal gene expression during foregut development and provide a means to predict gene expression patterns based solely on genomic sequence.We have investigated the  C. elegans foregut - a model for organogenesisComputational analysis combined with validation by reporter gene studies is uncovering the code for temporal gene regulation in the  Caenorhabditis elegans midgut  A family of transcription factors illustrates the second strategy of developmental control. These proteins are critical to form the digestive tract in  defects . For exapidermis . This dr enzymes . FoxA me enzymes .C. elegans PHA-4 recognizes sequences that conform to the consensus TRTTKRY (where R = A/G, K = T/G, and Y = T/C)  . SequencY = T/C) . MoreoveY = T/C) . These delopment . These ocis-regulatory network for temporal gene expression within the pharynx. We identify sites that function in combination with PHA-4 elements to distinguish early from late expression. These elements can be used to build synthetic promoters with the expected expression profiles and to identify previously undiscovered pharyngeal genes within the genome.In this study, we have combined bioinformatics and experimental approaches to investigate the Our goal was to discover new regulators of pharyngeal transcription that would function in combination with PHA-4 A. To achC. elegans genes  and green fluorescent protein (GFP) reporters enabled us to identify 37 early-onset and 34 late-onset pharyngeal genes  at the expense of Chromosomes I and IV . This construct expressed robustly in embryos and weakly in larvae and adults. To examine the Late-1 element for repressive activity, we inserted three copies of the Late-1 motif upstream of the PHA-4 sites. Remarkably, the presence of the Late-1 elements resulted in a marked decrease in embryonic GFP expression but had no effect on larval expression (pha-4P::GFP reporter were late-stage embryos in which the pharynx was nearly or completely developed. Conversely, the Late-1 element was not active when tested for enhancer activity with the Δpes-10 promoter (data not shown). Thus, Late-1 functioned as a repressor of early gut expression. Late-1 may also function in cells outside of the digestive tract, but this aspect of regulation was not tested.Tests of the Late-1 element revealed that this motif functions as a repressor rather than an enhancer . We demopression . EmbryosC. elegans and C. briggsae to enrich for functionally relevant versions of motifs, rather than those that arose by chance alone. For Early-1 and Early-2, we tested the activity of these elements in the promoter of the early-onset pharyngeal gene K07C11.4. For Late-1, we tested the activity of this element in the promoter of the late-onset pharyngeal gene R07B1.9.To characterize the temporal elements further, we examined their activity in endogenous pharyngeal promoters. We searched for examples of these motifs in predicted promoters that were conserved between C. elegans and C. briggsae revealed stretches of conserved sequences. These regions contain two predicted PHA-4 binding sites (at −151 to −157 and −141 to −147, relative to the ATG), an Early-1 motif (−114 to −123), and an Early-2 motif (−217 to −225). The distal predicted PHA-4 site had a relatively high affinity for PHA-4 in vitro, consistent with this gene being expressed early in pharyngeal development  within endogenous promoters. Second, we constructed synthetic promoters to examine the interplay between the temporal elements and PHA-4 binding sites. Third, we combined our observations from the synthetic and endogenous promoters to search the genome for genes containing similar configurations of the five C. briggsae orthologs. Interestingly, particular combinations of elements appear to be associated with Ph-E or Ph-L genes . In contrast, three copies of a low-affinity PHA-4 site were sufficient to activate pharyngeal expression later in development. Early expression was observed significantly less frequently with the low-affinity constructs than with the high-affinity constructs (n = 28/71). Notably, the strength of expression of both constructs was comparable in late embryos. We conclude that promoter activation depends on the affinity of PHA-4 for its binding sites and cannot be compensated for by multimers. This finding agrees with data that show FoxA factors bind DNA as monomers, not cooperatively  with a single temporal element (either Early-1 or Late-2) to determine how these elements behave in combination. The synthetic promoter constructs differed from our initial Δn = estimated number of transgenic embryos scored = 17/23; see n = 4/58). Because both artificial promoter constructs activated expression comparably in late embryos, we conclude that the differences in early expression reflect a genuine difference in the onset of expression between constructs rather than a difference in the strength or penetrance of expression. These data support a model in which the Early-1 element is able to mediate early pharyngeal gene expression but where the onset of gene expression is ultimately limited by the relative affinity of the PHA-4 binding site. We similarly tested Early-2 together with a high-affinity PHA-4 site, but did not observe any expression above background, suggesting that this combination or configuration of sites was not sufficient to activate pharyngeal expression.The onset of expression for artificial promoters containing an Early-1 site depended on the relative affinity of the accompanying PHA-4 site , as predn = 1/73 early embryos for constructs with a high-affinity PHA-4 site, n = 2/37 early embryos for constructs with a low-affinity site). Because the Late-2 element behaved as an enhancer, we hypothesize that the factor or factors that act through Late-2 are not available until late in development and that their absence in early development results in delayed expression of Late-2 dependent genes.Artificial promoters containing the Late-2 element were not expressed until mid-embryonic to late embryonic development, regardless of the relative affinity of the PHA-4 site . Furthermore, of the 14 pharyngeal genes identified by the genomic search, 86% (12/14) were expressed early in pharyngeal development, as predicted. The 14 pharyngeal genes included ten genes not identified in our microarray experiments, reflecting the power of bioinformatics in predicting gene expression.The combination of Early-1, Early-2, and PHA-4 sites provided a powerful approach to predict early pharyngeal genes. Of 40 genes with conserved copies of all three elements, 20 had available expression data, and 70% of these