DNA extraction methods for PCR-quality DNA from calluses and plants are not time efficient, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc. The need for a rapid and simple procedure is urgent, especially when hundreds of samples need to be analyzed. Here, we describe a simple and efficient method of isolating high-quality genomic DNA for PCR amplification and enzyme digestion from calluses, various wild-type and transgenic plants.We developed new rapid and reliable genomic DNA extraction method. With our developed method, plant genomic DNA extraction could be performed within 30 min. The method was as follows. Plant tissue was homogenized with salt DNA extraction buffer using hand-operated homogenizer and extracted by phenol:chloroform:isoamyl alcohol (25:24:1). After centrifugation, the supernatant was directly used for DNA template for PCR, resulting in successful amplification for RAPD from various sources of plants and specific foreign genes from transgenic plants. After precipitating the supernatant, the DNA was completely digested by restriction enzymes.This DNA extraction procedure promises simplicity, speed, and efficiency, both in terms of time and the amount of plant sample required. In addition, this method does not require expensive facilities for plant genomic DNA extraction. Molecular biological studies of plants require high-quality DNA. Several DNA extraction procedures for isolating genomic DNA from various plant sources have been described, including the salt extraction method and the cetyltrimethyl ammonium bromide (CTAB) method  and its e.g., the Wizard genomic DNA purification system; Promega, USA). However, these methods are not time efficient for consistently obtaining PCR-quality DNA from calluses and plants, since they require that the tissues be ground in liquid nitrogen, followed by precipitation of the DNA pellet in ethanol, washing and drying the pellet, etc.Most methods require the use of liquid nitrogen  or freez2) of leaf tissue.In our laboratory, we investigate the stability of transgenes expressed in calluses or plants transformed by nuclear or chloroplast transformation in tobacco, lettuce, potato, etc. In addition, we need high-quality genomic DNA for Southern blot analysis to confirm homologous recombination in chloroplast transformation . For our2O, and PCR was performed using 1 μl of the diluted supernatant as a template . An aliquot of the supernatant (~5 μl) was diluted 5 fold with sterile dH. An aliqDNA samples prepared using the three different extraction procedures  [To examine the presence of bar ,12 or thbar  at a dirme Table , lane 1,2450 bp)   were between 1.7 and 2.0. However, the DNA concentrations from rice, maize, and poplar were relatively low (< 3 μg). This may be because homogenization using a hand-operated homogenizer with a plastic tip is incomplete, since the leaves of these plants are stronger than the leaves of tobacco, potato, cabbage, lettuce, and Siberian ginseng. Genomic DNA from various plant sources was electrophoresed on 1% agarose gels, and high-molecular-weight DNA was obtained  is put in a 1.5-ml microfuge tube and homogenized directly; consequently, a very small sample is required for DNA extraction. There is no sample waste with our method, whereas much larger samples are required when plant samples are ground in a mortar and pestle with liquid nitrogen and transferred to a tube. Previously reported techniques require several steps [2 was used in the extraction, co-extracts  were extracted alongside the DNA and inhibited the PCR. On the contrary, our protocol does not require appropriate sample size to extract DNA. Warner et al. [There are many advantages in using our genomic DNA extraction method to obtain template for PCR amplification. Many different plants could be amplified using the same DNA extraction method and the same PCR protocol. Using this protocol, we successfully amplified DNA repeatedly from all eight plant sources examined. Our procedure is not only very simple, but is also time and cost effective. After homogenization in DNA extraction buffer using a hand-operated homogenizer, the template DNA for PCR could be extracted by phenol/chloroform/isopropyl alcohol treatment. Since this method does not require liquid nitrogen, expensive commercial DNA extraction kits, or ethanol precipitation to produce DNA template for PCR, we can save considerable time and expense. The time required for our DNA extraction method is less than 30 min, which is extraordinary compared with other genomic DNA extraction methods. With our procedure, leaf tissue , potato (Solanum tuberosum), cabbage (Brassica oleracea), rice (Oryza sativa), lettuce (Lactuca sativa), maize (Zea mays), poplar (Populus nigra), and Siberian ginseng (Eleutherococcus senticosus). The plants used for genomic DNA extraction were grown in a culture room or greenhouse. Tobacco, potato, cabbage, lettuce, and Siberian ginseng were grown in a culture room. Seeds were surface-sterilized with 70% ethanol for 3 min, and then with 10% sodium hypochlorite for 15 min. The seeds were washed five times in sterile water and placed in Petri dishes containing 4.6 g l-1 MS salts [-1 sucrose, and 7.5 g l-1 bactoagar at pH 5.7. The seeds were grown in a controlled environment at 25°C on a 16-h continuous light and 8-h dark daily cycle. Rice, maize, and poplar plants were grown in a greenhouse for genomic DNA extraction. Transgenic tobacco plants and Siberian ginseng calluses were also used to extract genomic DNA and to confirm foreign gene insertion by PCR amplification.We examined plant material from plant collections commonly used for foreign gene expression: tobacco , using a hand-operated homogenizer  with a plastic pestle, for 15~20 s. After an initial homogenization, another 150 μl of DNA extraction buffer were added and homogenized with the same homogenizer for 15~20 s. Then, 20 μl of 20% SDS were added and vortexed for 30 s. The samples were incubated at 65°C for 10 min for cell lysis. At this point, three different DNA extraction protocols were used for PCR amplification. Protocol 1: An equal volume of phenol/chloroform/isoamyl alcohol (25:24:1) was added to the samples, mixed by vortexing for 30 s, and then centrifuged at 10,000 g for 3 min at 4°C. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 2: The supernatant from protocol 1 was transferred to a fresh tube and extracted one more time with phenol/chloroform/isoamyl alcohol (25:24:1) and then with chloroform. The supernatant was diluted 5 fold, and 1 μl of the supernatant was used as the DNA template for PCR analysis. Protocol 3: The supernatant from protocol 2 was transferred to a fresh tube, and a double volume of ethanol was added to each sample, mixed well, and the samples were incubated at -20°C for 30 min. The samples were then centrifuged at 10,000 g for 10 min at 4°C. The pellet was washed with 70% ethanol, dried, and resuspended in sterile dH2O containing 20 μg/ml DNase-free RNase A. The concentration and purity were determined from the A260/A280 ratio using a spectrophotometer. Five micrograms of each genomic DNA sample were incubated at 37°C for 3 h for complete digestion with 20 U of EcoRI and HindIII  in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture.We tested three different variations of the genomic DNA extraction procedure. About 0.5 cmEcoRI and HindIII in a total volume of 100 μl and analyzed on 1.0% agarose gels using 15 μl aliquots of the reaction mixture. By using the genomic DNA isolated from the leaves or calluses of wild-type and transgenic plants, PCR amplifications were performed on a Perkin Elmer GeneAmp PCR System 2400  in a total volume of 25 μl containing 1 × PCR buffer, 0.2 mM dNTP, 10 pmol of each primer (Table Five micrograms of each genomic DNA sample measured by spectrophotometer were incubated at 37°C for 3 h for complete digestion with 20 U of TJK developed the method and performed majority of the experiments. MSY provided technical assistance, funding and supervision for the work. All authors have read and approved the final manuscript.


Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood.Eighteen patients (n = 12 breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve growth factor (NGF).TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness . Nerve Growth Factor, which up-regulates TRPV1 and induces nerve sprouting, was present basal keratinocytes: some breast pain specimens also showed NGF staining in supra-basal keratinocytes. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in keratinocytes in breast pain tissues; TRPV3, median [range] – no pain group, n = 6, 0.75 [0–2]; pain group, n = 11, 2 [8; p = 0.Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in keratinocytes deserve further study. Breast pain is a common problem, which can affect up to 70% of women . Breast The mechanisms of breast pain in the majority of women are not well understood at the cellular or molecular level. We hypothesized a relationship between clinical breast pain, nerve growth factor (NGF) and its regulated ion channels or receptors expressed by nociceptor fibres. Estrogens upregulate NGF receptor mRNA in sensory neurons , and enhEighteen patients were recruited  at Chelsea and Westminster, Charing Cross, Ravenscourt Park Hospitals in London and Broomfield Hospital in Essex were recruited. Breast reduction patients had no previous surgery. The breast reconstruction patients had Latissimus dorsi flap reconstructions after previous mastectomies, and had implants. Patients below 18 years or above 70 years, with any local skin inflammation, infection or cancerous skin changes were excluded. The Research Ethics Committee of Hammersmith Hospitals Trust and Mid Essex Hospitals Trust gave ethical permission for the study. Informed consent was obtained prior to the clinical examination and questionnaire administration.Age, parity, height, weight and menstrual data were collected. Details of current surgery, any previous breast surgery and breast disease were also recorded. A questionnaire which included questions on breast and period pain was administered, a diagram to indicate painful and tender areas and the 78 pain descriptors from the McGill Pain Questionnaire  were proFull thickness skin biopsies were collected from each patient along the incision line of about 2 mm depth. Samples were coded, frozen on site and stored at -70°C. The skin samples were mounted in embedding medium . Frozen tissue sections (10 μm) were collected onto poly-L-lysine-coated (Sigma Poole Dorset UK) glass slides and post-fixed in freshly prepared, 4% w/v paraformaldehyde in phosphate buffered saline . After washing in PBS, endogenous peroxidase was blocked by incubation with 0.3% w/v hydrogen peroxide in methanol. After a further wash in PBS the tissue sections were incubated overnight with affinity purified antibodies to TRPV1 , TRPV3 , TRPV4 , recombinant human NGF  or marker of large and some small calibre nerve fibres . Methodological controls included omission of primary antibodies, or their replacement with pre-immune serum. Specificity of antibodies has been described in previous publications ,20. SiteA non-parametric, two tailed test (Mann Whitney U) was used. Commercially available statistical software was used to perform the test (Prism 3™).Pain Rating Indices (PRI) and Visual Analogue Scores (VAS) were used to group patients with (n = 10) and without (n = 8) breast pain: pain group; PRI -Median (range) 12 (4–30); Mean (SEM) 13.44(3.07) and VAS – Median (range) 5(3.7–6.7); Mean (SEM) 5.14(0.31)]; in patients without breast pain, PRI and VAS were zero. Only 2 patients reported thermal pain descriptors , while most reported ache and tenderness. The numbers of breast reduction and reconstruction patients with pain were 7 out of 12 and 3 out of 6 respectively, and all reported pain of duration greater than 6 months. The presence (n = 8) or absence (n = 10) of dysmenorrhoea was also recorded. There was no association between breast pain and the presence of dysmenorrhoea.TRPV1-immunoreactive fibres were present mainly in the sub-epidermis in normal (pain free) skin Fig. . In breaTRPV3 immunoreactivity was detected in basal keratinocytes and occasional suprabasal cells throughout the epidermis. Quantification of immunostaining showed a significant (*P = 0.011) increase with breast pain  and the expression of the capsaicin receptor 1 (TRPV1) in nociceptor fibres, as these are key molecules in pain and hypersensitivity. Our finding, that TRPV1-positive intra-epidermal fibres were significantly increased in patients with breast pain and tenderness, is both novel and important. The increased and abnormal "clusters" of intra-epidermal fibres were shown in patients who had no previous breast surgery, and no known episodes of mastitis. Hence this may be a surrogate marker for "idiopathic" or macromastia related breast pain in some patients. Studies are in progress to correlate changes in TRPV1 nerve fibres with quantitative sensory perception thresholds.The cause of the increased intra-epidermal fibres is not known. Given the trend to thinning of the epidermis, normally associated with denervation , in the Our recent studies have demonstrated increased TRPV1-immunoreactive nerve fibres in inflammatory bowel disease , and in Little is known of the roles of TRPV3 and TRPV4 in human pain pathophysiology and keratinocyte function. While we have previously demonstrated TRPV3 in human sensory neurons , no TRPVBreast pain and tenderness appears to be associated with abnormal intra-epidermal innervation. This may reflect re-innervation of skin following nerve stretch damage, and/or collateral sprouting. While further studies are necessary to establish functional links between the TRPV1, TRPV3 and TRPV4 immunohistological changes and breast pain, our findings indicate a path for increasing understanding and treatment of breast pain.TRPV = transient receptor potential vanilloid; NGF = Nerve Growth Factor; VAS = Visual Analogue Score; PRI = Pain Rating IndexThe author(s) declare that they have no competing interests.PG and EW recruited patients, collected biopsies and participated in immunohistology studies. PF participated in immunohistology and coordination of the study. AH participated in design of the study and recruitment of patients. JD, GS and CB provided antibodies and helped draft the manuscript. PA conceived the original study, its design and coordination, and helped with the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


In response to the Blackburn and Rowley essay on the President's Council on Bioethics, several thought-provoking opinions on ethical challenges in biomedical research are expressed by prominent stakeholders It is indeed regrettable that a distinguished and thoughtful scientist such as Elizabeth Blackburn should have been dismissed from the President's Council on Bioethics. Scientific perspectives such as hers are surely needed on this committee.Her dismissal was apparently the consequence of her disagreement with some of the text of the Council's report, “Beyond Therapy: Biotechnology and the Pursuit of Happiness” (2003). The thrust of this report is that some of the directions of current biological research will, if carried to fulfillment, result in major changes in the nature of human life—changes that the report regards with foreboding.In their essay, Drs. I would suggest that as scientists we should face these issues forthrightly. We should not seek refuge in presentday uncertainties. The authors of the report are not naïve nor ignorant. Yes, if these lines of research are successful, their outcome will change the nature of human life.As an example, consider current research into the causes of aging. Clearly, we do not at present know how to achieve major increases in the human life span . But it is plausible that we will learn how to do so. And surely a, say, doubling of the human life span would change the nature of human life.Likewise, if we learn to modify the human gene pool so as to produce exceptional individuals or to alter human capabilities, or if powerful drugs are developed that may commandeer the human psyche, the nature of human life will be altered.But so be it. The nature of human life has changed repeatedly and profoundly in the past—with the invention of agriculture, with the invention of writing, with the development of machines and mechanical power, with the advent of modern science and medicine. The nature of human life is different in 2004 a.d. from what it was in 1000 a.d. or 46 b.c. or 5000 b.c. or 10,000 b.c., and it will change again in the future.The concerns expressed in the report are earnest, and they should be confronted in earnest.


They receive signals from various sources (e.g. cell surface receptors) and control the level of transcription initiation, which largely determines gene expression. In vertebrates, transcription start sites and surrounding regulatory elements are often poorly defined. To support promoter analysis, we present CORG The automated annotation of promoter regions integrates information of two kinds. First, statistically significant cross-species conservation within upstream regions of orthologous genes is detected. Pairwise as well as multiple sequence comparisons are computed. Second, binding site descriptions (position-weight matrices) are employed to predict conserved regulatory elements with a novel approach. Assembled EST sequences and verified transcription start sites are incorporated to distinguish exonic from other sequences.. The benefits of our framework are exemplarily shown in the context of phylogenetic profiling of transcription factor binding sites and detection of microRNAs close to transcription start sites of our gene set.As of now, we have included 5 species in our analysis pipeline . We characterized promoter regions of 16,127 groups of orthologous genes. All data are presented in an intuitive way via our web site. Users are free to export data for single genes or access larger data sets via our DAS server The CORG platform is a versatile tool to support analyses of gene regulation in vertebrate promoter regions. Applications for CORG cover a broad range from studying evolution of DNA binding sites and promoter constitution to the discovery of new regulatory sequence elements (e.g. microRNAs and binding sites). Applications range from grouping of sequences (e.g. protein sequences into families) to Speaking of gene regulation, it has been known for a long time that there is considerable sequence conservation between species in non-coding regions of the genome. A comprehensive explanation of this observation is still elusive. However, sequence conservation within promoter regions of genes often stems from transcription factor binding sites that are under selective pressure (see  for a remir-10 and mir-196 which are located in the (short) intergenic regions in the Hox gene clusters of vertebrates [Conserved sequence elements of other types have recently caught much attention. Not all non-coding conserved DNA in the vicinity of a gene's transcription start site necessarily functions at the level of transcriptional regulation. For example, most known methylation-guide snoRNAs are intronencoded and processed from transcripts of housekeeping genes . A few mtebrates -7.A second class of conserved sequence elements exert their function as regulatory motifs in the untranslated region (UTR) of the primary transcript or the mature mRNA. The UTRsite database , for exaThe CORG framework aims at detecting and describing regulatory elements that are proximal to the transcription start site. In this context, the comparison of upstream regions of orthologous genes is particularly valuable. This concept is called "phylogenetic footprinting" and an overview of this approach can be found in .Phylogenetic footprinting in a strict sense is carried out on orthologous promoter regions. Local sequence similarities can then be directly interpreted as related regions harboring conserved functional elements. We denote these similarities as Conserved Non-coding Blocks (CNBs).Homo sapiens, Mus musculus, Rattus norvegicus, Danio rerio and Fugu rubripes. Multiple alignments are built