were expressed in the pharynx , suggesting that, like Early-1, E1var in combination with other sites is predictive for pharyngeal expression. Of these pharyngeal genes, 70%  were expressed early, as predicted. Combining the results of the Early-1 and E1var searches, we identified a total of 35 pharyngeal genes, 73% of which (24/33) were expressed early.Given the possibility that E1var is a functional temporal element, we performed genomic searches for genes that contain conserved E1var plus Early-2 plus PHA-4 elements together within 500 bp of their predicted start codon. This search identified 120 genes with some overlap with the Early-1 search output. Of these 120 genes, 53 had expression data , and 47%p = 0.02). Strikingly, the Late elements accurately predicted the timing of expression in the pharynx, as all ten pharyngeal genes were expressed late in pharyngeal development. Considering the Early and Late genomic searches together, we identified 45 pharyngeal genes, 36 of which were not present in our microarray positives. Furthermore, the onset of expression of these 45 pharyngeal genes was predicted with 88% overall accuracy. There are three different reasons for our microarray experiments not identifying the 36 new pharyngeal genes: (1) some genes are expressed in both pharyngeal and non-pharyngeal cells and would not be significantly enriched in the par-1 versus skn-1 samples, (2) some genes are expressed post-embryonically and would not have been present in the embryonic samples used for the microarray experiments, and (3) some genes scored just below the 2-fold enrichment threshold for inclusion in our positive set.For late expression, 61 genes had conserved Late-1, Late-2, and PHA-4 sites, and these were also enriched for pharyngeal genes. Thirty of these genes had expression data, of which 33% (ten genes) were pharyngeally expressed , showingcis-regulatory motifs discovered here, combined with the PHA-4 binding site, establish a regulatory network that can account for the timing of activation of at least half of C. elegans pharyngeal genes.The advent of microarray technology and bioinformatics has provided a powerful tool to dissect the transcriptional regulatory circuits that guide complex developmental processes. We have analyzed pharynx organogenesis and defined novel regulatory elements that contribute to temporal gene expression. Our screens differ from previous examples of bioinformatic analyses in that the regulatory motifs were identified in unbiased searches of promoter sequences and subsequently tested for biological activity. We used three assays to demonstrate a function in vivo. First, we tested whether the regulatory elements were necessary for expression of native pharyngeal genes. Second, we determined whether they were sufficient for pharyngeal expression from synthetic promoters. And third, we did genome-wide searches based on these elements, a process that identified 19 new pharyngeal genes, of which 88% displayed the expected onset of expression. The myo-2 and R07B1.9).We previously proposed a model in which the relative affinity of PHA-4 for its binding sites controls the onset of pharyngeal gene expression. Because PHA-4 protein levels increase during development, we proposed that initially PHA-4 levels are low and only high-affinity sites are sufficiently occupied by PHA-4 to result in gene activation. As PHA-4 levels increase over time, lower-affinity sites also become occupied, leading to the expression of those genes. In addition, binding site affinity likely affects PHA-4 occupancy even at stable PHA-4 concentrations. The degree of PHA-4 occupancy would likely alter the probability of productive transcription of a target gene. However, the affinity model could not explain all temporal expression because some late-expressed pharyngeal genes had high-affinity PHA-4 binding sites (e.g., myo-2).In this study, we have identified additional regulatory elements that establish the onset of pharyngeal gene expression in combination with PHA-4. We suggest that the affinity of PHA-4 for its binding site determines the earliest possible time of pharyngeal gene expression but that other factors must be present for a promoter to be active. The Early elements likely represent binding sites for transcription factors that are available throughout embryonic pharyngeal development. In contrast, the Late-2 element is probably recognized by a transcription factor that is not available until midway through pharynx development, thereby delaying expression of these pharyngeal genes regardless of the quality of the PHA-4 binding sites. We note that our searches were restricted to genes with similar onset of expression, in order to identify temporal regulatory elements, and there may be yet more undiscovered elements that control temporal expression. For example, an element that confers cell-type-specific expression could also control onset of expression according to the availability of the relevant binding factor. Consistent with the idea of additional motifs, the synthetic promoters with two enhancer elements were functional but weak activators of expression compared to native promoters, which likely contain more regulatory motifs . Another possibility is that regulatory elements may be found within introns or the 3′ UTR (C. elegans and C. briggsae (data not shown). Thus, some of the genes whose onset of expression cannot be accounted for in our analyses likely have larger and more complex regulatory regions than the 500-bp window we used. Nonetheless, this limited sequence window allowed us to account for the onset of expression of roughly half of the Ph-E and Ph-L genes.The elements characterized here can account for the temporal expression patterns of many but not all known pharyngeal genes. One possible reason for this is that we do not yet have sufficient information to identify all functionally relevant occurrences of these elements. For example, some occurrences of the temporal elements may not be biologically meaningful, thereby generating false positives, while others may be hard to identify because of sequence heterogeneity or placement within e 3′ UTR . Consistncr-1; An interesting feature of the Early-1 element is that it is necessary for the expression of K07C11.4 in the proximal somatic gonad, as well as in the pharynx. Some, though not all, genes containing conserved Early-1 elements are expressed in the proximal somatic gonad , and LAG-1) have been discovered in C. elegans and Drosophila  IV;skn-1(zu67)/DnT1 IV;V;skn-1/DnT1 worms are uncoordinated, while skn-1 homozygotes are non-uncoordinated, allowing us to enrich for skn-1 homozygotes using a plate crawling assay. To synchronize, we performed a first round of hypochlorite embryo isolation and then allowed embryos to hatch overnight in liquid culture lacking food to obtain L1 larvae. We then added food to the cultures and grew the animals for 2–3 d to age them. For par-1, animals were aged to young adults, and embryos were collected from these by hypochlorite treatment. For the skn-1/DnT1 strain, animals were aged to L4 and then transferred to large plates for the crawling assay and later collected as young adults for embryo isolation. Collected embryos from either strain were aged another 3–6 h in small liquid cultures to ensure that par-1 and skn-1 embryos were at approximately the same stage of development, as determined by examining a sample of embryos under the light microscope. To extract total RNA from isolated C. elegans embryos, embryos pellets were frozen in microfuge tubes, crushed with a plastic pestle, and resuspended in RNA extraction buffer (1% lauroyl sarcosine, 0.1 M Tris base, 0.1 M NaCl, and 20 mM EDTA), followed by several rounds of phenol:chloroform extraction and ethanol precipitation  and three experiments using microarrays containing 94% of the genome (“full arrays,” experiments PS3, PS4, and PS5). Our previous microarray experiments (PS1–PS3) detected 242 positives, corresponding to 227 genes (We performed two microarray experiments using microarrays containing 62% of the 27 genes .2(par-1/skn-1) ≥ 1.00 in PS1–PS5 and were expressed above background in three of five experiments. In this selection, we observed considerable background of non-pharyngeal genes, primarily genes with maternally contributed transcripts or expression in pre-gastrulation embryos. We hypothesize that this background is the result of our par-1 embryos being harvested at an earlier stage than our skn-1 embryos in experiments PS4 and PS5. Because PS1–PS3 do not appear to exhibit this difference in staging, we applied an additional selection criterion to our data, requiring that all positives have a log2(par-1/skn-1) ≥ 0.58 on PS1, PS2, or PS3. This threshold reduces the inclusion of non-pharyngeal genes identified by PS4 and PS5 and identifies a total of 241 microarray positives. We further subtracted probable maternal genes from this set of positives using the C. elegans expression map of To extend the identification of candidate pharyngeal genes, we included data from PS4 and PS5. In this case, we selected genes that had an average logC. elegans and C. briggsae upstream and downstream sequences were extracted from the genome using the Ensembl EnsMart tool , which employs a variation of the expectation maximization algorithm  recurrently found variations of the motifs presented here. Other parameters of Improbizer were used at their default settings. For background sequences, we used three different sets of sequences  and gap parameter (g) for all motifs were zero and 35, respectively. The motif threshold (m) used for Early-1, Early-2, E1var, and Late-1 was six, for Late-2 was seven, and for PHA-4 was five. The random gene set referred to in C. elegans genes. Duplicates or splice isoforms of a gene were collapsed to a single selection, resulting in a total of 194 genes.Cluster-Buster finds the best possible occurrence of a motif (or motifs) in a given sequence and therefore requires the establishment of a threshold score to determine which occurrences are likely to be meaningful . We estaC. elegans and C. briggsae. We applied more stringent thresholds to the elements for these searches, to minimize the identification of non-pharyngeal genes. We optimized the Early element thresholds by searching the genome to identify known Ph-E and Ph-L genes and selecting the search parameters that maximized the ratio of Ph-E/Ph-L genes identified. The Cluster-Buster (C = 3.5, m = 6, and g = 35. The PHA-4 parameters were C = 1.9, m = 6, and g = 35. For E1var plus Early-2 combined the parameters were C = 1, m = 5.5, and g = 35. The PHA-4 parameters were C = 2, m = 6, and g = 35. Using these thresholds, our genomic searches identified four known Ph-E genes but no Ph-L genes. The same approach was used to optimize parameters for genome searches with the Late elements, maximizing the ratio of Ph-L/Ph-E genes identified. The parameters for Late-1 plus Late-2 combined were C = 2, m = 6, and g = 35. The PHA-4 parameters were C = 2.5, m = 6, and g = 35.For the genome searches, we searched for genes that contained a set of elements (e.g., Early-1 plus Early-2 plus PHA-4) within the first 500 bp of upstream sequence in both r-Buster  parametepes-10 promoter sequence present in pAP.10.To construct transcriptional reporters, we amplified promoter sequences from genomic N2 DNA using gene-specific primers that contained restriction endonuclease sites to facilitate cloning. PCR products were cloned into the vector pAP.10, which carries a GFP::HIS2B translational fusion, resulting in a nuclear-localized GFP . This clpes-10 promoter fragment. Clones were verified by restriction digests and sequencing. For the triplicate enhancer sequences, we used the following insert sequences : Early-1, Enhancer constructs and synthetic promoters were built using synthetic oligonucleotides that were cloned into pAP.10, upstream of the ΔFor the synthetic promoters, individual Early-1 or Late-2 sites were cloned in to the SphI and SalI sites of pAP.10, and individual PHA-4 sites were cloned in to the NheI and NsiI sites of pAP.10. Fragments from these single-site constructs were ligated to generate the constructs containing one temporal site and one PHA-4 site, with the sites separated by 95 bp of pAP.10 sequence. The sequences of the synthetic regions of these constructs were as follows : Early-1 plus PHA-4 (high), GCATGCTCGCe-pax-1 promoter; J. Stevenson and S. E. M., unpublished data). PWMs from Improbizer are listed in Sequences chosen were the best match to the position weight matrix (PWM) generated by Improbizer, except Late-1 , P-1 (which is based on two overlapping PWMs), and the PHA-4 sites  were placed in front of the Δpes-10 promoter , which confers a Roller phenotype (The transgenic marker that we used, henotype , does noDataset S1(14 KB PDF).Click here for additional data file.Table S1(57 KB PDF).Click here for additional data file.Table S2(14 KB PDF).Click here for additional data file.Table S3(22 KB PDF).Click here for additional data file.Table S4(18 KB PDF).Click here for additional data file.Table S5(30 KB PDF).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink) accession numbers for the genes and gene products discussed in this paper are act-1 (LocusID 179535), C49G7.4 (LocusID 178809), ceh-22/Nkx 2–5 (LocusID 179485), Ce-pax-1 promoter (LocusID 187105), D-pax2 (LocusID 43825), end-1 (LocusID 179893), F30H5.3 (LocusID 175207), F47D12.7 (LocusID 175891), K07C11.4 (LocusID 179198), LAG-1 (LocusID 177373), M05B5.2 (LocusID 187451), myo-2 (LocusID 181404), ncr-1 (LocusID 180719), par-1 (LocusID 179912), pha-4 (LocusID 180357), R07B1.9 (LocusID 181201), rol-6(su1006) (LocusID 174397), skn-1 (LocusID 177343), T05E11.3 (LocusID 178014), vestigial (LocusID 36421), and Drosophila Su(H) (LocusID 34881).The LocusLink (


Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories.Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human – Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species.Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research. Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. Expressed sequence tags (EST) are particularly useful genomic resources because they enable multiple lines of research and can be generated for any organism: ESTs allow the identification of molecular probes for developmental studies, provide clones for DNA microchip construction, reveal candidate genes for mutant phenotypes, and facilitate studies of genome structure and evolution. Furthermore, ESTs provide raw material from which strain-specific polymorphisms can be identified for use in population and quantitative genetic analyses. The utility of such resources can be tailored to target novel characteristics of organisms when ESTs are isolated from cell types and tissues that are actively being used by a particular research community, so as to bias the collection of sequences towards genes of special interest. Finally, EST resources produced for model organisms can greatly facilitate comparative and evolutionary studies when their uses are extended to other, closely related taxa.th century. At their pinnacle, salamanders were the primary model for early vertebrate development. Embryological studies in particular revealed many basic mechanisms of development, including organizer and inducer regions of developing embryos [Salamanders (urodele amphibians) are traditional model organisms whose popularity was unsurpassed early in the 20 embryos . Salaman embryos . In addi embryos -15. All Ambystoma mexicanum and the eastern tiger salamander, A. tigrinum tigrinum. These two species are members of the Tiger Salamander Complex [Ambystoma mexicanum has a long research history of over 100 years and is now principally supplied to the research community by the Axolotl Colony [A. t. tigrinum is obtained from natural populations in the eastern United States. Although closely related with equally large genomes (32 × 109 bp)[A. mexicanum is a paedomorphic species that retains many larval features and lives in water throughout it's life cycle while A. t. tigrinum undergoes a metamorphosis that is typical of many amphibians. Like many other traditional model organisms of the last century, interest in these two species declined during the rise of genetic models like the fly, zebrafish, and mouse [Here we describe results from an EST project for two ambystomatid salamanders: the Mexican axolotl,  Complex , a group Complex . Ambystol Colony , while A× 109 bp), these tnd mouse . HoweverA. mexicanum and several non-regenerating larval tissues in A. t. tigrinum.Eleven cDNA libraries were constructed using a variety of tissues Table . Pilot sA. mexicanum and A. t. tigrinum , we havA. mexicanum and A. t. tigrinum EST datasets at a BLASTX threshold of E = 10-7. The majority of contigs were supported at more stringent E-value thresholds  yielded different sets of highly redundant contigs. Only 25% and 28% of the A. mexicanum and A. t. tigrinum contigs, respectively, were identified in common  to be potential homologues. In this sense, the sequencing effort between these two species was complementary in yielding a more diverse collection of ESTs that were highly similar to human gene sequences.For the 10,592 contigs that showed significant similarity to sequences from the human RefSeq database, we obtained Gene Ontology (23) information to describe ESTs in functional terms. Although there are hundreds of possible annotations, we chose a list of descriptors for molecular and biological processes that we believe are of interest for research programs currently utilizing salamanders as model organisms , we identified DNA sequences corresponding to 69 of these 164 SCI rat genes  ,25; immefter SCI . Thus, after SCI  and Songfter SCI  against A. mexicanum limb regeneration ESTs against UNIGENE zebrafish fin regeneration ESTs  that corresponded to 1058 unique zebrafish ESTs. We then asked whether any of these potential regeneration homologues were represented uniquely in limb and fin regeneration databases (and not in databases derived from other zebrafish tissues). A search of the 1058 zebrafish ESTs against > 400,000 zebrafish ESTs that were sampled from non-regenerating tissues revealed 43 that were unique to the zebrafish regeneration database  that may be expressed in progenitor cells that proliferate and differentiate during appendage regeneration. Overall, our results suggest that regeneration is achieved largely through the reiterative expression of genes having additional functions in other developmental contexts, however a small number of genes may be expressed uniquely during appendage regeneration.Similar gene expression programs may underlie regeneration of vertebrate appendages such as fish fins and tetrapod limbs. Regeneration could depend on reiterative expression of genes that function in patterning, morphogenesis, and metabolism during normal development and homeostasis. Or, regeneration could depend in part on novel genes that function exclusively in this process. We investigated these alternatives by searching s Figure . This seA. mexicanum and A. t. tigrinum is needed to develop DNA markers for genome mapping [A. mexicanum and 18,765 base positions for A. t. tigrinum. Two classes of polymorphism were considered in this analysis: those occurring at moderate (identified in 10–30% of the EST sequences) and high frequencies (identified in at least 30% of the EST sequences). Within the A. mexicanum contigs, 0.49% and 0.06% of positions were polymorphic at moderate and high frequency, while higher levels of polymorphism were observed for A. t. tigrinum (1.41% and 0.20%). Higher levels of polymorphism are expected for A. t. tigrinum because they exist in larger, out-bred populations in nature.The identification of single