from pairwise CNBs as described in the subsequent section.Comparative approaches gain power from the inclusion of sequences from more than two species . Multi-sIn this work, we take a gene-centered view of phylogeny. Homology among proteins and thus genes is often concluded on the basis of sequence similarity. The EnsEMBL database  allows tVarious recent experimental efforts supply information about the position of transcriptional start sites in the human and mouse genome. Table Some repositories offer genomic coordinates for their start site entries. Existing genomic mapping information was incorporated unless the underlying genome assembly build differed. The remaining data were projected onto the genome with SSAHA (Sequence Search and Alignment by Hashing Algorithm), a rapid near-exact alignment algorithm .The notion of "promoter region" deserves some further explanation in the context of our approach. Typically, though not exclusively, we expect conserved regulatory regions to appear in the vicinity of the transcription start site of a gene. Since we do not know the precise location of the start of transcription for each and every gene, we chose to compare the sequence regions upstream of the start of translation from orthologous genes. If verified transcription start sites are known, we define a sequence window that is large enough to hold both, translation and transcription start sites, plus 5 kB upstream sequence. In case we lack this information, our observations on known transcription start sites indicate that most promoter regions should be captured in a sequence window of 10 kb size  in two sequences are computed with an implementation of the Waterman-Eggert algorithm. We have already given an account of the algorithm and statistics in ,20. The Q is the transition rate matrix. We left out the elements on the diagonal, which are constrained by the requirement that the sum of all elements in a row equals zero.where πi are the stationary nucleotide frequencies, their sum is constrained to be one. Although the two genomes under consideration are in general not in their stationary state with respect to the substitutional process we take the mean  of the two observed nucleotide frequencies, , to be the best estimate of the stationary base composition.The πi we have to allow for deviation from those ratios. We do this by choosing for example α ∝ (R(A → T)/πT + R(T → A)/πA)/2, where R(i → j) is either 1, 3, or 5 depending on the process under consideration. At the end we scale the matrix Q, such that the PAM distance [From other studies we have further knowledge about the relative rates between transversions, the transition A:T→G:C, and the transition G:C→A:T, which occur in roughly in the ratio 1:3:5 along vertebrate lineages . These rdistance  of the sSince we were mainly interested in highly conserved regulatory elements, we demanded an average similarity level at least as high as the average exon conservation between the species under comparison.i and j is then s = log/(πiπj)) where P is the probability of finding the pairing of i and j under the above substitution model [The score for aligning two nucleotides on model .O =  with vertices V and edges E is constructed, which represents the species-internal overlap of CNBs on the genomic coordinate level. Each vertex a ∈ V represents a footprint, which is a pairwise local alignment between two species. An undirected edge is placed between two vertices if the corresponding CNBs have only one species in common and show an overlap of at least 10 bp on the sequence level.All CNBs from pairwise sequence alignments are split up into groups as defined by gene homology. For each group a graph O, cliques of minimal size three are detected with an implementation of the Bron-Kerbosh algorithm [In our graph lgorithm . Only thlgorithm  who applN and edges E, which are one-way edges and form no cycles.Partial order graphs belong to the class of directed acyclic graphs (DAGs). A DAG is a graph consisting of a set of nodes The multiple alignment problem is then reduced to to subsequent alignment steps of individual sequences to a growing multiple alignment graph. If the sequences to be aligned share substantial sequence similarity, the number of bifurcation points within the POG stays low and allows rapid computation of the multiple alignment.Alignment results are subsequently trimmed to encompass the leftmost and rightmost ungapped block of at least 6 nucleotides.GENENEST [-4 is applied.Promoter regions in CORG always extend upstream from the most downstream coding start (ATG). As a consequence, promoter regions may contain exons that are not translated. Our way of detecting such exons is a similarity search of man-mouse footprints versus GENENEST , a databGENENEST . An E-vaThe TRANSFAC database  is a repS: one is given by the signal profile F and the other one by the background model B. Under both models the distribution of weight matrix scores can conveniently be calculated by convolution, since the score is a sum of independent random variables. Probability mass distributions of PF (Score(S)) as well as PB(Score(S)) can be computed by dynamic programming if column scores are reasonably discretized. This allows a fine tuning of the proportions of false positives and negatives for each TRANSFAC weight matrix. Both error levels were set to be equal. All details are given in [Potential binding sites are detected with TRANSFAC weight matrices by the method of . Here, tgiven in .We now present an overview of the web interface of the database and several example applications. and offers a redesigned web interface. From the search page one can quickly jump to gene loci via EnsEMBL or other standard identifers . The search query is processed according to the chosen reference source and a list of all matching database entries is returned to the user. This list serves as a springboard to a summary page where the genomic context of the selected gene and its similarities to other upstream regions is visualized as in Figure The CORG database is accessible via its home page Pairwise as well as multiple comparisons are displayed on demand at this stage with a JAVA applet that complies with the JDK 1.1 standard. Alternatively, upstream region sequence and corresponding annotation can be exported in EMBL format . The JAVA applet should run on all JAVA-compatible web browsers. Detailed information about the conserved non-coding block structure are simultaneously shown for multiple upstream regions of different species. If available, annotation information on putative binding sites of transcription factors and EST matches are displayed for the query sequence. The applet facilitates zooming into sequence and annotation. In addition, web links are assigned to sequence features that relate external data sources to the corresponding annotation.DAS, [ constitutes such an external data source. Position information on all conserved non-coding blocks and mapped promoters is accessible from this DAS server. Each DAS sequence feature provides a link to the corresponding CORG database entry. New DAS sources can be easily added to the ENSEMBL display. A small tutorial on installing external DAS data sources is available on our web page .CORG data may be also embedded into other viewers or programs via the distributed annotation system . DAS faAdditionally, tools for on-site batch retrieval of CORG data will be added to the web portal in the near future.One potential application of CORG is phylogenetic profiling of promoter regions. We define phylogenetic profiling in the context of gene regulation as comparative analysis of presence/absence patterns of binding sites in promoter regions. Here, we consider conserved predicted binding sites and contrast them with validated ones.6GG) in target gene promoters and by recruiting different co-factors. SRF regulates transcription downstream of MAPK signaling in association with ternary complex factors (TCFs) . TCFs bFigure We get a better idea on the cause of sequence conservation by browsing the multiple alignment. Textual information can be obtained by clicking on the alignment boxes. Then, the alignment appears in a pop-up window and may be copied to another destination. In Figure L ≥ 50 against the Rfam database [Non-coding RNA can be classified as transcribed regulatory elements. Non-coding RNAs are also accessible to the user via the CORG database. Since we were primarily interested in non-coding RNAs rather than small mRNA motifs we restricted our search here to long CNBs. A blast search of our multiple alignments with length database  and the database  identifiThe snoRNA U93 is an unusual mammalian pseudouridinylation guide RNA which accumulates in Cajal (coiled) bodies and it is predicted to function in pseudouridylation of the U2 spliceosomal snRNA . It appemir10 and the mir196 precursors are located at specific positions in the Hox gene clusters [mir-196 family regulates Hox8 and Hox7 genes, the function of mir10 is unknown.The known microRNAs belong to four different groups. The clusters -7. The mmir10 is a good example of this typical substitution pattern, which gives rise to a hairpin structure. The pairwise correlation structure of nucleotides is depicted on top of the multiple alignment in Figure SLCA1, a member of the sodium transporter family. This time the substitution pattern does not meet the minimal length of the microRNA definition above. Nevertheless, it is conserved across all vertebrate species as shown in Figure For a microRNA we expect a subsequence of about 20 nt that is almost absolutely conserved among vertebrates (the mature miRNA) and a well-conserved complementary sequence forming the other side of the stem from which the mature microRNA is excised. In contrast, the substitution rate should be much larger in the loop region of the hairpin . mir10 iWe have improved and extended our framework of comparative analysis and annotation of vertebrate promoter regions over previous releases (see ). The fo• Mapping of validated promoter regions and proper adjustment of the extent of upstream regions.• Multiple alignments from significant local pair wise alignments.• Novel approach to predict transcription factor binding sites.• Web site offers now a genomic context view .We presented selected application examples from the realm of vertebrate gene regulation. Conserved regulatory elements of different kinds  are readily accessible to CORG users. New genomes and annotation will be continuously added to CORG.. Programs, scripts and MySQL database dumps are available from the authors upon request.The database is freely accessible through the website Christoph Dieterich built the entire pipeline and some parts of the web interface. Steffen Grossmann annotated transcription factor binding sites and provided parts of the web interface. Andrea Tanzer analyzed known and novel RNA elements in the multiple alignments of the CORG database. Stefan Röpcke set up our database of binding site descriptions. Peter F. Arndt worked on an appropriate alignment scoring scheme. Peter F. Stadler and Martin Vingron initiated this work and provided all necessary infrastructure.Distribution of distance between start of transcription and translation. Histogram of observed genomic distances between start sites of transcription and translation in man for 1,700 entries from the EPD. The red and blue line indicates the 90% and 95% quantiles, respectively. Distances greater than 106 bp were exluded from the analysis as they mostly occur due to mismappings in the ENSEMBL database.Click here for file


Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families.GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA).Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene 67 was detected, which segregated with CP in affected individuals.A novel amino acid mis-sense mutation in GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts.This result is interesting because auto-antibodies to GAD Cerebral palsy (CP) is a term used to define a group of disorders  characteDepending on the overall clinical picture, CP can be sub-classified into a number of phenotypic groups ,9. DyskiKuban and Leviton  suggesteGAD1, which codes for the 67 kDa isoform of L-glutamate decarboxylase (GAD EC:4.1.1.15).A non-progressive, autosomal recessive, symmetrical spastic CP locus has been mapped to a 5 cM region between D2S124 and D2S333, at 2q24-31.1 (LOD score of 5.75) in consanguineous families originating from the Mirpur region of Pakistan (OMIM:603513) . AffecteGAD1 and GAD2 , have presumably arisen by duplication and been conserved during evolution, as indicated by their sequence homology [N-termini demonstrate ~23% homology, but their C-termini, which contain the catalytic site, have ~73% amino acid sequence identity between the isoforms [GAD1 in familial spastic CP probands and unaffected family members.GAD requires the cofactor pyridoxal 5'-phosphate (PLP) and catalyses the production of Gamma-aminobutyric acid (GABA) from glutamate . Two sephomology  and the homology . Their Nisoforms . GABA anisoforms . Both isisoforms . GAD actThe oldest affected male diagnosed with non-progressive, spastic CP, demonstrated global developmental delay, with no associated neurological abnormalities and moderate mental retardation. His affected younger sister was also diagnosed with spastic CP, global developmental delay and moderate mental retardation.The oldest non-progressive, spastic CP female has severe mental retardation, and is confined to a wheelchair. The next oldest spastic CP male has severe mental retardation, mild hypertonia and ataxia of the upper limbs. The next oldest spastic CP male is a dizygotic twin born by Caesarean section. This patient on presentation demonstrated developmental delay, mild hypertonia and ataxia of the upper limbs. The youngest affected female is not able to walk or stand unaided and has severe developmental delay. Details of the clinical picture in these pedigrees have been described previously ,14.. Clones were grown up in casamino acid selective broth overnight and harvested by centrifugation. The pellet was then washed twice in 0.5 ml 100 mM Tris-HCl, pH7.5, 0.5 M EDTA buffer. After a second round of centrifugation the pellet was resuspended in molten 1% LMP agarose in 5 mM Tris-HCl, pH7.5, 0.05 M EDTA, 10 mM NaCl with 100 μg of Zymolase. The agarose was cooled and the resultant plugs were incubated overnight at 37°C in 50 ml of 0.5 M EDTA, 10 mM Tris-HCl, pH7.5, 10 mM NaCl. The buffer was replaced with fresh solution to which 100 μl of 40% Sarkosyl NL30 and 50 μl Proteinase K (1 mg/ml) was added and the plugs were incubated overnight at 50°C.ICI and CEPH YAC libraries were screened by PCR amplification of STSs that were mapped between D2S2157 and D2S385. The positive clones  were obtained from the UK HGMP Resource Centre The YAC chromosomal DNA was purified and separated for sizing by CHEF electrophoresis. The switching angle was 120°, using a CHEF™ electrophoresis tank (Bio Rad), run for 16 hours at 6 V/cm, 10°C with an initial pulse time of 30 sec to a final pulse time of 90 sec. DNA was stained with (20 mg/ml) ethidium bromide solution (BDH) for 2 hours and visualized under ultra-violet illumination. Southern blotting and membrane hybridisation of CHEF gels were performed on Hybond-N™ membranes (Amersham). DNA was immobilised by heating the membrane to 80°C in vacuum for 1 hour in a gel dryer (Bio-Rad). Radio-labelled YAC vector-specific probes were generated using the Megaprime random primer kit (Amersham) as described in the manufacturer's instructions, using PCR products as a template. Sizes were estimated based on comparisons with the known sizes of the native yeast chromosomes., , . These primers were used to amplify microsatellite marker alleles from individuals by PCR. Individual alleles were identified by denaturing polyacrylamide gel electrophoresis on an ABI Prism 377 sequencer and analysed using Genescan™ and Genotyper™ (version 1.1.1) software (Applied Biosystems).The microsatellite markers: Cen-D2157, D2S124, D2S2330, CHLC.GATA71B02 (D2S1776), D2S2345, CHLC.GATA71D01, D2S294, AFMA109YC1, D2S376, D2S2284, D2S2177, D2S2194, D2S333, D2S2302, D2S2381, D2S326, AFMA304WB1, D2S138, D2S148, D2S300-Tel, were amplified using fluorescently labelled primers (Lifetech) previously designed by the Whitehead Institute or on the GDB database GAD1 exon sequences were amplified by PCR using the primers described in Table GAD1 were designed from the sequence of BAC RP11-570c16 and obtained from Lifetech  and RP-9 PAC (HGMP Resource Centre) contig for the interval D2S2157 to D2S385 was constructed C , onto our YAC/PAC contig. ESTs were then collated according to their expression profiles to produce a "binned" EST map, on which candidate gene selection could be based. This reduced the number of hypothetical genes in the region and allowed the combination of genetic, physical mapping and expression data, into a single comprehensive map.The Goldenpath Human Genome Working Draft Assembly 2001, is an annotation of the Washington BAC contig, combining sequence data of BACs, ESTs, known genes and hypothetical genes. We mapped ESTs and known, uncharacterised or hypothetical genes on the basis of sequence homology . The 67 kDa (594 AA residues) form [GAD1) [25) produced from an alternatively spliced GAD1 transcript of 2 kb that contains an in-frame stop codon. This GAD1 splice variant has only been found in human islets, testis and adrenal cortex, although the homologue is present in fetal mouse brain [67 and GAD65 consist of two major sequence domains  demonstrate ~23% homology. These N-terminal domains are thought to be responsible for sub-cellular targeting and the formation of GAD65–GAD67 heterodimers [C-terminal domains (AA residues 96–585 in GAD65 and 102–594 in GAD67) contain the catalytic site, with ~73% sequence identity between the isoforms [es) form  is localm [GAD1) . There is Figure . The N-trodimers . The C-tisoforms  , which responds to short-term changes in neuronal activity and is more responsive to levels of PLP [67 predominantly exists bound to the PLP cofactor (holoGAD), providing a constitutive level of GABA production [et al. [25 is expressed in a temporally controlled manner, in the developing striatum and cortex in rodents, suggesting this may provide a mechanism of regulating GABA production in differentiating neurons.In the CNS, GADe neuron . This sue neuron . This iss of PLP . On the oduction . Bond et [et al.  showed tet al. [Gad2-/- mice are slightly more susceptible to seizures, consistent with an excitatory increase in the relative ratio of glutamate/GABA. However, they showed no obvious overall change in neuronal GABA content. Therefore GAD67 alone appears to produce sufficient GABA for effective neurotransmission [Gad1-/- mice demonstrated a decrease of ~20% in total glutamate decarboxylase activity at birth. This was assayed by the conversion of 14C-labelled glutamate to 14CO2 in the presence of PLP. There was also a marked (7%) reduction in total GABA content in cerebral cortex homogenate measured by liquid chromatography [67 in non-neural tissues [Asada et al.  undertoosmission . Gad1-/-tography . Unfortu tissues . It is o tissues ,30.GAD1 mutation was previously suspected of being the cause of PDE. Linkage of pyridoxine-dependent epilepsy has however been reported to 5q31.2-31.3, with GAD1 and GAD2 excluded [Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder OMIM:266100), characterized by generalized seizures during the first hours of life. The associated pathology may result from an alteration in the binding of the co-factor PLP to GAD. Interestingly epilepsy is commonly associated with CP and grand mal epilepsy developed at age six months in the two linked pedigrees described here [0, charac65 and GAD67 have been identified as auto-antigens in "Stiff Person Syndrome" , and in cerebellar ataxia [65 and GAD67 [GADr ataxia -34. GABAnd GAD67 -37. Thisnd GAD67 . Hyperexnd GAD67 .65 auto-antibodies in the CSF of ataxic and SPS patients selectively suppress GABA-mediated transmission in cerebellar Purkinje cells, without affecting glutamate-mediated transmission [A receptors [Anti-GADsmission ,40. Low smission . The exaeceptors . This prCLN3 gene are thought to be responsible for the neurodegenerative disorder Batten disease (OMIM:204200). In cln3-knockout mice autoantibodies to GAD65 have been reported to be associated with brain tissue and result in inhibition of GAD activity [cln3-knockout mice and along with anti-GAD65 autoantibodies in Batten disease patients may contribute to the associated preferential loss of GABAergic neurons.Mutations in the activity . These mDrugs, which potentiate the action of GABA, such as benzodiazepines and baclofen, ameliorate muscle rigidity and spasticity. These GABA agonists are thought to counter disinhibition of the velocity-dependent increase in skeletal muscle during stretch reflexes, observed in spasticity, which is the result of inadequate presynaptic inhibition of the muscle spindles ,35,37. γGAD1 sequence change G(36)C, which segregates with autosomal recessive spastic CP in 4 affected siblings. This nucleotide substitution causes a missense mutation, changing serine (12) to a cysteine in the N-terminal domain. This serine residue is conserved between all mammals (human/mouse/rabbit/pig) for which data are available. The association of GAD67 with membranes requires formation of heteromeric links with GAD65, which are mediated via their N-terminal domains. The N-terminus of GAD65 is palmitoylated and binds to the cellular membrane. The first 27 amino acids appear to be essential in this function [65, and the inability of residues 1–29 of GAD67, to substitute for this region, highlights the potential impact on cellular localisation of a nucleotide substitution in this domain [65 also undergoes phosphorylation of the first four serine residues in the N-terminal domain. These post-translational modifications highlight the importance of the flexibility and accessibility of this domain. N-terminal epitopes of GAD65, in the region corresponding to the residue, which undergoes mutation in GAD67, are particularly prominent autoantigens [We have identified a function . The pals domain . GAD65 aantigens .Gad1 knockout where complete loss of Gad1 enzymatic function  resulted in a cleft palate phenotype and neonatal death [25 splice variant of GAD67, also contains the S(12)C amino acid substitution in affected CP patients. This truncated variant is identical to the first 213 amino acids of GAD67, with the addition of an extra 11 C-terminal residues. It lacks the binding site for the cofactor PLP and is believed to lack any GAD activity [25 is not known, but it may compete with GAD67 for incorporation into protein complexes. Therefore the presence of an N-terminal mutation would affect both GAD25 and GAD67, and may disrupt a complex regulatory mechanism for GAD67.S(12)C amino acid substitution may thus produce subtle effects on cellular localisation, protein-protein interactions and/or protein processing, with a subsequent effect on GABA production. This is not inconsistent with the mouse al death ,30. Ther67 (S12C) and GAD25 (S12C), as recombinant proteins, to assess catalytic activity and binding properties with respect to their normal counterparts. However, it may well prove difficult to detect subtle effects based on sub-cellular localisation changes in the mutant proteins, or changes in post-translational modification patterns.This study illustrates the difficulty of gene cloning in rare autosomal recessive diseases mapped in small, consanguineous pedigrees. Identification of an ancestral haplotype allows refinement of the locus, but this has not been possible in this example. Within the minimal linkage region, any sequence change will segregate with the disease phenotype. Detection of a nonsense mutation leading to a protein truncation would provide compelling support for a mutation as causative. However, in the present example we have not seen such a mutation in the candidate gene so far examined. We are now expressing the variant forms of GADGAD1 (G36C), into the Gad1-/- mouse to see if this can rescue the cleft palate phenotype and reveal a CP-like