nucleotide polymorphisms (SNPs) within and between orthologous sequences of  mapping , quantit mapping , and pop mapping . We estiA. mexicanum and A. t. tigrinum assemblies, and the resulting alignments were filtered to retain only those alignments between sequences that were one another's reciprocal best BLAST hit. As expected, the number of reciprocal 'best hits' varied depending upon the E value threshold, although increasing the E threshold by several orders of magnitude had a disproportionately small effect on the overall total length of BLAST alignments. A threshold of E<10-80yielded 2414 alignments encompassing a total of 1.25 Mbp from each species, whereas a threshold of E<10-20 yielded 2820 alignments encompassing a total of 1.32 Mbp. The percent sequence identity of alignments was very high among presumptive orthologues, ranging from 84–100% at the more stringent E threshold of E<10-80. On average, A. mexicanum and A. t. tigrinum transcripts are estimated to be 97% identical at the nucleotide level, including both protein coding and UTR sequence. This estimate for nuclear sequence identity is surprisingly similar to estimates obtained from complete mtDNA reference sequences for these species (96%, unpublished data), and to estimates for partial mtDNA sequence data obtained from multiple natural populations [A. tigrinum complex.To identify SNPs between species, we had to first identify presumptive, interspecific orthologues. We did this by performing BLASTN searches between the ulations . These rulations . From a A. mexicanum and A. t. tigrinum ESTs and extended this information to develop informative molecular markers for a related species, A. ordinarium. Ambystoma ordinarium is a stream dwelling paedomorph endemic to high elevation habitats in central Mexico [Ambystoma ordinarium shares approximately 98 and 97% mtDNA sequence identity with A. mexicanum and A. t. tigrinum respectively [Relatively little DNA sequence has been obtained from species that are closely related to commonly used model organisms, and yet, such extensions would greatly facilitate genetic studies of natural phenotypes, population structures, species boundaries, and conservatism and divergence of developmental mechanisms. Like many amphibian species that are threatened by extinction, many of these ambystomatid salamanders are currently in need of population genetic studies to inform conservation and management strategies [e.g. ]. We chal Mexico . This spectively .A. ordinarium, A. mexicanum and A. t. tigrinum EST contigs were aligned to identify orthologous genes with species-specific sequence variations (SNPs or Insertion/Deletions = INDELs). Primer pairs corresponding to 123 ESTs (Table A. ordinarium populations. Seventy-nine percent (N = 97) of the primer pairs yielded amplification products that were approximately the same size as corresponding A. mexicanum and A. t. tigrinum fragments, using only a single set of PCR conditions. To estimate the frequency of intraspecific DNA sequence polymorphism among this set of DNA marker loci, 43 loci were sequenced using a single individual sampled randomly from each of the 10 populations, which span the geographic range of A. ordinarium. At least one polymorphic site was observed for 20 of the sequenced loci, with the frequency of polymorphisms dependent upon the size of the DNA fragment amplified. Our results suggest that the vast majority of primer sets designed for A. mexicanum / A. t. tigrinum EST orthologues can be used to amplify the corresponding sequence in a related A. tigrinum complex species, and for small DNA fragments in the range of 150–500 bp, approximately half are expected to have informative polymorphisms.To identify informative markers for Ts Table  were scrXenopus (37) that is currently providing the bulk of amphibian genome information. Here we show the utility of ambystomatid ESTs for identifying chromosomal regions that are conserved between salamanders and other vertebrates. A region of conserved synteny that corresponds to human chromosome (Hsa) 17q has been identified in several non-mammalian taxa including reptiles (38) and fishes (39). In a previous study Voss et al. (40) identified a region of conserved synteny between Ambystoma and Hsa 17q that included collagen type 1 alpha 1 (Col1a1), thyroid hormone receptor alpha (Thra), homeo box b13 (Hoxb13), and distal-less 3 (Dlx3) . Our results show that BLAST-based definitions of orthology are informative between salamanders and human. All other presumptive Hsa 17 loci mapped to Ambystoma chromosomal regions outside of the previously defined synteny group. It is interesting to note that two of these loci mapped to the same ambystomatid linkage group (Cgi-125, Flj20345), but in human the presumptive orthologues are 50 Mb apart and distantly flank the syntenic loci in Figure Hsa 17 orthologues during vertebrate evolution.Salamanders occupy a pivotal phylogenetic position for reconstructing the ancestral tetrapod genome structure and for providing perspective on the extremely derived anuran s et al. 0 identif fishes 3. In a prs et al. 0 identifAmbystoma. Genome resources in Ambystoma can be extended to multiple, closely related species to explore the molecular basis of natural, phenotypic variation. Such extensions can better inform our understanding of ambystomatid biodiversity in nature and draw attention to the need for conserving such naturalistic systems. Several paedomorphic species, including A. mexicanum, are on the brink of extinction. We can think of no better investment than one that simultaneously enhances research in all areas of biology and draws attention to the conservation needs of model organisms in their natural habitats.Ambystomatid salamanders are classic model organisms that continue to inform biological research in a variety of areas. Their future importance in regenerative biology and metamorphosis will almost certainly escalate as genome resources and other molecular and cellular approaches become widely available. Among the genomic resources currently under development (see ) are a cA. mexicanum and A. t. tigrinum). An approximately equivalent number of contigs were identified for each species, with 21,091 unique contigs identified overall. The strategy to sequence cDNAs from a diverse collection of tissues from normalized and non-normalized libraries yielded different sets of highly redundant contigs. Only 25% and 28% of the A. mexicanum and A. t. tigrinum contigs, respectively, were identified in common. To demonstrate the utility of these EST resources, we searched databases to identify new probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human/Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. Over 100 new probes were identified for regeneration research using informatic approaches. With respect to comparative mapping, 13 of 15 EST markers were mapped successfully, and 6 EST markers were mapped to a previously defined synteny group in Ambystoma. These results indicate a high probability of mapping success using EST markers developed from the joint assembly of A. mexicanum and A. t. tigrinum contigs. Finally, we found that primer sets designed for A. mexicanum / A. t. tigrinum EST orthologues can be used to amplify the corresponding sequence in a related A. tigrinum complex species. Overall, the EST resources reported here will enable a diversity of new research areas using ambystomatid salamanders.Approximately 40,000 cDNA sequences were isolated from a variety of tissues to develop expressed sequence tags for two model salamander species . The V1 (A. mex), V2 (A. tig), V4-5 (A. tig), and V6-7 (A. mex) libraries were made from an assortment of larval tissues  to spike PCR reactions: . All successful amplifications with inserts larger than ~500 bp were sequenced . Sequencing and clean-up reactions was carried out according to manufacturers' protocols. ESTs were deposited into NCBI database under accession numbers BI817205-BI818091 and CN033008-CN045937 and CN045944-CN069430.A. mexicanum (AJ584639). Finally, all sequences less than 100 bp were removed. The average length of the resulting ESTs was 629 bp. The resulting high quality ESTs were clustered initially using PaCE [The PHRED base-calling program  was useding PaCE  on the Uing PaCE  with an Xenopus laevis and X. tropicalis UNIGENE ESTs (Mar. 2004) using TBLASTX. Zebrafish ESTs were downloaded from UNIGENE ESTs (May 2004). BLAST searches were done with an E-value threshold of E <10-7 unless specified.All contigs and singletons were searched against the human RefSeq database (Oct. 2003 release) using BLASTX. The subset of sequences that yielded no BLAST hit was searched against the non-redundant protein sequence database (Feb. 2004) using BLASTX. The remaining subset of sequences that yielded no BLAST hit was searched against A. mexicanum and A. t. tigrinum assemblies were joined into a single assembly using CAP3 and the following criteria: an assembly threshold of 12 bp to identify initial matches, a minimum 100 bp match length, and 85% sequence identity. To identify putatively orthologous genes from A. mexicanum and A. t. tigrinum assemblies, and generate an estimate of gene sequence divergence, assemblies were compared using BLASTN with a threshold of E <10-20. Following BLAST, alignments were filtered to obtain reciprocal best BLAST hits.All low quality base calls within contigs were masked using a PHRED base quality threshold of 16. To identify polymorphisms for linkage mapping, contigs from A. mexicanum and A. t. tigrinum assembly. Polymerase chain reaction (PCR) primers were designed using Primer 3 [2, 0.25 U Taq, and a 3-step profile . DNA fragments were purified and sequenced using ABI Big Dye or Amersham Dye terminator chemistry. Single nucleotide polymorphisms were identified by eye from sequence alignments.Polymorphic DNA marker loci were identified by locating single nucleotide polymorphisms (SNPs) in the joint Primer 3  to ampliHsa 17) were identified by comparing the joint A. mexicanum and A. t. tigrinum assembly to sequences from the human RefSeq (NCBI) protein database, using BLASTX at threshold E<10-7. Linkage distance and arrangement among markers was estimated using MapManager QTXb19 software [Primer 3 [Array Designer2 (Premier Biosoft) software. Species-specific polymorphisms were assayed by allele specific amplification, restriction digestion, or primer extension, using the reagent and PCR conditions described above. Primer extension markers were genotyped using the AcycloPrime-FP SNP detection assay (Perkin Elmer). See Table Putative salamander orthologues of genes on human chromosome 17 (software  and the software . All PCRPrimer 3  and ArraA. mexicanum and A. t. tigrinum ESTs; JS: comparative mapping and polymorphism estimation; DW: extending ESTs to A. ordinarium; JM, KK, AS, NM: PCR and gel electrophoresis; BH and ET: cDNA library construction and sequencing for spinal cord regeneration ESTs; MR, SB, DG: cDNA library construction and clone management for limb regeneration ESTs; DP and SV conceived of the project and participated in its design and coordination. All authors read and approved the final manuscript.SP and DK: bioinformatics; JW: clone management and sequencing in support of


Feeding related peptides have been shown to be additionally involved in the central autonomic control of gastrointestinal functions. Recent studies have shown that ghrelin, a stomach-derived orexigenic peptide, is involved in the autonomic regulation of GI function besides feeding behavior. Pharmacological evidence indicates that ghrelin effects on food intake are mediated by neuropeptide Y in the central nervous system.In the present study we examine the role of ghrelin in the central autonomic control of GI motility using intracerobroventricular and IP microinjections in a freely moving conscious rat model. Further the hypothesis that a functional relationship between NPY and ghrelin within the CNS exists was addressed.-1 BW and saline controls) decreased colonic transit time dose related. Central administration of the NPY1 receptor antagonist, BIBP-3226, prior to centrally or peripherally administration of ghrelin antagonized the ghrelin induced stimulation of colonic transit. On the contrary ICV-pretreatment with the NPY2 receptor antagonist, BIIE-0246, failed to modulate the ghrelin induced stimulation of colonic motility.ICV injections of ghrelin  decreased the colonic transit time up to 43%. IP injections of ghrelin (0.3 nmol – 3.0 nmol kg1 receptor dependent mechanisms.The results suggest that ghrelin acts in the central nervous system to modulate gastrointestinal motor function utilizing NPY NPY binds preferentially with high affinity to Y1 and Y2 receptors, and there is evidence suggesting that these two receptor subtypes are involved in CNS regulation of digestive function by NPY action in arcuate nucleus and the paraventricular nucleus of the hypothalamus [The presence or absence of food in the gut stimulates the release of several regulatory peptides. These orexigenic (NPY, AGRP, ghrelin, MCH, Orexin-A, ...) and anorexigenic (CRF, CCK, CART, GLP-1, leptin, insulin, ...) peptides participating in the hypothalamic control of feeding behavior and satiety have been shown to be additionally involved in the autonomic control of gastointestinal (GI) functions like secretion and motility. For example fasted motor activity of the GI tract, e.g. the colon, is observed after intracerebroventricular (ICV) injection of neuropeptide Y whereas CRF ICV-treatment cause the disruption of fasted colonic motor activity -6. Thereeceptors ,17. Thuseceptors . NPY actthalamus .Taken together there is overwhelming evidence that ghrelin, beside its satiety modulatory capacity, is involved in the CNS control of digestive function of the upper gastrointestinal tract. In the CNS ghrelin and NPY, the most potent orexigenic neuropeptides known, are anatomical associated and functionally related. Moreover hypothalamic NPYergic neurons are downstream mediators of feeding related ghrelin action.In the present study we scrutinize the hypothesis that central neuropeptide Y receptor activation is involved in the ghrelin induced modulation of gastrointestinal motility using a microinjection-model in conscious fed and freely moving rats.All experimental components described were performed in accordance with the requirements of German legislation for the protection of animals and were licensed and supervised by the appropriate government body.®, Lage, Germany) an tap water ad libitum. After surgical procedures, rats were housed individually. During experimental procedure the animals had continuous access to food and water.Male Sprague-Dawley rats with a mean body weight of 350 ± 50 g were maintained on a 12 : 12 h photoperiod. They were housed in colony cages under conditions of controlled humidity and temperature (22 ± 2°C) for at least 7 days prior to the surgical procedure. The animals were fed a standard rat diet  [see Ref.: [2 receptor antagonist, BIIE-0246  [see Ref.: [Ghrelin (Bachem, Heidelberg, Germany) doses of 0.03 nmol (100 ng), 0.3 nmol (1 μg) and 3 nmol (10 μg)/5 μl were dissolved in 0.15 M sterile saline (B. Braun, Melsungen, Germany). The NPYee Ref.: ] and theee Ref.: ] were diee Ref.: ,20. Probee Ref.: .-1 i.p., Parke-Davis, Freiburg, Germany) and xylazine (5 mg kg-1 i.p., Bayer AG, Leverkusen, Germany). Animals were positioned in a stereotactic apparatus (David Kopf Instruments, Tujunga, CA). The head was fixed in a nose-down-position (-3 mm) and the skull exposed. Then trepanation of the skullcap was performed according to coordinates obtained from Paxinos and Watson [For surgical procedures, rats were anesthetized with a mixture of ketamine (75 mg kgd Watson  (mm from-1) and xylazine (5 mg kg-1). After laparatomy a polyethylene microcatheter  was chronically implanted into the proximal colon 1 cm distal from the caecocolonic junction. The catheter was fixed at the colonic wall by a purse-string suture and routed subcutaneously to the interscapular region, where it was exteriorized through the skin and secured. The animals were allowed 7 days recovery after abdominal surgeries before the beginning of habituation training sessions. Experiments were performed in fed, conscious rats.This method was performed as described elsewhere . Prior t-1/1 ml 0.15 M saline and the high dose of ghrelin was 3 nmol kg-1/1 ml saline. NPY receptor antagonists were injected ICV 15 min before ghrelin was given peripherally at doses of 200 nmol/rat (BIBP-3226) and 120 nmol/rat (BIIE-0246) respectively.The doses of ghrelin were calculated according to the lowest effective doses to stimulate food intake [see Ref.: ]. For IPFor ICV injections a 1 μl micro syringe (Hamilton, Reno, NV, USA), attached to a 32 G injection needle via a PE-50 tube-catheter was used. The stainless steel injection cannulas (32 G) were cut to protrude 2 mm beyond the tips of the guide cannulas. The conscious animals were gently restrained by hand, the injection needle was inserted through the guide cannula, and vehicle  or NPY receptor antagonists  and ghrelin (0.03 nmol, 0.3 nmol or 3 nmol/5 μl), were consecutively injected ICV slowly over a 60 s period. We used a 15 min time interval between ICV injection of receptor antagonist or vehicle and ICV ghrelin administration. The injection needle was left in place for 2 min after injection to allow diffusion of the solution and to prevent back flow. Then dummy cannulas were reinserted into the guide cannulas. After the last experimental testing session, the rats were anesthetized and 5 μl of alcian blue 8GX were injected ICV.Colonic transit time was calculated by using an enteral dye-marker. Trypan blue, a non-absorbable dye, was injected in 0.2 ml volume through the catheter positioned in the proximal colon and followed by a 0.2 ml saline flush immediately after the ICV or IP microinjection. Colonic transit time was evaluated as the time interval between dye injection and the discharge of the first blue pellet. Faecal pellet output was monitored continuously by a self-developed, automated observation system that mechanically registers the time of all bowel movements for 24 h. The device consists of a conveyor belt placed under the mesh bottom cage, which transports faecal pellets with defined velocity to a collector.-1 i.p.) and xylazine (5 mg kg -1 i.p.), and 0.05% alcian blue 8GX was microinjected intracerebroventricular under the same conditions as vehicle or peptide. The anaesthetized animals were transcardially perfused with phosphate buffered saline (PBS) buffer (0.1 M, pH 7.4) followed by Zamboni's fixative . The brains were removed and cryoprotected in 25% sucrose. The site of injection was confirmed by inspection of intracerebroventricular dye distribution. Animals that received injections outside of the 3rd ventricle were excluded from data analysis.The methods were performed as described in previous studies . When ex-1 BW and the high dose of ghrelin was 3.0 nmol kg-1 BW. After injections, rats were subsequently returned to their home cages and maintained in a non-stressful environment to monitor colonic transit time. In order to minimize interindividual variation, and to reduce the number of animals needed to perform this study, animals were tested twice in this study. In randomized order, each rat received vehicle and a single dose of ghrelin or vehicle ICV or IP. The time interval between the experiments performed on the same animal was at least 4 days.The aim of the first experiment was to determine whether exogenous ghrelin would alter colonic motor function. Thus in experiment I, dose response effects of ghrelin in the cerebrospinal fluid (ICV) and the periphery (IP) on colonic transit time were examined. Ghrelin or saline as a vehicle was administered