picture. These experiments will be reported elsewhere. However, the possibility that reduced GAD67 activity may cause CP in the patients studied herein, in a manner reminiscent of that seen in SPS, leads us to report our findings at this stage. The success reported in treating SPS with intravenous immunoglobulin [Stability of the mRNA transcripts from these variants will be assessed by transfection studies in neuronal cells. Eventually, it would be of interest to attempt knock-in experiments with globulin ,46, suggBAC Bacterial artificial chromosomeBLAST Basic local alignment search toolCEPH Centre d'Etude du Polymorphisme HumainCHEF Contour-clamped homogenous electric fieldCNS Central nervous systemCP Cerebral PalsyCSF Cerebrospinal fluidDNA Deoxyribonucleic acidEST Expressed sequence tagGABA Gamma-aminobutyric acidGAD L-Glutamate decarboxylaseGVG Gamma-vinyl-gamma aminobutyric acidHGMP Human genome mapping projectIDDM Type 1 Insulin-Dependent Diabetes MellitusMRI Magnetic resonance imagingNCBI National Centre for Biotechnology InformationPAC Plasmid artificial chromosomePCR Polymerase chain reactionPDE Pyridoxine-dependent epilepsyPFGE Pulse field gel electrophoresisPLP Pyridoxal 5'-phosphatePVL Periventricular leukomalaciaSPG Spastic paraplegiaSPS Stiff Person SyndromeSSCP Single-strand conformational polymorphismSTS Sequence tagged siteYAC Yeast artificial chromosomeThe author(s) declare that they have no competing interests.CNL carried out the molecular genetic studies, sequencing and drafted the manuscript. The YAC mapping work was undertaken by JPL and CNL. AFM, DTB and IMC conceived the study and participated in the design and coordination, they also secured financial sponsorship from the Wellcome Trust and MRC. RA, SM, ERM and CGW recruited and gained consent from the families detailed in this study. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Coevolution between pairs of antagonistic species is generally considered an endless "arms race" between attack and defense traits to counteract the adaptive responses of the other species.When more than two species are involved, diffuse coevolution of hosts and parasitoids could be asymmetric because consumers can choose their prey whereas preys do not choose their predator. This asymmetry may lead to differences in the rate of evolution of the antagonistic species in response to selection. The more long-standing the coevolution of a given pair of antagonistic populations, the higher should be the fitness advantage for the consumer. Therefore, the main prediction of the hypothesis is that the consumer trophic level is more likely to win the coevolution race.We propose testing the asymmetry hypothesis by focusing on the tritrophic system plant/aphid/aphid parasitoid. The analysis of the genetic variability in the virulence of several parasitoid populations and in the defenses of several aphid species or several clones of the same aphid species could be compared. Moreover, the analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation may complement the detection of differences between host and parasitoid trophic specialization.Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots. Thus this hypothesis profoundly changes our understanding of coevolution and may have important implications in terms of pest management. Coevolution is the result of reciprocal selective pressures exerted by interacting species. Many studies have been devoted to the hypothesis of an endless "arms race" between antagonistic species, in which each species develops escalating attack and defense traits to counteract the adaptive responses of the other species. In reference to Lewis Carroll's book "Through the Looking Glass", Van Valen  named thThe RQH was initially developed in the context of multiple species interactions, to account for the constant probability of species extinction. However, as modeling the coevolution of many species involves a number of difficulties, later studies based on the RQH have mostly been limited to interactions between pairs of species. From this restricted situation, two main predictions can be made: first, arms races induce an advantage of rare genotypes of which resistance or virulence is more efficient and thus, frequency-dependent fluctuations of resistance and virulence may be predicted. Second, a geographic view of the coevolutionary process suggests a dynamic mosaic structure, with local and temporary "hot spots" of antagonistic species coevolution .However, for plant-pathogen interactions  and for Nevertheless, the RQH continues to lie at the heart of the debate concerning the coevolution of antagonistic species, probably because of the lack of plausible alternative hypotheses. Although metapopulation structures or time lags between the responses of antagonistic species to selective pressures have been evoked as reasons for the lack of clear experimental evidence in favor of the RQH , the addAdaptive responses to selective pressures exerted by antagonistic organisms are not necessarily the same for the target species and the consumer species. There is a first level of potential evolutionary asymmetry between them because the failure of virulence for a consumer is a delay in fitness acquisition whereas the failure of defense for a prey is the loss of its entire fitness. Such a difference was underlined by Dawkins  noting AExtending the field of interest to interactions and coevolution between more than two species (i.e. diffuse coevolution) may offer new perspectives. Considering the reciprocal selective pressures exerted by many species leads us to take into account the specificity of virulence and resistance. Until now, few works have been devoted to this subject (but see ). HoweveA recent model described the evolution of resistance of one host subjected to the attacks of two types of parasitoids differing in their virulence and specificity (different genotypes of a species or different species) and the evolution of virulence of one parasitoid attacking two types of hosts differing in their resistance and specificity . The resThe asymmetry hypothesis (AH) may lead to differences in the rate of evolution of the antagonistic species in response to selection. For a specialist consumer capable of target choice, chosen targets constitute a more or less "constant environment". On the other hand, for a generalist defender, the diversified, facultative and fluctuating attacks by a set of enemies constitute a "variable environment". The constancy of the environment may lead to the faster adaptation of specialists (mostly consumers under AH) than of generalists (mostly targets under AH) . Thus, uDrosophila species to encapsulate parasitoid eggs. In this case, it can be also noted that the resistance trait is not specific  when virulence traits (reviewed in [The asymmetry hypothesis seems to fit well with numerous data concerning geographic structures published in the literature (reviewed in  but see iewed in ) are divLysiphlebus testaceipes and the aphid Aphis gossypii. To measure this variability, several populations of the parasitoid collected on different clones of the aphid will be confronted to different clones of this aphid. The different clones of the aphid will be confronted to the different parasitoid populations. This system that considers intra specific genetic variability for each trophic level will be used to evaluate the fitness of the consumers and of the targets in the different parasitoid /aphid combinations. An important point is that virulence will be estimated from the success rate of parasitism (i.e. the production of offspring) whereas defense will be estimated through the measure of the aphid fitness, i.e. the number of offspring produced by the aphid whatever the outcome of the parasitism (success or failure). Two key factors of the host fitness will be considered: 1) the host survival rate in case of parasitism failure ; and 2) the number of offspring produced by the host after parasitism: after a parasitoid's sting, the host may produce offspring until mummification in case of parasitoid embryo development or until a variable date in the case of parasitism failure. B- The analysis of the neutral population genetic structure of the parasitoid as a function of the aphid host, the plant host and geographic isolation. The specialization of L. testaceipes on different aphid clones can lead to a neutral genetic differentiation of the parasitoid as a function of the host and the plant because the reproduction of the parasitoid occurs soon after the emergence of the adults from their host. The genetic differentiation between populations of the parasitoid sampled from different aphid clones and from different plant species could be evaluated and compared to a putative effect of isolation by distance.We propose a dedicated test of the hypothesis of the specialization of the parasitoid virulence and the absence of specialization in the defense of the aphids, against the RQH. The study will focus on the tritrophic system plant/aphid/aphid parasitoid. Two complementary experimental approaches could be considered: A- The analysis of the genetic variability of virulence and defense of the parasitoid This test may allow local verification or rejection of the predictions of the AH . When performed several times on animals from diverse geographic origins, this should eventually allow rejection of the classical interpretation of RQH and the host spots theory of coevolution .The consequences of the asymmetry hypothesis are important. Genetic structures induced by the arms race between antagonistic species may be disturbed by asymmetry in coevolution, producing neither rare genotype advantages nor coevolutionary hotspots . HoweverThe AH cannot be applied to every situation. In the case of host-parasitoid associations, the high level of genetic variability in resistance observed within some local host populations ,21 is beAsymmetry may be suspected in cases involving successive trophic levels other than host-parasitoid associations, such as plant-insect or parasitoid-hyperparasitoid combinations, as soon as individuals of the upper trophic level can choose their target. However, random attacks, such as those due to plant pathogens, may favor more generalist traits of virulence, but the specificity of consumers, and therefore asymmetry, may be restored through indirect choices: pathogens transmitted by vectors may use or manipulate the specificity traits of the vector. Large sets of species interactions may thus lead to asymmetric coevolution.Acyrthosiphon pisum is parasitized by the hymenopteran Aphidius ervi and is specialized on different plant species. Hufbauer & Via [A. ervi than is the clover host population. If the association they studied dealt with two aphid and one parasitoid populations, under the AH, virulence specialization rather than resistance level variations can explain the observations: the parasitoid population is specialized on the clover host population, but keeps a partial virulence on the alfalfa aphid biotype. This suggests that short-term parasitoid specialization may be a key factor in biological control efficiency. For instance, consumers introduced to control a pest could rapidly specialize against non-target hosts.The AH may have consequences in terms of pest management. For instance, it is generally thought that variations in parasitism outcome result from a variability of host resistance due to the selection for higher resistance ,22. Howeer & Via  observeder & Via  also obsThe asymmetry hypothesis thus provides food for thought concerning diffuse coevolution and could be applied to domains beyond host-parasitoid coevolution. Similar thoughts may be applicable to the durability and efficiency of plant resistance or immunological responses to diseases transmitted by vectors. Its theoretical implications and its consequences in terms of population management are potentially important and remain unexplored.RQH: Red Queen HypothesisAH: Asymmetry HypothesisBoth authors have been involved in the elaboration of the hypothesis and the evaluation of its consequences, and in the drafting of the paper.


Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset.Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum .To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds  of the major African vector Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes.These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria. Plasmodium parasites in their mosquito vectors [Daphnia-bacterium [In the last few years, exciting advances in the biology and molecular genetics of the development of  vectors ,2 have l vectors , bringin vectors ,5. A cru vectors ,7, givin vectors ,9. Unfor vectors . One of  vectors . Because vectors  and the  vectors , it is c vectors , snail-s vectors , bumble- vectors , Daphniaacterium ), the ouacterium . In othePlasmodium falciparum and Anopheles gambiae. Malaria genotype by mosquito genotype interactions were tested with a standard procedure of quantitative genetics from measurements of the resistance of different genetic backgrounds of the mosquito A. gambiae, a major malaria vector in sub-Saharan Africa, to different isolates of the human malaria parasite P. falciparum. The parasite isolates were obtained from naturally infected children in western Kenya that harboured gametocytes, the infective stage of the parasite. The genetic backgrounds of mosquitoes were 'mosquito families' generated as the F1 offspring of single egg-laying females. Each mosquito family was challenged with each parasite isolate, and all mosquitoes were simultaneously fed on the blood of the gametocyte carriers via membrane-feeding. This basic design was repeated three times throughout three successive experimental blocks that involved different families and isolates, giving a total of 18 mosquito families, 11 parasite isolates, and 62 specific interactions. As in previous studies [Despite its potentially important role for malaria epidemiology and control, such a genetic specificity of host-parasite compatibility between malaria parasites and their insect vectors have never been investigated. This study examines the potential for genotype by genotype interactions in the combination that is most important for the epidemiology of malaria –  studies , the res studies ,19. Thes studies .A. gambiae s.s. caught in the area surrounding Mbita, a small village on the shore of Lake Victoria in Suba District (western Kenya). These mosquitoes had been initially adapted to feed on a Parafilm® membrane, and then maintained in standard insectary conditions using a rabbit as a blood source for routine maintenance. Females of the colony were blood-fed on a rabbit and allowed to lay eggs in individual vials. Immediately after hatching, each larva was individually placed in one well of a 12-well plate with three mL of filtered lake water. They were fed daily on a standard diet of Tetramin® fish food . Adults were kept in an insectary and supplied with a 6% glucose solution and cotton soaked with distilled water. The temperature and humidity in the insectary followed the daily environmental fluctuations. So that mosquito age at pupation did not affect the success of infection, only females that pupated seven days after hatching were used. The wing length of the mosquitoes, measured from the tip (excluding the fringe) to the distal end of the allula with a precision of 0.02 mm, was used as an indication of body size [The mosquitoes used in this study came from a colony that had been established in 2001 from ody size . Where bP. falciparum carriers were recruited from the two- to 10-year old children in the rural area around Mbita, from December 2003 to January 2004. Finger-prick blood samples were collected and thick blood smears were air-dried, stained with 8% Giemsa during 15 minutes, and examined microscopically for the presence of P. falciparum. Children with asexual parasitemia  were immediately treated with sulfadoxine-pyrimethamine according to national guidelines. Asymptomatic gametocyte-positive children were recruited for the study after their parents or guardians had signed an informed consent form. The Kenyan and the United States National Institute of Health ethical review committees approved this recruitment procedure.P. falciparum isolates were collected from gametocyte carriers that had been identified one or two days before, and used to feed the mosquitoes on the same single day . The gametocyte densities were assessed just before blood withdrawal on a blood smear (as described above) by counting against 500 leukocytes, and converted to numbers of parasites per μL by assuming a standard leukocyte count of 8,000/μL. Although gametocyte densities in the venous blood and the peripheral finger-prick blood might differ, potential differences were assumed to be proportional among the isolates. A sample of five mL of venous blood was collected from each gametocyte carrier in a heparinized tube, 400 μL of which were stored at -20°C for further parasite genotyping. So that the importance of human factors such as transmission-blocking immunity [g and the autologous serum was replaced with the same volume of a pool of AB serum from two French blood donors without any malaria exposure . The mixture was used to feed mosquitoes, which had been starved for 12–16 h before blood feeding, with a standard membrane-feeding system [For logistic reasons, the experiment was repeated three times, and within each experimental block infections were done simultaneously. For each of the three blocks, immunity  was reduP. falciparum DNA was extracted from the blood samples using the QIAamp DNA blood kit following the manufacturer's instructions . The isolates were typed using the semi-nested PCR method slightly modified from a previous study [us study  . In this analysis, the square root of the number of oocysts was used, so that the assumptions of the statistical tests  were satisified. As the study was run in three successive experimental blocks, both analyses included the effect of block, and the effects of family, isolate and their interaction. The effect of wing length was also included as a potential confounder . As diffP. falciparum oocysts, and the number of oocysts in infected mosquitoes ranged from one to 97 . The prevalence and the number of oocysts differed among blocks  of the 455 mosquitoes of the study were infected by 51 of theWhile the crude variation among families  was substantial , most of this variation was due to differences among blocks  and the effect of the interaction on the number of oocysts was significant in one of the cases and showed a tendency in the other case .Separate analyses of the data for the two numbers of clones (two or three) ensured that the number of clones contained in each isolate did not confound the interpretation. The effect of the mosquito family by parasite isolate interaction on the likelihood of infection was significant in both cases  and the number of oocysts . In conclusion, the two potential confounders – number of clones per isolate and gametocyte density – had no qualitative influence on the results of the analysis.In addition, differences in gametocyte density between isolates Table  may be ePlasmodium species and strains [Plasmodium parasites [While the specificity of mosquito-malaria interactions at the species level is well documented , the pre strains . The prearasites ,31. The Plasmodium recognition by mosquitoes are still largely unknown, a group of thioester-containing proteins (TEPs) represents a promising family of candidate recognition molecules. One of them, the complement-like protein TEP1, has recently been shown to bind to and mediate the killing of the rodent malaria parasite P. berghei by the mosquito A. gambiae [TEP1 gene are associated to susceptible and refractory strains of A. gambiae [The specificity of host-parasite interactions is often postulated to occur at the level of parasite recognition. While the molecular mechanisms of  gambiae . Moreove gambiae . It is tThe mosquito genotype by parasite genotype interactions shown in this paper may help to understand some puzzling aspects of the epidemiology of malaria. Thus, even in areas with intense transmission, the probability that a mosquito becomes infected is generally low ,32. FurtPlasmodium might be misleading for the development of malaria control strategies. Indeed, most of these laboratory-based studies focus on the response of one mosquito line or colony against a single parasite strain and thus do not represent the genetic diversity of mosquitoes and parasites in natural populations [The epidemiological consequences of mosquito genotype by malaria genotype interactions are perhaps most obvious in the context of malaria control with mosquitoes transformed to be resistant against malaria. The strong genetic specificity of compatibility between parasite isolates and individual insect vectors suggests that most studies on the mechanisms underlying the resistance of mosquitoes against ulations ,13. Howeulations .P. falciparum development, a major component of its vector competence, varies considerably between different combinations of parasite isolates and individual, genetically variable, vectors. Optimal transmission may thus require some specific compatibility between the insect's and the parasite's genotypes. This result has important consequences for the epidemiology of malaria. Overall, it suggests that conclusions from a particular subset of mosquito and malaria genotypes will not necessarily hold for other combinations of genotypes. Therefore, field studies taking into account the full diversity of mosquito and parasite populations are necessary to reach valid conclusions concerning the technologies developed in laboratories for the control of malaria.This study demonstrated that the resistance of an anopheline mosquito to LL participated in the design and the coordination of the study, carried out the fieldwork, participated in the molecular analysis, performed the statistical analysis, and wrote the manuscript. JH participated in the fieldwork. PD carried out the molecular analyses and helped to draft the manuscript. LCG supervised and coordinated the fieldwork. JCK conceived and designed the study, performed the statistical analysis, and wrote the manuscript.


Lung disease in patients with cystic fibrosis is thought to develop as a result of airway inflammation, infection, and obstruction. Pulmonary therapies for cystic fibrosis that reduce airway inflammation include corticosteroids, rhDNase, antibiotics, and high-dose ibuprofen. Despite evidence that high-dose ibuprofen slows the progression of lung disease in patients with cystic fibrosis, many clinicians have chosen not to use this therapy because of concerns regarding potential side effects, especially gastrointestinal bleeding. However, studies have shown a low incidence of gastrointestinal ulceration and bleeding in patients with cystic fibrosis who have been treated with high-dose ibuprofen.The described case illustrates a life-threatening upper gastrointestinal bleed that may have resulted from high-dose ibuprofen therapy in a patient with CF who had undergone a pneumonectomy. Mediastinal shift post-pneumonectomy distorted the patient's esophageal anatomy and may have caused decreased esophageal motility, which led to prolonged contact of the ibuprofen with the esophagus. The concentrated effect of the ibuprofen, as well as its systemic effects, probably contributed to the occurrence of the bleed in this patient.This report demonstrates that gastrointestinal tract anatomical abnormalities or dysmotility may be contraindications for therapy with high-dose ibuprofen in patients with cystic fibrosis. Airway inflammation is thought to cause much of the lung damage in patients with cystic fibrosis (CF) ,2. Such Recent therapies for CF lung disease have been shown to preserve lung function by decreasing airway exposure to inflammation. For example, airway clearance, antibiotics , rhDNase, and oral corticosteroids have been used to help decrease airway inflammation -6. High-Pseudomonas aeruginosa pneumonia. The consolidation was unresponsive to intensive therapy including intravenous (IV) antibiotics, chest physiotherapy, systemic steroids, and rhDNase. The patient developed recurrent severe pulmonary exacerbations necessitating IV antibiotic therapy 4 times per year between the ages of 14 and 15 years. A computerized tomography scan of the chest when the patient was 15-years-old revealed complete collapse of his right lung. A ventilation/perfusion scan revealed only 8% of his ventilation and perfusion in his right chest, while the remaining 92% was in his left chest. In addition, the left lung had begun to herniate into his right chest. It was felt that the right lung was a recurrent source of infection, which was causing serious morbidity. Therefore, the patient underwent a total right pneumonectomy that he tolerated well.The patient was a teenager with CF who suffered for several years from right lower lobe consolidation, as a result of recurrent Approximately one year later, in preparation for initiating high-dose ibuprofen therapy, the patient's stool was checked for occult blood. He was found to have occasional guaiac positive stools. An upper gastrointestinal endoscopy revealed chronic esophagitis, esophageal ulcerations, and Barrett's esophagus thought to be attributable to gastroesophageal reflux. A 24-hour pH probe revealed significant gastroesophageal reflux despite anti-reflux therapy. Therefore, a fundoplication was performed in order to prevent further esophageal damage. Subsequently, it was believed that his esophageal ulcerations resolved as multiple stools were documented to be guaiac negative.High-dose ibuprofen therapy was initiated when the patient was 17-years-old, based on published recommended dosage and pharmacokinetic protocols . The patFollowing this episode the patient's ibuprofen was discontinued, and his oral medications  were switched to liquid form, whenever possible. His pancreatic enzymes were administered after removing the enzyme microspheres from their capsule and mixing them in applesauce.Reported complications of pneumonectomy include mediastinal shift with herniation of the remaining lung, cardiac herniation, cardiac arrhythmias, bronchopleural fistula, esophageal motility disorders, and development of scoliosis -11. It iIn our patient, esophageal dysmotility along with the distortion of esophageal anatomy probably combined to slow the esophageal transit time of the ingested ibuprofen, which may have led to development of ulcerations as a result of prolonged concentrated contact within the esophagus. Also, the ibuprofen could have contributed to the development of ulcerations by inhibiting cyclooxygenase systemically, which decreased prostaglandin E production . In turnDelayed esophageal transit may have occurred with the patient's other oral medications prior initiation of ibuprofen. For example, his pancreatic enzymes likely were retained in the same esophageal segment, which placed him at risk of developing esophageal damage akin to the development of fibrosing colonopathy and strictures described in patients with CF who received high-dose pancreatic enzymes . The enzAccording to the 2002 CF Foundation registry, only 3.8% of patients in North America with CF were treated with high-dose ibuprofen . In 1999The initial randomized, double-blind, placebo control study of high-dose ibuprofen in CF did not demonstrate serious side effects during 4 years of therapy with ibuprofen sufficient to achieve peak plasma concentrations of 50–100 mcg/L . Seven oThe incidence of gastrointestinal disease in patients reported to the CF Foundation registry from 1996–2000, was compared between patients who were and were not taking ibuprofen . She wasThe risk of developing gastrointestinal side-effects from high-dose ibuprofen therapy is low for patients with CF. However, ibuprofen may be contraindicated for those who are at increased risk because of gastroesophageal reflux, history of gastrointestinal ulcerations, or abnormal gastrointestinal motility or anatomy.None declared.JM wrote the case report. RA treated the reported patient, and edited the report.The pre-publication history for this paper can be accessed here:


Neisseria meningitidis is a serious infection which is most common in young children and adolescents. This study investigated the relationships between the incidence and age distribution of meningococcal disease, and socioeconomic environment.Meningitis caused by An ecological design was used, including mapping using a Geographical Information System (GIS) at census ward level.Incidence of meningococcal disease was highest in the most deprived wards, with a relative risk of 1.97 (1.55 – 2.51). Mapping revealed geographical coincidence of deprivation and meningococcal disease, particularly in urban areas. Two-thirds of the increased incidence was due to cases in the under fives.The results suggest that area deprivation is a risk factor for meningococcal disease, and that its effects are seen most in young children. Neisseria meningitidis are still an important cause of morbidity and mortality in the United Kingdom, with 1548 cases notified in England and Wales in 2001[Infections caused by The infection is most common in infants and adolescents, with peaks of incidence at 0–5 years and 15–19 years. OutbreaIt is accepted that many infectious diseases preferentially affect the most disadvantaged in society. This has been discussed at historical, global, and natThis study was prompted by an impression, gained from the practice of a consultant in communicable disease control (CCDC), that infant cases of meningococcal disease came from families of low socioeconomic status, whilst older cases tended to be drawn from families of higher socioeconomic status. Studies in northeast Thames, southweThe study was ecological in design, and used census-derived area data, map data, and individual case data as described below. The geographical unit of analysis was the 1991 census ward, and the study population was the entire Eastern region population of the UK (1999 estimate 5.3 million).The Eastern region of England is made up from the counties of Hertfordshire and Essex (both adjoining Greater London to the south), Suffolk and Norfolk along the east coast, Cambridgeshire centrally and Bedfordshire to the north. Much of the area is rural, although there are several medium-sized urban areas in Essex and Hertfordshire, with links to the capital. Away from the London area, the major urban centres include Cambridge and Norwich (both with large university populations), the ports of Ipswich and Harwich, and Peterborough and Luton.Data on cases came from the Eastern region Communicable Disease Surveillance Centre (CDSC Eastern) regional database of enhanced surveillance of meningococcal disease. To be iThe regional population data were projections for 1999 based on 1991 census data, obtained from the compendium of clinical and health indicators 2001[The deprivation index used was the Townsend score, which combines local measures of unemployment, car ownership, overcrowding, and housing tenure. This meThe shape files used in the MapInfo (© MapInfo Corporation) and EpiMap2000  maps were digitised ward boundaries from the 1991 census, obtained from the EDINA (Edinburgh data and information access) UKBORDERS service. Vector-Ethical approval was obtained from the West Hertfordshire health authority local research ethics committee. An application form was submitted, along with a copy of the project protocol, and written approval was returned.Cases were mapped to census wards using their postcode georeference (NHS postcode database). Microsoft Access was used to assign the deprivation scores, to divide and manipulate ward data, and to link the geographical and attribute files. The age distribution of cases and incidences was calculated using Microsoft Excel, which was also used to create the charts.©StatsDirect Ltd), apart from the relative risk confidence intervals, which were calculated using Microsoft Excel[Statistical testing was performed using StatsDirect  enhanced meningococcal database during 1999 and 2000. Of these, 524 had some postcode details and 499 had the full postcode. 458 cases had a ward assigned, and, of these, 451 had a Townsend score. These data losses were due to postcodes not being recorded (the major factor) or incorrect, discrepancies within the NHS postcode file, and incomplete ward deprivation data . Further analysis is therefore restricted to the 451 cases with Townsend score. Where incidence rates are given, they will generally be underestimates of the true incidence , due to the loss of case data.It was assumed that the data losses were random with respect to the variables of interest. The age structure was well preserved despite the loss of around 40% of the initial cases. Figure Of the 1184 wards included in the analysis, 325 (27%) had at least one case of meningococcal disease in the two-year period. In these wards, the maximum number of cases was 6, and the median 1.The overall incidence for 1999 and 2000 was 7.4 cases per 100,000 per year. Within the wards containing more than one case, the median incidence was 12.9 per 100,000 per year, and ranged from 3.7 to 60.0. Given the small numbers involved, Poisson confidence intervals for these incidences are wide. In a high incidence ward , the confidence interval was 11.4 to 106.8 cases per 100,000 per year. In a low incidence ward , the confidence interval was from 0.1 to 22.5 cases per 100,000 per year. The ward incidences will be underestimates due to the loss of cases described above.Figure Table Poisson regression using population density and Townsend score as predictors revealed population density to be a non-significant contributor to the variation in incidence (p = 0.086). A second model, setting incidence against Townsend score alone, suggested that ward incidence rises by 12% (9 – 16%) for every unit increase in deprivation score.The age-specific incidence rates are shown in figure The Poisson confidence intervals for these incidences are narrower than for individual wards. Two were calculated: 21.3 to 35.1 for an incidence of 27.6 per 100,000 per year, and 0.3 to 4.9 for a lower incidence of 1.7 per 100,000 per year.Figure Figure Figure The results for the regional surveillance data support the theory that meningococcal disease is associated with socioeconomic deprivation. Compared to the Welsh study this stuThe analyses of the age distribution of cases and incidences suggest that there is variation between the deprivation groups. There was a significant difference between the age-specific incidence rates between the groups, and a median age difference of five years between the most and least deprived groups (non-significant). The relative risk of disease in the most deprived wards compared to the least deprived , is greater than that observed in the northeast Thames study  age groups experienced higher age-specific rates in the most deprived group, but the highest case numbers were seen in the under 5 age group. The increased incidence in the under fives in the most deprived group, compared to the least deprived, accounted for 68% of the total difference in age-specific incidence. This suggests that children under five are more vulnerable to meningococcal disease in the most deprived areas. The continuation of a similar pattern when case counts, percentage and age-specific incidence were considered improves the robustness of this conclusion.Several environmental factors might contribute to the increased risk of childhood meningococcal disease in more deprived areas. Childcare arrangements, necessitated by either personal or environmental circumstances, might expose a child in a more deprived area to more potential carriers. Smoking is also known to be more common in people from disadvantaged backgrounds and a number of studies have identified passive smoking as risk factors for both nasopharyngeal carriage and meningococcal disease ,28-30.An area with a relatively greater number of under fives might have a higher overall incidence rate, simply because there are more susceptible individuals. Analysis of the percentage age distribution of the populations in each deprivation group does not reveal any marked variations. There is a slight excess of 20–29 year olds in the most deprived wards compared to the other wards, but not an excess of under fives.Mapping of the incidence rates and deprivation indices by ward definitely added value to the routine surveillance data, emphasising the focal nature of disease, and the relationship of these foci to areas of higher deprivation. Mapping incidence at ward level, rather than pinpointing individual cases as in the Welsh study, both coThis is an ecological study, so the associations shown may not be valid at an individual level. Population density might still be a factor, as the regression is subject to some autocorrelation, the population variable occurring in the incidence (dependent) and population density (predictor) terms. A separate analysis, of urban and rural areas, might show whether this is the case. The paucity of cases in rural areas might make this difficult unless several more years' data were included. The north east Thames study did, howGeographical bias in postcoded data might have contributed toward the results, as the health authority with the lowest rate of postcode inclusion, and therefore the greatest loss of data, includes many of the more affluent parts of the region. The cases supplied by this authority only accounted for 7.4% of all cases and 4.4% of postcoded cases, so this potential bias is unlikely to have had a major effect.Routine childhood immunisation with meningitis C conjugate vaccine started in November 1999, so shouMapping of deprivation indices and meningococcal cases is a useful tool in the analysis of routine surveillance data. Mapping of incidence rates revealed an association between areas of high incidence and areas of higher deprivation by Townsend score. High incidence and deprivation often coincided in urban areas. Mapping of deprivation indices also reveals areas of rural deprivation, such as the coastal band in north Norfolk.Yearly mapping of routine surveillance data can help to target control strategies for meningococcal disease locally. Analytic studies would be helpful in elucidating the mechanisms by which socioeconomic conditions influence the risk of meningococcal disease in the region. Along with the study described here, knowledge gained from such investigations could inform the work on health inequalities, and try to reduce such inequalities through health promotion and community infection control.None declared.CJW obtained and analysed the case and geographical data, performed the statistical analyses and mapping, and wrote the text of the paper. PH suggested the idea for the study and helped develop the methods by which the question was addressed. LW provided the data from the enhanced meningococcal surveillance database and advised on data quality and sources. IL provided expertise in geographical information systems and postcode geography.LW, PH and IL all advised on the design of the study, and the analysis and interpretation of the results. All authors contributed to drafting the paper and have read and approved the final draft.The pre-publication history for this paper can be accessed here:


However, a recent report suggests that E2-BSA does not compete for E2 binding to purified ER in vitro. To resolve this apparent discrepancy, we performed competition studies examining the binding of E2 and E2-BSA to both purified ER preparations and ER within intact cells. To eliminate potential artifacts due to contamination of commercially available E2-BSA preparations with unconjugated E2 , the latter was carefully removed by ultrafiltration.The classic model of estrogen action requires that the estrogen receptor (ER) activates gene expression by binding directly or indirectly to DNA. Recent studies, however, strongly suggest that ER can act through nongenomic signal transduction pathways and may be mediated by a membrane bound form of the ER. Estradiol covalently linked to membrane impermeable BSA . Staining was restricted to the cell membrane when E2-BSA-FITC was incubated with stable transfectants of the murine ERα within ER-negative HeLa cells and with MC7 cells that endogenously produce ERα. This staining appeared highly specific since it was competed by pre-incubation with E2 in a dose dependent manner and with the competitor ICI-182,780.As previously reported, a 10-to 1000-fold molar excess of E2-BSA does bind to purified ER in vitro and to ER in intact cells. It seems likely that the size and structure of E2-BSA requires more energy for it to bind to the ER and consequently binds more slowly than E2. More importantly, these findings demonstrate that in intact cells that express ER, E2-BSA binding is localized to the cell membrane, strongly suggesting a membrane bound form of the ER.These results demonstrate that E The stu2-BSA is commonly composed of approximately 10 molecules of E2 attached to every BSA molecule. An E2-BSA solution equimolar in estradiol to a solution of estradiol alone would contain one-tenth the molarity of E2-BSA with respect to the concentration of estradiol alone. However the rate of E2-BSA binding is dependent upon the concentration of E2-BSA (rate = k [E2-BSA] [ER]). Even if every collision between E2-BSA and ER produced binding as successful as collisions between E2 alone and the ER, an E2-BSA solution equimolar in E2 would have approximately one-tenth the rate.Commercially available E2-BSA to ER compared with free E2. However, once binding has occurred, the stability of the E2 molecule in the ER binding pocket may be only modestly impaired. This may explain how pre-incubation with E2-BSA results in successful binding, whereas immediate addition of E2-BSA does not have sufficient time to establish successfully bound forms. A similar rationale may explain our results and those of other investigators [2-BSA-FITC only to cells that express ERα. These studies typically employ at least a 30-minute incubation time with E2-BSA-FITC, which may be sufficient to result in significant binding. These factors strongly suggest that the rate of binding is an important consideration in experiments assessing potential interactions between E2-BSA and ER.Taken together, these factors reduce the binding efficiency of Etigators  that dem2-BSA activation of non-genomic effects has recently been reported that strongly supports the existence of a membrane ER. Immunocytochemistry using antibodies specific to several domains of the ERα stained only on the membrane of GH3 cells [2 conjugated to peroxidase also bound only to the membrane of pancreatic islet cells and this binding was competed by E2 [2-BSA-FITC was shown to stain only the membrane of ER deficient CHO cells transiently transfected with ERα and ERβ [2-BSA-FITC membrane staining is absent with ER deficient HeLa cells and present only on the membrane of cells that endogenously produce ER or HeLa cells that stably express mERα. Taken together these data strongly suggest that non-genomic effects of E2 are at least partially mediated by a membrane associated ER. However, whether the receptor is the classical nuclear ER translocated to the membrane or an ER unique to the membrane remains unanswered.Although an estrogen receptor has not been directly isolated and characterized from the cell membrane, evidence other than EH3 cells . Membraned by E2 . The mem and ERβ . Moreove and ERβ . Lastly,2-BSA can bind to the estrogen receptor but the rate of binding is impeded due to steric and other considerations. Commercially available forms of the reagent are contaminated with dissociable E2 and should be purified prior to studies designed to demonstrate effects mediated through a membrane ER. Although we demonstrate that classical nuclear ERs can be translocated to the membrane, the conclusive identity of the endogenous membrane receptor awaits purification and sequencing of the putative membrane ER protein.The results presented here suggest that EFull-length cDNA encoding the mouse ERα was cloned into a vector containing the CMV promoter driving the neomycin resistance gene (pcERα). HeLa cells maintained in MEM containing 10% fetal bovine serum under 5% CO2 were transfected with pcERα and successful transfectants (HeLa-ERα) were selected by survival in media containing the neomycin analog, G418 (400 ug/ml).2-BSA  was added to a centrifugal filter unit with a MW cut-off of 3,000 (Millipore) and centrifuged at 14,000 × g until 50 ul of retentate remained. The retentate was washed 3 times with 350 ul of buffer, recovered and volume adjusted to 400 ul.400 ul of E3H-labeled E2  was incubated with recombinant ERα  for four hours at room temperature in binding buffer . The binding of labeled E2 to ERα was competed by various concentrations of ultrafiltered E2-BSA or E2 (10-9 to 3.5 × 10-6 M in E2) added four hours prior to or concurrently with the addition of labeled E2. ERα was precipitated by the addition of a hydroxyapatite slurry (50% v/v in TE) and centrifugation at 10,000 × g. The pellet was washed three times with wash buffer  and 3H-E2 binding determined by liquid scintillation counting.-8 M (in estrogen) E2-BSA conjugated to FITC  or BSA-FITC  for 30 minutes at 4°C. Binding of E2-BSA-FITC to MC7 cells was competed by a 30 minute pre-incubation with E2, ICI-182,780, or E2-BSA . Cells were fixed and FITC staining visualized by confocal microscopy. Images were digitized, inverted to black on white, and pixel density for each cell determined by averaging the density across the cell membrane at four orthogonal points .HeLa-ERα cells or mammary tumor cells  were plated on glass cover slips and incubated with 500 ul of 102-BSA-FITC during the incubation with MC7 cells was assessed by HPLC. E2-BSA-FITC  was added to empty wells and to wells containing MC7 cells prepared as above for 30 minutes at 4°C. 10 ul of supernatant was resolved using a C-18 reverse phase column . A multiple solvent deliver system  provided a methanol-water gradient from 80% methanol to 50% methanol over 30 minutes at a flow rate of one ml/minute. Peaks were detected by a scanning fluorescence detector  at an excitation wavelength of 495 nm and emission wavelength of 519 nm. Area under the curve was calculated using standard algorithms (Millenium Software). Assays were performed in triplicate.The stability of E6 cells) incubated with several concentrations of 3H 17β-estradiol in the presence and absence of a 200-fold excess of cold 17β-estradiol for 30 min at 37°. Cells were then incubated on ice for 15 min, washed three times with 2 ml of ice cold 0.2% BSA-saline and pelleted by centrifugation at 1,5000 rpm for 10 min at 4°C. Cells were lysed by the addition of 100 ul of lysis buffer, vortexed and counted. Data was analyzed by Scatchard analysis.Subconfluent HeLa-ERα or native HeLa cells were trypsinized and aliquots (2 × 10None declared.DB wrote the manuscript and performed binding assays. MK generated the stable cell lines. YT performed binding assays.