ICV or IP in conscious lightly restrained rats as previously described. For IP injection the low dose of ghrelin administered peripherally was 0.3 nmol kgvia a NPY receptor dependent pathway was addressed. Therefore, we determined if a pretreatment with selective NPY1- (BIBP-3226) and NPY2 (BIIE-0246) receptor antagonists administered into the cerebrospinal fluid would block the alterations of colonic motor activity induced by centrally and peripherally administered ghrelin. The animals were pretreated with the NPY-Y1 and -Y2 receptor antagonists, injected ICV or vehicle , 15 min prior to ICV or IP ghrelin injections. Thereafter colonic transit time was assessed as described above.In experiment II the hypothesis that ghrelin acts at the CNS to modulate colonic motor function The criterion used to include results in the data analysis of the ICV-injected group was the correct placement of the ICV cannulas.Results are expressed as mean ± SEM. The data of all studies were analyzed by ANOVA and differences between groups were evaluated by the Student-Newmann-Keuls test. P < 0.05 was considered significant.In experiment I, dose response effect of peripherally and centrally administered ghrelin on colon transit time in fed and freely moving rats were examined. As demonstrated in Fig. via NPY receptor dependent mechanisms was addressed. As shown in Fig. 1 receptor antagonist 15 min prior to ICV application of 0.3 nmol ghrelin totally blocked the ghrelin induced effect on colonic transit. Application of BIBP-3226 into the CSF of the control group that was microinjected with vehicle, had no effect. Changes in colonic transit time induced by IP injection of ghrelin (3.0 nmol kg-1 BW) were canceled by ICV injection of NPY1 receptor antagonist, BIBP-3226. . Two of these, NPY1- and NPY2 receptors, are found in high density in the hypothalamus. There is compelling evidence that, in particular, NPY1 and NPY2 receptors are involved in the CNS regulation of gastrointestinal function. [1 and Y2 receptor pathways in the present study and did not investigate the role of neuropeptide Y receptor subtypes Y4 and Y5 which are also expressed in the hypothalamus and are also involved in the autonomic control of feeding behavior and GI function. In the present study pretreatment with the NPY1 receptor antagonist, BIBP-3226, blocked stimulation of colonic motility induced by systemic microinjection of exogenous ghrelin (ICV and IP). In our hands BIIE-2046, which is a selective antagonist of the NPY Y2 receptor, failed to affect the ghrelin induced induction of fasted motor activity. It was previously described that knocking out NPY significantly decreases ghrelin stimulated feeding [1 receptors in the hypothalamus, e.g. the PVN, and results in the stimulation of GI motor function [via activation of hypothalamic, in particular by using NPYergic pathways via activation of NPY-Y1 receptors, in the arcuate nucleus. This hypothesis is in good agreement with our observation that the effect of peripherally (IP) administered ghrelin on colonic motility is blocked by ICV pretreatment with the specific NPY1 receptor antagonist, BIBP-3226. The NPY2 receptor antagonist BIIE-0246 injected in the 3rd ventricle at the equipotent dose as BIBP-3226 was not effective to antagonize the ghrelin effect on GI motility significantly. This data suggests that ghrelin unfolds a stimulatory effect on colonic motility primarily by acting on central NPY1 and not via NPY2 receptors. This interpretation is confirmed by the observation that Y1 receptors acts rather postsynaptically and the Y2 receptor rather presynaptically [4 or Y5 receptors in the CNS are involved in the CNS control of gastrointestinal function should be examined in future studies.There is convincing evidence that the most effective appetite stimulating peptides, NPY and ghrelin, act in the CNS and the periphery to simulate not only feeding but also GI function such as gastric acid secretion and gastric motility ,8-10. Stfed rats . Furthereceptors ,16,26. Feceptors . Taken tunction.  For this feeding ,28. In t feeding . The res feeding . Thus we feeding . Further feeding . Compell feeding ,18. For function . Pretreafunction ,17. Withfunction . Thus weptically ,32 The qrd ventricular injection of ghrelin this could simply mean that the peptide gained access to the 4th ventricle and reached further caudal brain sites, e.g. NTS, DVC and medulla oblongata. With respect to the distribution of GHS- and NPY receptors in the CNS this hypothesis is possible, but how ghrelin action on any of these brain sites would modulate digestive function is not known. This question should be examined in future studiesWe hypothesize that circulating ghrelin exhibits its effect by activating hypthalamic neurons, in particular neurons in the arcuate nucleus bearing GHS- and NPY-Y1 receptors. Further this ghrelin induced neuronal activation leads to stimulation of GI motor function by activation of higher hypothalamic brain sites, e.g. activation of neuronal projections within the paraventricular nucleus of the hypothalamus. On the other hand it is possible that the site of action of circulating ghrelin is not the hypothalamus but other brain sites. In our model using 3In summary, we presented evidence that ghrelin is involved in the CNS control of GI function. Apart from humoral pathways ghrelin acts into the CNS to control GI function by a mechanism of action involving neuropeptide Y1 receptor pathways. Further this study support the hypothesis giving by Chen et al. that ghrelin has an absolute requirement for neuropeptide Y pathways to unfold its physiological effects .AGRP agouti-related peptideARC arcuate nucleusCART cocain- and amphetamine-regulated transcriptCCK cholecystokininCNS central nervous systemCSF cerebrospinal fluidCRF corticotropin releasing factorDVC dorsal motor nucleus of vagusGHS-R growth hormone secretagogue receptorGLP-1 glucagon like peptide-1ICV intracerebroventricularMCH melanocortin hormoneMI microinjectionNPY neuropeptide YNTS nucleus of the solitary tractPOMC proopiomelanocortinPVN paraventricular nucleus of the hypothalamusThe author(s) declare that they have no competing interests.JJT participated in the design and coordination of the study, performed the microinjection studies and drafted the manuscript. CGT was the surgeon in charge and participated in the animal experiments. M-KHS and SM were involved in the design and coordination of the study. MR participated in the analysis and interpretation of data and revised the manuscript critically.The pre-publication history for this paper can be accessed here: