At a fundamental level, the continuity of life depends on cell division. Humans generate many millions of cells per second just to stay alive, with most cell types dividing and multiplying repeatedly during a lifetime. Details of cell division vary from cell to cell and organism to organism, but certain features are universal, including what is arguably a cell's most crucial task: the faithful duplication and segregation of its genetic material.During mitosis, a cell copies its nuclear DNA, then splits into two identical daughter cells, a process that involves moving the replicated chromosomes  toward opposite ends of the cell. After chromosomes replicate, a protein complex called cohesin binds the sister chromatids together. Cohesion helps the cell distinguish between the copies, which in turn aids proper distribution. Improper sister chromatid segregation can yield an abnormal number of chromosomes  in the daughter cells, a condition associated with cancer. During meiosis—the cell division that produces egg and sperm cells—aneuploidy causes a number of congenital disorders, including Down's syndrome.To end up in their appropriate positions, sister chromatids must establish attachments to tentacle-like protein polymers called spindle microtubules, which emanate from spindle poles at opposite ends of a cell. Cohesion between the chromatids makes these bipolar attachments possible and keeps sister chromatids from separating after they attach to the spindle. Cohesion occurs along the length of a chromosome and is particularly strong around centromeres, the pinched region of a chromosome. Centromeres, in turn, assemble another protein complex called the kinetochore, which mediates the attachment of chromosomes to spindle microtubules; together, they guide chromosomes to their respective destinations.Saccharomyces cerevisiae, a favorite system for cell biologists. In the first paper, Jennifer Gerton and colleagues generated a map for the entire yeast genome of locations where cohesin binds to chromosomes during meiosis and mitosis. In the second paper, Paul Megee and colleagues found that centromeres attract large concentrations of cohesin to their flanks and that the assembly of these cohesin domains is mediated by centromere–kinetochore complexes.Cohesin's binding locations were discovered by removing chromatin—the mass of DNA and proteins that forms chromosomes—from cells, and purifying the regions associated with cohesin. These studies looked at cohesin's binding distribution either genome-wide or at select regions of a few chromosomes. Here, two research groups use a similar approach to provide a broader picture in their analysis of cohesin binding in the budding yeast Gerton's group reports that large regions surrounding centromeres have “intense” cohesin binding. These binding sites correlate with DNA base composition—DNA is composed of four chemical bases, or nucleotides, that are referred to as A, C, G, and T—showing a strong association with AT-rich regions. In meiotic chromosomes, cohesin binding sites are interspersed between the DNA double-strand breaks that initiate the exchange of genetic information characteristic of meiosis, perhaps keeping the chromatids attached without interfering with genetic recombination.Most striking, the authors note, is the observation that cohesin binding changes according to the cell's gene transcription program. Cohesin prefers DNA that lies between active transcription zones and is unceremoniously displaced from regions where RNA transcripts are being made . This suggests that elongation through a region and cohesion binding may be incompatible. These observations support previous work indicating that DNA sequences required for the replication and segregation of chromosomes must be protected from transcription to function properly. Whatever the explanation, this finding begs the question of how more complicated genomes can accommodate these two seemingly contradictory processes.Megee's group investigated whether all yeast chromosomes have these large centromere-flanking cohesin regions and whether the centromeres and DNA sequences that surround them somehow facilitate the assembly of cohesin complexes. By removing centromeres and generating cells incapable of assembling kinetochores, the researchers show that the assembly of these cohesin regions is mediated solely by the centromere–kinetochore complex.What's more, inserting centromeric DNA sequences in abnormal chromosomal locations produced new cohesin-assembling regions around these “neo” centromeres. The kinetochores' influence appears to stretch over tens of thousands of DNA bases and serves chromatid segregation in two crucial ways: by recruiting high levels of cohesin to centromeres' sides, which attaches chromatids to their bipolar spindles, and by attaching chromatids to microtubules, which provides their passage to the cell's opposite sides. The maintenance of genomic integrity, the authors conclude, likely relies on the coordination of these essential functions.


We first identified groups of coregulated Saccharomyces cerevisiae genes enriched for genes with known upstream or downstream cis-regulatory sequences. Reasoning that many of these gene groups are coregulated in related species as well, we performed similar analyses on orthologs of coregulated S. cerevisiae genes in 13 other ascomycete species. We find that many species-specific gene groups are enriched for the same flanking regulatory sequences as those found in the orthologous gene groups from S. cerevisiae, indicating that those regulatory systems have been conserved in multiple ascomycete species. In addition to these clear cases of regulatory conservation, we find examples of cis-element evolution that suggest multiple modes of regulatory diversification, including alterations in transcription factor-binding specificity, incorporation of new gene targets into an existing regulatory system, and cooption of regulatory systems to control a different set of genes. We investigated one example in greater detail by measuring the in vitro activity of the S. cerevisiae transcription factor Rpn4p and its orthologs from Candida albicans and Neurospora crassa. Our results suggest that the DNA binding specificity of these proteins has coevolved with the sequences found upstream of the Rpn4p target genes and suggest that Rpn4p has a different function in N. crassa.Relatively little is known about the mechanisms through which gene expression regulation evolves. To investigate this, we systematically explored the conservation of regulatory networks in fungi by examining the  A systematic examination of the gene regulatory elements in ascomycete fungi reveals striking conservation along with some examples of the ways in which regulatory systems can evolve The diversity of modern organisms reflects and arises from an underlying molecular diversity that is only beginning to be understood. In recent years, much focus has been given to the evolution of protein coding regions, under the assumption that diversification of protein function has driven the evolution of organismal form and function. Nevertheless, the relative dearth of species-specific genes, and the seeming abundance of functionally homologous proteins in many different genomes, suggest additional mechanisms of diversification. One mechanism likely to play a significant role is variation in gene expression . Subtle cis-regulatory elements in alignments of orthologous regulatory regions, consistently showing that these elements evolve at a slower rate than the nonfunctional DNA that surrounds them  and four unannotated MEME matrices that may represent novel S. cerevisiae regulatory sequences, for a total of 42 S. cerevisiaecis-elements in 35 unique gene groups.We next identified and manually annotated 264 partially redundant groups of genes that are predicted to be coregulated in  element , suggest element . All butS. cerevisiae regulatory elements were shown to be conserved in orthologous regulatory regions from four closely related saccharomyces species  in the 14 fungal species (shown in each column in the figure).  and 27 (64%) were found in the related species S. kluyveri and Kluyveromyces waltii, respectively, and 21 (50%) and 14 (33%) were found in Ashbya gossypii and Candida albicans, respectively. Outside of the hemiascomycete group, we identified three to four (7%–10%) of these elements in the euascomycete fungi and two (5%) in Schizosaccharomyces pombe. Notably, when an identical procedure was performed using randomized consensus sequences, zero sequences were enriched with p < 0.0002 in their respective gene group from any species   . This serevisiae  as well h. pombe , stronglnidulans . The exp factors , as it irevisiae , consistS. cerevisiae upstream elements in the orthologous gene groups from other species, particularly in the more distantly related fungi. One possible explanation for this observation is that, although the genes are still coregulated in these species, thecis-regulatory mechanisms that control their expression have evolved. We therefore searched the upstream regions from each group of orthologous genes for novel sequence motifs, using the program MEME . In the case of the proteasome genes, MEME identified the same motif upstream of orthologous genes from the related euascomycete Histoplasma capsulatum, for which partial genome sequence is available ( http://www.genome.wustl.edu/projects/hcapsulatum/) (unpublished data). That these sequences were identified in the same gene groups from multiple euascomycetes (but not the other species) implies that they are clade-specific. Although future experiments will be required to elucidate the exact roles of these sequences, our observations suggest that the identified cis-sequences are functionally relevant and conserved across species.This procedure identified 19 unique MEME matrices that were not identified in the se fungi . More thS. cerevisiaecis-regulatory elements are restricted to a narrow region upstream of their target genes . The same consensus sequence was also enriched in the orthologous upstream regions of all of the hemiascomycete fungi, but not in the upstream regions retrieved from fungi outside of the hemiascomycete group. We noticed that, in addition to the Rpn4p consensus site, a number of related hexameric sequences were also highly enriched in the orthologous upstream regions from C. albicans (unpublished data). This hinted at the possibility that a slightly different set of regulatory sequences governs the expression of the C. albicans proteasome genes.We were particularly interested in exploring patterns of revisiae . For theS. cerevisiae and C. albicans. To identify these sequences in an unbiased way, we first generated a species-independent “meta-matrix” based on a limited subset of the proteasome upstream regions from both species  contributed to the differences in these sets of sequences. Examination of the nucleotide frequencies in each genome ruled out the possibility that the differences in cis-sequences arose simply by drift in the underlying genomic base composition (unpublished data). To further distinguish between the above models, we cloned and purified S. cerevisiae Rpn4p (Sc_Rpn4p) and the orthologous protein from C. albicans (Ca_Rpn4p) and measured their binding properties in vitro. The interaction of each protein with three different DNA sequences (each representing one of the three classes of upstream sequences described above) was measured using the Biacore 3000 affinity system, which measures biomolecular interactions between proteins and DNA , the C. albicans-specific Sequence B (GAAGGCAAAA), and Sequence C (AGTGGCAACA), which represents sequences found in both species. Sc_ Rpn4p bound preferentially to Sequence A and, to a lesser extent, to Sequence C; however, the binding of Sc_Rpn4p to Sequence B was barely detectable . For example, the proteasome subunits and the ribosomal proteins are among the most highly conserved proteins, and the genes that encode them are expressed with similar patterns in S. cerevisiae, C. albicans, and Sch. pombe  and Swi4-Swi6 cell-cycle box, or SCB (CGCGAAA) regulatory elements are found upstream of G1-phase cell-cycle genes, similar to the E2F element found in these genes in worms, flies, humans, and plants (Sch. pombe, and the other higher eukaryotes. While some of the genes regulated by these elements are well conserved across organisms (namely, the DNA replication proteins), genes whose products are involved in budding are also expressed in G1 phase and regulated by these elements in S. cerevisiae ; however, Sc_Rpn4p could transcribe a reporter gene to higher levels if Sequence A was present in its promoter compared to when Sequence B or a minimal promoter was placed upstream of the reporter gene see . These rThe altered specificity of Sc_Rpn4p is due to amino acid differences in its DNA-binding domain, since the hybrid Rpn4p (containing the Ca_Rpn4p DNA binding domain) bound to Sequence B as well as it did to Sequence C see C. DetermRPN4 in all three euascomycete species contained upstream Rpn4p elements, raising the possibility that this gene is autoregulated at the level of expression in these fungi. Future experiments will test the function of this factor in N. crassa as well as the role of the novel sequence in mediating proteasome gene expression.An extreme example of the previously discussed modes of evolution is the complete alteration of a regulatory system's target genes . This macis-sequences upstream of the proteasome genes from the hemiascomycete and euascomycete fungi. Another example can been seen in the ribosomal protein genes, which must also be expressed to the same relative levels   , As. nidulans  , and Sch. pombe  were identified by tBLASTn and manually added to the list of orthologs for these species.Genome sequence and open reading frame (ORF) annotations for the saccharomycete species were obtained from P. Cliften, M. Kellis, and the Database . Sequenc. waltii , A. gossgossypii , C. albiandida/) , N. crash. pombe . A conseS. cerevisiae and S. paradoxus, S. mikatae, and S. bayanus (Saccharomyces Genome Database ( http://www.yeastgenome.org/) . All other orthologs to S. cerevisiae genes were assigned using the method of Wall et al.  was applied such that at least 20 genes were selected in each group. Transcripts that are bound by RNA binding proteins were taken from  was deemed statistically significant for the consensus sequences, although if the sequence was enriched in the known group of target genes, we relaxed the cutoff to p = 0.01. A cutoff of p ≤ 2 × 10–5 was applied to sequences that matched the MEME matrices. For the Mig1p and GATA binding sequences, which are sufficiently short and occur frequently in each genome, we also scored the enrichment of genes whose upstream region contained two or more examples of the known binding sites.where S. cerevisiae genes or their orthologs in each species, using a two-component mixture model both with and without a motif-width specification of 8 bp. Unless otherwise noted, we used 500 bp upstream (for the hemiascomycetes) or 1,000 bp upstream (for the euascomycetes and Sch. pombe) of the genes in each group. Thus, for each group of coregulated genes, we performed 14 MEME analyses (each identifying three matrices) on the upstream regions of the genes from a given species. Matrices that matched known S. cerevisiae regulatory elements were identified by manual and automated comparisons, similar to that previous described . The score S′ was assigned to each matrix, equal to 0.75× the average S of the motif examples, using the base frequency from each genome as the background model . This score was used as a cutoff to identify genomic examples of the matrix.In this formula, S of each 8-bp sequence within the 1,000 bp upstream region of each gene. The background model was based on the genomic nucleotide frequency in the 50 bp upstream window corresponding to the position of the sequence being assessed. We used this model to overcome the species-specific positional nucleotide biases immediately upstream of coding sequences (A. M. M., A. P. G., D. Y. C., and M. B. E., unpublished data). A sequence was considered a match to the matrix if S > S ′. The enrichment of genes that contained each motif was scored using the hypergeometric distribution, as described above. A p ≤ 1 × 10–5 (0.01 divided by the number of matrices tested in each species) was considered statistically significant. Out of the MEME matrices trained on the non-S. cerevisiae species, 53 were enriched in the gene group in which they were identified. Of these elements, 28 were similar to S. cerevisiae elements shown in S. cerevisiae genes. An additional six matrices were redundantly identified in nearly identical gene groups  from the same species, and two elements were very similar and identified in the same gene group from As. nidulans andM. grisea. Thus, in all, 19 novel elements were identified. The complete list of matrices is available in To identify genes whose upstream regions contained examples of each motif, we calculated the log-likelihood S. cerevisiae position-weight matrices were identified as described above. We then calculated the frequency of each sequence in 50-bp windows upstream of the potential target genes and compared it to the frequency of that element in the corresponding upstream window for all of the genes in that genome. To identify distributions that were statistically different from the background, we identified 50-bp windows that contained a disproportionate number of the cis-sequences in the target upstream regions compared to the background, using the hypergeometric distribution presented above, where i was the total number of elements identified upstream of the genes in each group, M was the number of those elements that fell within a given 50-bp window, l was the total number of elements upstream of all of the genes in that genome and N was the number of those elements that fell within the same 50-bp window. We considered an element's distribution to be significant if there was at least one 50-bp window with p ≤ 0.01; only 5%–10% of the elements had distributions that met this criterion in gene groups other than their putative target genes. We calculated the correlation between element positions in S. cerevisiae and each of the other species by taking all possible pairwise combinations of a cis-element's positions in a given S. cerevisiae upstream region and in the orthologous region from other species and plotting these values for each group of coregulated genes .To compare the upstream sequences identified in proteasome genes fromhttp://rana.lbl.gov) as shown in This set of elements was organized by sequence similarity as follows. Each basepair was represented by a four-dimensional binary vector of indicator variables: G = 1,0,0,0; A = 0,1,0,0; C = 0,0,1,0; T = 0,0,0,1. Each basepair in each 10-mer sequence was replaced by the corresponding vector of indicator variables, translating the 10-mer sequence into a 40-dimensional binary vector. The sequences were organized by hierarchically clustering the binary vectors that represented them, using the program Cluster . The orgS. cerevisiaeRPN4 ORF and its orthologs in C. albicans(orf6.4920) and N. crassa (NCU01640.1) were cloned by PCR from genomic DNA  using Bio-X-act DNA polymerase (BioLine, Boston, Massachusetts, United States). Primers that exactly spanned each ORF (excluding the first ATG) and introduced XmaI and NcoI sites at the 5′ and 3′ ends, respectively, of each PCR product, were used to amplify each ORF. The digested products were cloned into pCAL-n  to add an amino-terminal calmodulin-binding protein tag to each protein. In addition, a hybrid protein was generated from the amino-terminal portion of Sc_ RPN4 (corresponding to nucleotide position 4–1,247) and the DNA binding-domain from C. albicansorf6.4920 (position 1,235–1,611), guided by Clustal W , and the hybrid was cloned into pCAL-n as described above. The wild-type amino acid sequences of Sc_Rpn4p, Ca_Rpn4p, and the hybrid clones were verified by DNA sequencing.  Each plasmid was used to transform BL21DE3-RIL E. coli cells (Stratagene).The lustal W  alignmenSc_RPN4, Ca_RPN4, or Hybrid_RPN from the above plasmids and cloned into the GAL-inducible expression plasmid pRS-TAP (provided by D. Nix) by homologous recombination and gapped plasmid repair. Reporter constructs were generated by cloning 40-bp fragments that contained either one or five copies of Sequence A or Sequence B upstream of the HIS3 minimal promoter in pDC204 (provided by D. Y. C.). Yeast strain BY4741 (MATahis3Δ1 leu2Δ0 met15Δ0 ura3Δ0, provided by M. Kobor) was transformed with each overexpression construct and each reporter construct. Liquid cultures were grown to mid-log phase and washed three times with synthetic-dropout medium lacking histidine and glucose. Serial culture dilutions were spotted onto solid SC medium lacking uracil, leucine, and histidine, with 2% galactose, and containing 0–15 mM 3-amino triazole (Sigma, St. Louis, Missouri, United States). Photos were taken after growth for 3 d at 30 °C.Yeast overexpression plasmids were constructed by PCR amplification of 600 of approximately 1.0. The cells were induced with 0.3 mM IPTG (Sigma) at 30 °C for 1 h, collected by centrifugation at 4 °C, and flash-frozen in liquid nitrogen. The cells were resuspended in ice-cold 8V calcium binding buffer  and lysed on ice by sonication. The lysate was cleared by centrifugation, and the soluble extract was loaded onto 0.5 ml of calmodulin resin (Stratagene) in a 2-ml column  at 4 °C. The column was washed with 8V calcium binding buffer followed by 8V binding buffer adjusted to 0.5 M NaCl. The resin was eluted with elution buffer (50 mM Tris-Cl [pH 7.5], 0.5 M NaCl, and 2 mM EGTA), and the eluates were flash-frozen and stored at –80 °C.The proteins were purified from bacteria by affinity purification. 250 ml of LB medium containing 50 ng/ml carbenicillin (Sigma) was inoculated with 8 ml of saturated cultures and grown at 37 °C to ODC. albicans orf6.8078 gene: Sequence A (GCGTGCCAGATAATCGGTGGCAAAACGGAAGAAAAAGTGA); Sequence B (GCGTGCCAGATAATCGAAGGCAAAACGGAAGAAAAAGTGA); and Sequence C (GCGTGCCAGATAATCAGTGGCAACACGGAAGAAAAAGTGA).  The HPLC-purified oligonucleotides were combined at a ratio of 2:1 unbiotinylated:biotinylated oligonucleotides in 10 mM Tris-Cl (pH 7.4), 1 mM EDTA, and 50 mM NaCl, heated to 95 °C for 10 min, and incubated at room temperature overnight. Each double-stranded, biotinylated sequence was bound to one flow cell of an SA sensor chip (Biacore) in HBS buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% P20) at a flow rate of 10 μl/min. Each cell was coated with roughly the same DNA (approximately 46–56 response units) according to the manufacturer's instructions. The fourth flow cell was not coated with DNA and served as a control. A single cell on a second SA chip was coated in the same way with double-stranded, biotinylated Sequence I (ACTTGTTCCCGCTCGCTGGAGCTCCTCCAACGACACGGGC), representing an instance of the GGAGCT site and flanking sequence from the N. crassa proteasome gene NCU06712.1.The interaction of each purified protein with three predicted Rpn4p binding sites was measured using a Biacore 3000 system (Biacore, Piscataway, New Jersey, United States). Complementary 40-nucleotide oligonucleotides were designed, with one oligonucleotide containing a 5′ biotinylated group . Each of the three sequences contained a different 10-bp core flanked by the same 15 bp that flanked a natural Rpn4p site from the Protein was diluted to 10–100 nM in ice-cold HBS buffer and maintained on ice until injection into the Biacore system. Proteins were passed through four flow cells at a flow rate of 10 μl/min for 90 s at room temperature, then HBS buffer was flowed over the chip at 10 μl/min for 180 s. The protein was desorbed by flowing 0.5% SDS over the chip for 30 s followed by HBS. The kinetics of binding were examined using the Biacore software, and the fit of each calculation was acceptable according to the manufacturer's instructions.GGTGGCAAAACGGAAGAAAA), Sequence E (CCAGATAATCAGTGGCAAAACGGAAGAAAA), and Sequence F (CCAGATAATCGGTGGCAACACGGAAGAAAA); the fourth sequence, Sequence G, (CCAGATAATCCTGCATTTGGCGGAAGAAAA) was chosen as the worst-scoring sequence to the Sc_Rpn4p position-weight matrix and served as a negative control. Each fragment was mixed with 50 nM protein at a 1:1 or 5:1 molar ratio (or buffer was added for mock experiments), incubated for 50 min on ice, then injected into the Biacore system, as described above. The maximum response units of each protein binding to the three sequences on the chip were measured using the Biacore software.Double-stranded competitor DNA was generated by mixing equimolar amounts of complementary 30-nucleotide fragments, heating to 95 °C for 10 min, and allowing the mixture to cool to room temperature overnight. The DNAs were quantified before and after annealing by replicate absorbance measurements. Four different competitor fragments were used: Sequence D (CCAGATAATCDataset S1(2 KB TXT).Click here for additional data file.Dataset S2(339 KB XLS).Click here for additional data file.Dataset S3(22 KB XLS).Click here for additional data file.Dataset S4(47 KB XLS).Click here for additional data file.Dataset S5(90 KB TXT).Click here for additional data file.Dataset S6(64 KB TXT).Click here for additional data file.Dataset S7(69 KB TXT).Click here for additional data file.Dataset S8(53 KB TXT).Click here for additional data file.Dataset S9(74 KB TXT).Click here for additional data file.Dataset S10(70 KB TXT).Click here for additional data file.Dataset S11(63 KB TXT).Click here for additional data file.Dataset S12(64 KB TXT).Click here for additional data file.Dataset S13(54 KB TXT).Click here for additional data file.Dataset S14(433 KB XLS).Click here for additional data file.Dataset S15(433 KB XLS).Click here for additional data file.Dataset S16(433 KB XLS).Click here for additional data file.Dataset S17(433 KB XLS).Click here for additional data file.Dataset S18(433 KB XLS).Click here for additional data file.Dataset S19(433 KB XLS).Click here for additional data file.Dataset S20(416 KB XLS).Click here for additional data file.Dataset S21(359 KB XLS).Click here for additional data file.Dataset S22(411 KB XLS).Click here for additional data file.Dataset S23(414 KB XLS).Click here for additional data file.Dataset S24(411 KB XLS).Click here for additional data file.Dataset S25(411 KB XLS).Click here for additional data file.Dataset S26(416 KB XLS).Click here for additional data file.Dataset S27(398 KB XLS).Click here for additional data file.Dataset S28(406 KB XLS).Click here for additional data file.Dataset S29(405 KB XLS).Click here for additional data file.Dataset S30(404 KB XLS).Click here for additional data file.Dataset S31(404 KB XLS).Click here for additional data file.Dataset S32(389 KB XLS).Click here for additional data file.Dataset S33(399 KB XLS).Click here for additional data file.Dataset S34(410 KB XLS).Click here for additional data file.Dataset S35(383 KB XLS).Click here for additional data file.Dataset S36(384 KB XLS).Click here for additional data file.Dataset S37(381 KB XLS).Click here for additional data file.Dataset S38(405 KB XLS).Click here for additional data file.Dataset S39(378 KB XLS).Click here for additional data file.Dataset S40(403 KB XLS).Click here for additional data file.Dataset S41(408 KB XLS).Click here for additional data file.Dataset S42(402 KB XLS).Click here for additional data file.Dataset S43(382 KB XLS).Click here for additional data file.Dataset S44(399 KB XLS).Click here for additional data file.Dataset S45(372 KB XLS).Click here for additional data file.Dataset S46(379 KB XLS).Click here for additional data file.Dataset S47(42 KB TXT).Click here for additional data file.Dataset S48(11 KB TXT).Click here for additional data file.Dataset S49(19 KB XLS).Click here for additional data file.Figure S1p < 2 × 10–4 in the denoted gene group from any species.Consensus sequences identified by enrichment in (1.1 MB TIF).Click here for additional data file.Figure S2p < 2 × 10–4 in any gene group. However, the enrichment was not consistent across species. Only two randomized sequences were enriched in the same gene group from two species, although the enrichment pattern did not correlate with the species tree. Thus, randomized sequences are enriched with different characteristics than the functional consensus sequences shown in Fifteen of the randomized sequences shown in (898 KB TIF).Click here for additional data file.Figure S3cis-regulatory elements in other species requires identification of orthologs of the coregulated S. cerevisiae genes. We wondered how our enrichment-based method would be affected if incorrect orthologs were assigned to individual S. cerevisiae genes, thereby producing “noise” in the gene groups. To test the sensitivity of our method to this type of noise, we performed the following gene replacement control: For each group of S. cerevisiae genes, we performed 100 trials in which 0%–100% of the genes in each group were randomly selected and replaced with random S. cerevisiae genes. The number of trials in which the p of enrichment was below our cutoff of p < 2 × 10–4 was scored with an orange box, according to the key shown at the bottom of the figure. Nearly all of the cis-elements could be identified in their respective gene groups despite some amount of “noise” in the gene group.Our ability to detect conserved (864 KB TIF).Click here for additional data file.Figure S4S. cerevisiae proteasome gene (x axis) were plotted against the positions of Rpn4p elements upstream of the orthologous proteasome gene from each of the other species (y axis). The linear fit is shown in the upper right corner of each plot.Positions of Rpn4p elements upstream of each (685 KB TIF).Click here for additional data file.Figure S5S. cerevisiae G1-phase genes (x axis) were plotted against the positions of MCB elements upstream of the orthologous G1-phase gene from each of the other species (y axis). The linear fit is shown in the upper right corner of each plot.Positions of MCB elements upstream of (767 KB TIF).Click here for additional data file.Figure S6S. cerevisiae or C. albicans proteasome genes that matched the species-independent meta-matrix were identified as described.Sequences within 500 bp upstream of the cis-sequences from S. cerevisiae (top) or from C. albicans (bottom). The height of each letter represents the frequency of that base in that position of the matrix. Positions in the matrices that are statistically different (see (A) The identified sequences were used to generate sequence logos  to repreS. cerevisiae proteasome genes (shown in red) and C. albicans proteasome genes (shown in blue) were pooled and organized by a hierarchical clustering method, as described in S. cerevisiae genes only (red bar), C. albicans genes only (blue bar), or both the S. cerevisiae and C. albicans proteasome genes (black bar) are indicated, along with the consensus sequence representing each denoted group.(B) Examples of the species-independent meta-matrix found upstream of (1.1 KB TIF).Click here for additional data file.Figure S7S. cerevisiae cells harboring a HIS3 reporter gene with either a minimal promoter (left), minimal promoter + Sequence A (middle), or minimal promoter + Sequence B (right), and overexpressing Sc_Rpn4p from a galactose-inducible promoter, were grown on 0 mM, 1 mM, 5 mM, or 15 mM His3p inhibitor 3-amino-triazole. Two serial dilutions of each strain were plated for each drug concentration. The level of drug resistance is indicative of the level of HIS3 expression (pression .(631 KB TIF).Click here for additional data file.


Nearly a dozen varieties of interdependent cells work in harmony to protect the body from infectious pathogens. Dendritic cells and B-cells carry remnants of pathogens to nearby helper T-cells , which coordinate a threat response by signaling killer T-cells to destroy the intruder. Yet another class of cells cleans up the debris such an encounter inevitably creates. Each of these cell types is further classified based on the combinations of surface proteins they express. Such proteins may simply identify the cell type or may help define its function.An immune response can activate millions of cells; once these cells perform their respective jobs, it's crucial that they retire from active duty. That's where regulatory T-cells, or Treg cells, come in: an immune system that fails to retreat from an immune response can be just as dangerous as one that fails to organize.+CD25+ T-cells) and that suppress T-cell activation in mice and humans. Treg cells appear to be key immune system regulators, as their absence results in autoimmune and allergic diseases. Now a team led by Derya Unutmaz at Vanderbilt University report that human Treg cells are highly vulnerable to HIV infection.Research has recently focused on a subset of Treg cells that express CD4 and CD25 proteins . FOXP3 is required for the development of Treg cells in mice. Though it's not clear what role the human form of the gene plays in human Treg cell development, FOXP3 mutations cause an autoimmune disease associated with hyperactive T-cells. Here the authors demonstrate that FoxP3 transforms T-cells into Treg cells. The FoxP3-engineered T-cells behaved just like naturally occurring Treg cells: when exposed to a population of resting naïve CD4 T-cells, the engineered cells suppressed their activation. Overexpression of FoxP3 also made activated T-cells more susceptible to infection.Treg cells isolated from healthy volunteers were not only highly susceptible to HIV infection but were also killed by the virus. Because Treg cells account for only about 1% of human T-cells and are difficult to grow in a test tube, Unutmaz and colleagues developed a way to manufacture sufficient quantities for study by introducing a transcription factor called FoxP3-expressing CD4+CD25+ T-cells—a marker for Treg cells. The finding that HIV targets cells that normally suppress immune function is significant, given that HIV infection is characterized by chronic T-cell hyperactivation. Disruption of Treg cells, Unutmaz and colleagues conclude, could in turn disrupt the delicate balance of immune system function, setting the stage for hyperactivation—and a chronically hyperactive immune response could eventually exhaust the immune system. With a method to generate large numbers of Treg cells for study, Unutmaz and colleagues have paved the way for identifying mechanisms that mediate Treg cells' suppressive function and provided another resource for determining how HIV tips the scales toward disease progression.Since Treg cells are so susceptible to HIV, the researchers reasoned that these cells might be compromised in HIV-infected patients and that loss of Treg cells could lead to T-cell hyperactivation. Their logic was borne out by the finding that a portion of HIV patients with low CD4 counts and high levels of activated T-cells also had greatly depleted numbers of


Flavonoids, one of the major groups of secondary metabolites, play important roles in the physiology, ecology and defence of plants. Their wide range of activities is the result of their structural diversity that encompasses a variety of functional group substitutions including hydroxylations. The aromatic hydroxylation at position 6 of flavonols is of particular interest, since it is catalyzed by a 2-oxoglutarate-dependent dioxygenase (ODD), rather than a cytochrome P450-dependent monooxygenase. ODDs catalyze a variety of enzymatic reactions implicated in secondary metabolite biosynthesis.Chrysosplenium americanum using internal peptide sequence information obtained from the native plant protein. This novel clone was functionally expressed in both prokaryotic and eukaryotic expression systems and exhibited ODD activity. The cofactor and cosubstrate requirements of the recombinant proteins are typical for ODDs, and the recombinant enzymes utilize 3,7,4'-trimethylquercetin as the preferred substrate. The genomic region encoding this enzyme possesses two introns at conserved locations for this class of enzymes and is present as a single copy in the C. americanum genome.A cDNA fragment encoding an ODD involved in the 6-hydroxylation of partially methylated flavonols, flavonol 6-hydroxylase (F6H), was isolated and characterized from Recombinant F6H has been functionally expressed and characterized at the molecular level. The results demonstrate that its cofactor dependence, physicochemical characteristics and substrate preference compare well with the native enzyme. The N-terminal region of this protein is believed to play a significant role in catalysis and may explain the difference in the position specificity of the 6-hydroxylation reaction. Chrysosplenium americanum (Saxifragaceae), a semi-aquatic weed, accumulates a variety of tetra- and penta methylated flavonol glucosides substituted at various positions of the flavonol ring [O-methyltransferases and two distinct O-glucosyltransferases in a highly ordained sequence  constitute a class of non-heme, iron-containing cytosolic enzymes that utilize an oxoacid as a cosubstrate and a reducing agent for the reactive iron moiety, typically ascorbate. This widespread class of enzymes has been implicated in a variety of plant metabolic pathways, including the biosynthesis of some amino acids, hormones, signalling molecules and a variety of secondary metabolites [.11.- conC. americanum cDNA library and the isolation of a F6H clone. The identity of this clone was confirmed after identifying internal peptide sequences in the translated amino acid sequence of the isolated cDNA clone, as well as by functional expression of F6H in both prokaryotic and eukaryotic systems. The molecular characterization of this gene, with respect to its genomic organization and phylogenetic relationship to other ODDs involved in plant secondary metabolism, contributes to the growing pool of information on this class of enzymes.Using the peptide sequence information obtained from the purified plant protein , degenerC. americanum bacteriophage expression library [cF6H, that contained the 3'-end of this putative cDNA clone, including an in-frame stop codon (TAA), a putative polyadenylation signal (ATATAA) and a short polyA tail (Accession # AY605048), although it lacked the translation start site. The same cDNA library was also screened with an oligonucleotide probe derived from the F6H·1 sequence. Putatively positive clones were isolated and characterized, but none yielded a full-length F6H cDNA clone, although several clones were isolated with homology to related sequences, particularly F3H and aminocyclopropanecarboxylate oxidase (ACCO). Further attempts to isolate a complete F6H ORF were unsuccessful, including the use of inverse PCR and the GenomeWalker technique (ClonTech).Several peptides were obtained from tryptic digestion of the native protein, one in particular, Micro1 – DNGWILLHIGDSNGHR, exhibited significant similarity to other known flavonoid ODDs such as the flavanone 3-hydroxylase (F3H) , flavono library . The PCRcF6H, translates into a 376 amino acid-long sequence with a predicted molecular mass of 40.9 kDa and a pI of 5.1. The fact that the native protein exhibits a molecular mass of 43 to 45 kDa following gel filtration or SDS-PAGE analysis respectively [cF6H sequence. The amino acid residues involved in iron (His222, His285, Asp224) and α-KG-binding (Arg295, Ser297), as well as other conserved residues are present within the cF6H sequence  fusion protein were assessed over a 4-h period and recombinant F6H enzyme activity was assayed. The level of expression of the fusion protein was monitored by Western bolt analysis of cell lysates using anti-(His)n Figure . This con Figure . The lev6-tag, the fusion protein was cleaved by incubation with enterokinase (EK). The enzyme activity of the cleaved protein was significantly reduced compared to the Ni-NTA-purified rF6Hb Hb Table . The loscF6H sequence possesses two potential glycosylation sites at Ser139 and Ser305 that may contribute to catalytic activity, perhaps by stabilizing a favorable tertiary conformation or through the modulation of interactions with other proteins. The effects of such post-translational modifications and protein folding were taken into account by expression of the recombinant protein in the eukaryotic expression system Pichia pastoris. This system is relatively easy to manipulate and exhibits many of the advantages of eukaryotic expression, including protein processing, folding and modification. Recombinant protein expression was both intracellular (rF6Hy) and secreted (rF6Hys). The chosen culture media (BMGY/BMMY) contained protein stabilizing factors, such as peptone and yeast extract, shown to reduce proteolysis, particularly of secreted proteins [6 antibodies, was maximal at 3 to 4 days post-induction in both intracellular and secreted systems  indicates the presence of a nonpolar region at the very end of the protein sequence, which may participate in dimerization or aggregation processes.In both prokaryote and eukaryote expression systems rF6H was soluble, exhibited similar physicochemical properties to the native protein and was no more susceptible to proteolytic degradation. Interestingly, as with the native plant protein , the reco Figure . In the The substrate specificity of recombinant F6H was tested using the Ni-NTA-purified enzyme with different flavonol substrates Table . The resCitrus unshiu bifunctional dioxygenase [Chrysosplenium F6H exhibits a specificity for partially methylated flavonoids involved in polymethylated flavonol biosynthesis characteristic of the species.Given the fact that neither of the recombinant proteins utilized naringenin as a substrate, it seems unlikely that F6H catalyzes a side reaction of F3H or a bifunctional activity as F3H-FLS, as has been demonstrated with the xygenase . Therefoxygenase . Taken tKm values of rF6Hb for the flavonol substrate (63 μM) suggests that the binding affinity has been reduced 17-fold compared to the native protein (0.27 μM) [The cofactor requirements for the bacterially expressed recombinant protein were similar to those of the native plant protein . Ferrousbstrate 6 μM suggeThe hydroxylation of an aromatic carbon may be the result of substrate positioning in relation to the oxidizing moiety within the active site. Mehn and colleagues  in attemC. americanum DNA was used as a template for amplification reactions with primers designed to the outermost regions of the cF6H sequence. This produced a fragment of ~3.1 kbp (gF6H) that contained two potential intronic sequences , indicating the existence of related sequences within the genome.Southern analysis of genomic DNA, probed with the F6H is present as a single copy in the C. americanum genome. The deduced amino acid sequence of cF6H exhibits homology to other ODDs, particularly at the C-terminal, and this conservation can be extended to the organization of the gene as a whole. The fact that gF6H possesses two introns at conserved locations for ODDs [Medicago truncatula [O-methylation at this position by a flavonol 6-O-methyltransferase (F6OMT). Immunolocalization of both enzymes, F6H and F6OMT, should provide further evidence in support of this view.The ORFs of the clones isolated using different approaches were identical in sequence, indicating that they represent the same protein. This is in agreement with the results obtained from Southern analysis suggesting that for ODDs , suggestuncatula . It has uncatula . It is lcF6H from C. americanum exhibits highest similarity, at the amino acid level, to various F3H homologs. Phylogenetically, it is evident that homologous genes encoding biochemically related proteins in single pathways are clustered into related subgroups , was collected from St. Anicet, Québec, and was maintained in the greenhouse under conditions simulating its natural habitat regarding light intensity, temperature and humidity.Flavonol substrates were from our laboratory collection. All other chemicals were of analytical reagent grade. Buffers: Assay buffer, 50 mM Tris-HCl (pH 7.3), 10 mM DTT, 150 mM NaCl, 10% glycerol (v/v); Wash solution I, 2 × SSC, 0.1% SDS (w/v); Wash solution II, 0.1 × SSC, 0.1% SDS (w/v); Blocking solution, 5% SDS (w/v), 125 mM NaCl, 25 mM sodium phosphate (pH 7.2); Blot washing solution, 0.5% SDS (w/v), 12.5 mM NaCl, 2.5 mM sodium phosphate (pH 7.2). FPLC buffers were filtered, degassed and stored at 4°C.The ligand-affinity purified F6H protein  was subjC. americanum cDNA Lambda UniZap XR library was screened by PCR as described in [6 pfu) per reaction. The following degenerate oligonucleotide primers were derived from two internal peptide sequences obtained from the native protein, Micro1 and Micro2;A ribed in  with 5 μMicro1For-5'-GCTGGATCCTCCTTCATATA-3';Micro1Rev-5'-(GC)GATAC(AT)GG(TC)AA(TC)(CG)TTAG(ATGG(CT)TATAC-3';Micro2For-5'-AATTGTTGAAGCATGTGAAG-3';Micro2Rev-5'-(TC)GGGGTTAG(AG)AG(TC)GT(AC)CG(AG)AA-3';T3 + 6 - 5'-GCT CGA AAT TAA CCC TCA CTA AAG GG-3'T7 + 6 - 5'-GAA TGG TAA TAC GAC TCA CTA TAG GGC G-3'cF6H. Given the lack of fresh C. americanum tissue and its disappearance from its natural habitat, for use in 5'-rapid amplification of cDNA ends (RACE) experiments or primer extension, genomic DNA-based techniques such as inverse PCR and GenomeWalker (ClonTech) were employed in attempts to isolate the remaining 5'-end of the putative F6H sequence. Nevertheless, neither of these approached proved successful.Primer combinations: i) Micro1Rev and Micro2For; ii) Micro1For and T3; iii) Micro1Rev and T7; iv) Micro2For and T3; v) Micro2Rev and T7. PCR products were subjected to DNA sequencing for characterization after cloning into the pGEM-T vector (Promega). DNA sequencing was carried out using the dideoxy-mediated chain termination method . FragmencF6H, C. americanum genomic DNA (0.1 to 1.0 μg) was employed as a template for PCR using primers designed to the ends of the cF6H sequence. Putatively positive fragments were subsequently cloned into the pGEM-T vector for DNA sequencing.To isolate the genomic region coding for the F6H, genomic DNA (15 μg) was digested overnight and subjected to Southern analysis according to [cF6H, was labeled using the BioPrime Kit (Amersham) designed for the preparation of biotinylated nucleotide probes for blotting with the IRDye800-conjugated Streptavidin  as the detection system. The membrane was pre-hybridized using UltraHyb-OS (Ambion) at 42°C for 2–3 h. Hybridization was carried out with denatured, biotinylated probe solubilized in a fresh aliquot of UltraHyb-OS overnight at 42°C. The membrane was washed according to manufacturer's instructions using wash solutions I and II. The membrane was blocked with blocking solution for one hour at room temperature, prior to probing with IRDye800-labelled streptavidin (1:10,000) in blocking solution for 1 h at room temperature. The membrane was washed with blot washing solution three times, 15 min each, prior to detection.To determine the copy number of rding to . The procF6H was cloned into an E. coli expression system  The fragment was amplified by PCR using gene-specific primers, containing BamHI and HindIII recognition sites, respectively. The DNA insert is positioned downstream and in-frame with a sequence encoding the (His)6-tag and an enterokinase cleavage recognition site. The pTrc-His-F6H construct was transformed into Top 10 cells by heat shock, and selected on ampicillin (50 μg/mL) containing media. Recombinant protein production in E. coli strain Top 10 was induced by the addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 4 h at 37°C. After sonication of the cell pellet and centrifugation, the supernatant was assayed for F6H enzyme activity or purified on Ni-NTA resin (Qiagen). The affinity-purified protein was eluted in presence of 250 mM imidazole and subjected to buffer exchange on a PD-10 column against the assay buffer. Enzyme assays and analysis of reaction products were carried out as previously described [The cDNA fragment encoding the near-full length F6H, Pichia pastoris expression system  can be either intracellular (pPicZ) or secreted (pPicZα), depending on the presence of an in-frame signal peptide. The cF6H fragment was amplified by PCR using gene-specific primers, containing KpnI and SacII recognition sites, respectively. For the pPicZ construct, the initiation ATG was part of the yeast consensus sequence (AATAATGTCT) included in the 5' gene-specific cloning primer. For the pPicZα construct, the insert was cloned in-frame with the N-terminal signal sequence and C-terminal His-tag. The pPicZ-cF6H and pPicZα-cF6H constructs were transformed into Pichia cells by electroporation and putative multi-copy recombinants were selected on Zeocin-containing media according to manufacturer's instructions (Invitrogen).Heterologous expression in the Recombinant F6H production was induced by the addition of 0.5% methanol (in the absence of glucose) every 24 h for 3 to 4 days. A buffered culture medium  was used for cell growth and protein induction in order to enhance protein stability and limit enzyme inactivation. In the case of intracellular recombinant protein production, cells were lysed using glass beads and extracts were prepared according to manufacturer's instructions (Invitrogen). Secreted proteins were collected and concentrated by ammonium sulfate precipitation (35 to 70% saturation), and used for activity assays after desalting or for subsequent affinity purification.Recombinant proteins obtained from both prokaryotic and eukaryotic expression systems were subjected to SDS-PAGE  and WestFifteen ODD genes involved in secondary metabolism where biochemical information was available, belonging to different plant species, were selected from the PubMed and GenBank searches. The amino acid sequences were aligned using CLUSTAL-W  and PHYLcF6H F6H cDNAF6H flavonol 6-hydroxylasegF6H genomic F6HODD 2-oxoglutarate-dependent dioxygenaseα-KG α-ketoglutarateORF open reading framerF6H recombinant F6HE. coli)rF6Hb recombinant F6H expressed in bacteria (P. pastoris) – intracellularrF6Hy recombinant F6H expressed in yeast (P. pastoris) – secretedrF6Hys recombinant F6H expressed in yeast (TMQ 3,7,4'-trimethylquercetinTMQg 3,7,4'-trimethylquercetagetinDA carried out experiments in the project, participated in the design and coordination of the project, as well as the writing of the manuscript. RKI conceived of the project and contributed to the writing of the manuscript. All authors read and approved the final manuscript.


Ritter's method is a tool used to optimize AV delay in DDD pacemaker patients with normal left ventricular function only. The goal of our study was to evaluate Ritter's method in AV delay-interval optimization in patients with reduced left ventricular function.Patients with implanted DDD pacemakers and AVB III° were assigned to one of two groups according to ejection fraction (EF): Group 1 (EF > 35%) and Group 2 (EF < 35%). AV delay optimization was performed by means of radionuclide ventriculography (RNV) and application of Ritter's method.For each of the patients examined, we succeeded in defining an optimal AV interval by means of both RNV and Ritter's method. The optimal AV delay determined by RNV correlated well with the delay found by Ritter's method, especially among those patients with reduced EF. The intra-class correlation coefficient was 0.8965 in Group 1 and 0.9228 in Group 2. The optimal AV interval in Group 1 was 190 ± 28.5 ms, and 180 ± 35 ms in Group 2.Ritter's method is also effective for optimization of AV intervals among patients with reduced left ventricular function (EF < 35%). The results obtained by RNV correlate well with those from Ritter's method. Individual programming of the AV interval is fundamentally essential in all cases. Cannon waves may be induced by programming excessively short AV intervals, and diastolic mitral regurgitation may occur with excessively long programmed AV intervals. The AV interval is considered optimal . Patient heart rate remained constant during the measurement period at programmed pacemaker lower rate (60 – 70 beats/min).We performed radionuclide ventriculography (RNV) after in vivo marking of erythrocytes with tin DTPA and 10 MBq/kg KG Tc-99m, using a single-head Gamma camera  with a high-resolution, medium-energy collimator. For RNV we applied the equilibrium technique at 16 frames per cycle with patients at rest, and ventricular pacing at programmed AV intervals. We acquired a minimum of one million counts per image, and stored the data in a 64 × 64 matrix.We calculated left ventricular ejection fraction (LVEF) semi-automatically after spatial and temporal smoothing and background subtraction. After Fourier analysis of ventricular stimulation progression, we recorded (with examiner definition) a region of interest (ROI) around the end-diastolic contour of the left ventricle and calculated the LVEF as follows:By 1994 a method developed by Ritter et al. had become established for optimizing the AV interval [Ritter's method employs the following formula for calculation of the optimal AV interval:AVopt = AVlong - (a - b)We applied the following procedure in application of this formula see Fig. :The first step involves programming for the pacemaker a nonphysiologically short AV interval, followed by determination of "a". This value "a" is the temporal interval between the ventricular contraction spike and the end of the A wave. "a" designates the electromechanical delay between right ventricular stimulation and the beginning of the left ventricular systole .AVlong), followed by determination of "b". This value "b" is the temporal interval between the ventricular contraction spike and the end of the A wave. AVlong - b defines the duration of the undisturbed maximal diastolic left ventricular filling.The next step is programming for the pacemaker a long AV interval  as well as for reduced left ventricular EF (intra-class coefficient of 0.9228).Since Ritter's initial publication in 1994, AV interval optimization on the basis of the mitral valve inflow profile has been reported in one additional study . This stIn comparison to time-consuming and expensive RNV, and AV-interval optimization by Swan-Ganz catheterization (with the associated risks of an invasive procedure), Ritter's method offers the following advantages: it is non-invasive and can be quickly performed (approx. 5 min.). It does not require long years of echo experience, and it is cost-effective. Even with patients not readily amenable to sonographic detection, the mitral valve inflow profile is almost always qualitatively satisfactory enough to allow application of Ritter's method. The only noteworthy disadvantage of this method is the necessity for continuous ventricular stimulation: which means that it can be used only for patients with a complete AV block. Patients with only intermittent high-grade AV blocks are accordingly not suited for Ritter's method.opt = 88 ms ± 35 ms with atrial triggering, and AVopt = 143 ms ± 41 ms for the AV sequential mode. Knorre [opt = 100.5 ± 27.8 ms for atrial triggering, and AVopt = 169 ± 24.5 ms for the AV sequential mode. Haskel [opt = 144 ± 48 ms with atrial triggering, and AVopt = 176 ± 44 ms for the AV sequential mode. Ishikawa [opt = 161 ± 26 ms.In our patients, the mean optimal AV interval in Group 1 EF > 35%) was 190 ± 28.5 ms. In comparison, the optimal AV interval among the patients with chronic heart failure in Group 2 was 180 ± 35 ms. Data in the literature are not consistent on the duration of the optimal AV delay. Kindermann  consider% was 190Ishikawa  has deteOur results on the length of the optimal AV delay lie within the range found in the literature. The variance in data observed in some cases emphasizes the highly individual nature of the optimal AV delay: indeed, it results from the interatrial conduction period specific to each patient, and the potential delays induced by pacing versus intrinsic depolarization and conduction in a given patient -4.As a result, the mean optimal AV intervals determined by us cannot be applied to other patient cohorts with the same basic disease. Individual programming of the AV interval is therefore necessary.In summary, our findings confirm that Ritter's method can be reliably applied for patients with normal and with reduced left ventricular pump function. The only prerequisite is a continuous ventricular stimulation.


Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i.Differentiating 2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced.We analyzed Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance.We conclude that there is a link between the second messengers cAMP and Ca Dictyostelium discoideum amoebae form a multicellular organism by chemotactic aggregation. The signaling molecule that mediates aggregation and development is cAMP. Aggregation proceeds in a rhythmic fashion; cAMP is secreted periodically by cells in the center of the aggregate. Cells in the neighbourhood respond by an oriented inward movement and secrete cAMP themselves to relay the signal. In cell suspensions periodic synthesis and release of cAMP leads to rhythmic shape changes that cause alterations in light transmittance and spike-shaped and sinusoidal light scattering oscillations [2+, K+ or H+ (for review see [2+]i were postulated to comprise the (or at least a part of the) master oscillator controlling oscillation patterns [2+]i-transient induced by addition of CaCl2 or calmodulin antagonists alters light scattering oscillations and can even reset the oscillation phase [2+]i-increase determines whether light scattering and the accompanying cAMP oscillations are abolished or augmented: large [Ca2+]i-transients inhibit cAMP and light scattering oscillations [2+]i-elevations enhance oscillations of both parameters [2+ exerts a dual control over the production of the first messenger cAMP  that islium see ,11. RegA pathway ,13. Chem pathway . We foun2+]i we analyzed extracellular Ca2+-fluxes in cell suspensions and studied [Ca2+]i in single amoebae. cAMP-induced Ca2+-influx in suspensions of regA- cells occurred with a similar time course as in wild type. Yet, influx was reduced by approximately 40% . Addition of the PDE inhibitor IBMX (up to 200 μM) to wild type cells affected neither the amount nor the characteristics of cAMP-activated extracellular Ca2+-fluxes.To test whether the absence of the main cAMP-specific phosphodiesterase affects regulation of [Ca40% Fig. . The los2+]i and cAMP-activated [Ca2+]i-changes of regA- to wild type cells in the absence and intracellular presence of IBMX. The inhibitor should affect the activity of both cAMP phosphodiesterases, RegA and PDE-E [2+]i was similar in both strains . After ium Fig. , Table 12+]i-regulation in regA- amoebae might be caused by a change in the characteristics of Ca2+-fluxes of internal stores. A positive influence of cAMP via PKA-mediated phosphorylation of both, IP3-receptors and ryanodine receptors on release of stored Ca2+ has been reported . In wild type amoebae a cAMP-stimulated [Ca2+]i-increase is not detectable in the presence of BAPTA; the occurrence of a transient [Ca2+]i-elevation in regA- cells indicates an augmented release of Ca2+ from stores in the mutant. Support for an effect of cAMP via PKA came from experiments where we incubated cells with the membrane permeant activator of PKA, Sp-5,6-DCl-cBIMPS [2+]i was increased in regA- cells upon treatment with 30 μM Sp-5,6-DCl-cBIMPS for 60 min ; agonist-induced [Ca2+]i-transients in nominally free Ca2+-buffer were unaltered in height  as compared to control cells. In addition, we found that preincubation of wild type amoebae with 30 μM Sp-5,6-DCl-cBIMPS reduced cAMP-activated Ca2+-influx in cell suspensions by 26 ± 8% (mean ± s.e.m. of 3 independent experiments).The effect of the increased basal cAMP concentration on the [Caview see ). We the+]i Fig. . The elel-cBIMPS ,21. Basa2+]i, either by affecting Ca2+-stores or by activating Ca2+-influx alter light scattering oscillations [regA- suspensions two types of responses were observed. On one hand, regular oscillations with a phase length of 4.3 ± 1 min (mean ± s.d. of 61 determinations in 6 independent experiments) occurred  which indicates that the mutant strain is practically as sensitive as wild type. Measurement of [Ca2+]e in regA- cell suspensions revealed irregular [Ca2+]e oscillations, similar to the results obtained for light scattering ; in both strains chemotactic velocity was reduced. The effect was more pronounced in regA-: already in the presence of 2 mM EGTA cells chemotaxed more slowly than under control conditions  as compared to control cells; Mann Whitney rank sum test). Wild type cells were unaffected by preincubation with 5 mM EGTA for up to 1 hour whereas at 10 mM EGTA chemotaxis was reduced.It had been reported previously that ormation . In accoormation  we also 2+ was demonstrated to control light scattering oscillations by affecting the synthesis of cAMP; depending on the height of an artificial [Ca2+]i-transient the production of cAMP which in this case serves as first messenger was either augmented or blocked [2+ in Dictyostelium cells. We observed altered agonist-induced Ca2+-fluxes and [Ca2+]i-transients in the regA- mutant cell line where the absence of the main cAMP-hydrolyzing PDE led to a fourfold increased basal cAMP level. One could argue that the effect on [Ca2+]i was not a consequence of the increased basal concentration of cAMP but rather due to a potentially altered pattern of gene expression in the mutant strain. Indeed, this is possible and could result in a different signal perception and/or processing. However, we consider an alteration in gene expression unlikely to be responsible for the augmented [Ca2+]i-transients upon cAMP-stimulation since the same effect could be evoked in wild type amoebae by loading of the PDE inhibitor IBMX into the cytosol. In addition, IBMX evoked an increase in basal [Ca2+]i in both, wild type and mutant cells. In regA- the inhibitor should act on the additional cAMP-PDE (PDE-E) [2+]i might not be achieved as consistently as in the mutant since IBMX must act on both PDEs.The cytosolic concentration of Ca blocked ,4. The r (PDE-E) ,17 and t2+]i could be caused by several factors. Ca2+-flux characteristics can be changed by influencing Ca2+-channels and/or Ca2+-ATPases located on both, the plasma membrane and membranes of internal stores. When we analyzed Ca2+-fluxes with a Ca2+-sensitive electrode influx was reduced in the mutant while the rates of influx and efflux were unchanged. If the activity of the plasma membrane Ca2+-ATPase (PMCA) was altered then flux rates should be affected. Moreover, the reduced amount of Ca2+-influx precludes activation of a plasma membrane Ca2+-channel. In other cell systems activation of the PMCA and of Ca2+-channels by an increase in cAMP levels was shown [2+-channel or PMCA activity in Dictyostelium amoebae.The sensitizing effect of the increased amount of cAMP on [Caas shown -24 but oDictyostelium a cAMP-activated [Ca2+]i-elevation occurred in the extracellular presence of the Ca2+-chelator BAPTA. This argues for an alteration of Ca2+-uptake into and/or Ca2+-release from stores. An as yet unknown negative regulation of Ca2+-sequestration could cause accumulation of Ca2+ in the cytosol; until now, however, activation of SERCA-type Ca2+-ATPases was found only . On thear cells ; the samar cells . Stimulaemployed . In summdazolium  which wadazolium . Calcinereceptor . In regA2+]i to be responsible for the irregular light scattering and extracellular [Ca2+]-oscillations of regA- cells. Previously, Wessels et al. [regA- as compared to wild type. This effect is plausible, as the increased sensitivity of the Ca2+ second messenger system exerts a negative feedback on cAMP synthesis: large [Ca2+]i-transients inhibit production of cAMP [2+]i-oscillations and their mutual dependence has also been shown in neurons: absence of either, cAMP or [Ca2+]i-oscillations resulted in failure of the other component to oscillate [Dictyostelium the strong decrease in peak cAMP oscillation levels affected [Ca2+]e-oscillations which were irregular. The basis is probably an influence on [Ca2+]i-oscillations. Such oscillations were suggested to occur but have not been demonstrated in single cells until now, presumably due to the small size of the amoebae and the characteristics of the wave [We suggest the alteration in [Cas et al.  have sho of cAMP . An intescillate . In Dictthe wave .regA- cells and an essential role of RegA for a correct response in a natural cAMP wave and chemotactic migration was assigned [regA- cells to polarize was due to a difference in the developmental stage as compared to wild type cells. However, regA- develops much faster than wild type which suggests an even more efficient chemotaxis as this response increases during differentiation to aggregation competence. Alternatively, an altered or dampened signaling response caused by a lower number of cAMP receptors present on the cell surface could have caused the reduced chemotactic response. We consider this to be unlikely for the following reason. Aggregation-competent Dictyostelium amoebae possess roughly 50.000 cAMP receptors at the cell surface [regA- expressed less receptors than wild type this should not influence the accuracy of the response. We propose the reduced polarization capacity of regA- amoebae to be caused by their altered [Ca2+]i-regulation. In the mutant strain the threshold for generation of an agonist-induced [Ca2+]i-increase is lower than in wild type. The [Ca2+]i-elevation is not as tightly controlled and occurs even in the presence of BAPTA. The characteristics of a [Ca2+]i-increase are important for the resulting cytoskeletal rearrangements and whether pseudopods are formed correctly. Indeed, artificial induction of a small global [Ca2+]i-transient by incubation with calmidazolium caused overall pseudopod protrusion [2+]i-gradient at the rear end was shown [2+]i-pattern during chemotaxis. By contrast, a role of the [Ca2+]i-elevation for the chemotactic response was questioned by Traynor et al. [3-receptors of higher eukaryotes aggregated and differentiated almost normally but displayed no cAMP-activated global [Ca2+]i-change; yet, the existence of localized, small [Ca2+]i-transients in this particular mutant cell line that had escaped detection could not be excluded [With respect to chemotaxis, reduced suppression of lateral pseudopod formation was shown in assigned ; subsequassigned . When weassigned . In prin surface . Yet, fo surface . So evenotrusion . In migras shown  which inr et al.  because excluded .regA- cells we found that the mutant was more sensitive. When compared to wild type, lower doses of EGTA were sufficient to reduce chemotactic speed. The effect of EGTA treatment is most probably due to emptying of the storage compartments [2+ affects the Ca2+-content of stores [RegA- cells are more sensitive than wild type amoebae because of the lower threshold for Ca2+ release and thus a more rapid depletion of Ca2+ in the cells.When we analyzed the influence of pretreatment with EGTA on chemotactic behaviour of wild type and artments ; the pref stores ,36. RegA2+]i-elevations which in turn lead to uncontrolled pseudopod extension. [Ca2+]i regulates cAMP acting as first messenger in a negative feedback loop: when the [Ca2+]i response is increased the amount of cAMP synthesized upon stimulation is low as observed in regA- cells devoid of the phosphodiesterase RegA. The low level of cAMP relay results in improper light scattering oscillations. We conclude that intracellular cAMP acts on [Ca2+]i via PKA: phosphorylation of the system responsible for release of Ca2+ from stores leads to a greater sensitivity facilitating Ca2+ liberation. The cAMP activated [Ca2+]i-increase is due to Ca2+-release from internal stores which triggers subsequent extracellular Ca2+-entry. The fraction of the [Ca2+]i-elevation that is mediated by liberation of Ca2+ is thus larger in the mutant.Abnormal basal levels of cAMP impair chemotactic performance by augmenting agonist-activated [CaFura2-dextran and BAPTA were from MoBiTec (Göttingen, FRG). IBMX was purchased from Sigma (Munich, FRG) and cAMP was from Boehringer (Mannheim, FRG). Sp-5,6-DCl-cBIMPS was from Biomol (Hamburg, FRG).D. discoideum axenic wild type Ax2 was grown as described [regA- (kindly provided by Dr. P. Thomason) was grown in the presence of blasticidinS. Cells were washed by repeated centrifugation and resuspension of the cell pellet in cold Sørensen phosphate buffer . Amoebae were shaken at 2 × 107 cells/ml, 150 rpm and 23°C until use. The time, in hours, after induction of development is designated tx.escribed ; the mut2.5–t4 2 ml of cell suspension was pipetted into cuvettes and aerated. Light scattering oscillations were recorded at 500 nm with a photometer as described [At tThe total amount of cAMP was determined using the cAMP enzyme immuno assay (Biotrak, Amersham Pharmacia Biotech, Freiburg, FRG) according to the manufacturer's instructions. Samples were prepared as outlined previously .2+-concentration ([Ca2+]e) was measured in 2 ml of cell suspension (5 × 107 cells/ml in 5 mM Tricine, 5 mM KCl, pH 7.0) with a Ca2+-sensitive electrode (Möller, Zürich, Switzerland) as described [2+]e-oscillations were measured at a cell density of 1 × 108 cells/ml.The extracellular Caescribed . [Ca2+]e2+]-imaging was done as outlined in [7 cells/ml; 20 μl) were loaded at t3 with the Ca2+-indicator fura2-dextran (concentration in the loading solution: 5 mg/ml SP-buffer + 1 mM CaCl2) by electroporation (0°C, 850 V, 3 μF, 200 Ω). Immediately after electroporation, 80 μl of cold 5 mM MgCl2 was added and cells were incubated for 10 min on ice. Then cells were washed 3× with 5 mM Hepes, pH 7.0 (H5-buffer). Washed cells (2–5 μl) were placed on glass coverslips and incubated in a humid chamber until use. When experiments were done in nominally Ca2+-free medium, 85–88 μl of H5-buffer was added 1 min before the [Ca2+]-imaging experiment. To test the response of amoebae in the presence of BAPTA, 75–78 μl of H5-buffer was pipetted to the cells; 10 μl of 10 mM BAPTA was added during the [Ca2+]-imaging experiment and 10–12 sec later cAMP was given. When the response of cells was to be analyzed in the presence of extracellular CaCl2, H5-buffer (85–88 μl) with 1 mM CaCl2 was added to the cells 15 min before the [Ca2+]-imaging experiment to load stores  to the cells. To load cells with IBMX, they were electroporated with fura2-dextran in the presence of 250 μM of the inhibitor. The cytosolic concentration of IBMX is in the range of maximally 2–5% of the concentration present during electroporation [regA- was done at t4 and wild type [Ca2+]-imaging was done at t7–8. In another series of experiments we treated regA- cells with Sp-5,6-DCl-cBIMPS, a membrane permeant activator of PKA [2+]-imaging experiment.Cytosolic [Calined in . Cells . cAMP-sporation . Measure7 cells/ml were placed on a coverslip and allowed to settle for at least 30 min. The morphology of the cells was controlled microscopically: when elongated and thus aggregation competent cells were present, an aliquot of cells from the suspension was diluted for the chemotaxis assay. RegA- was tested at t4–t5, wild type was measured at t7–t10. 250 μl of cells in 5 mM Hepes, pH 7.0 (1 × 105 cells/ml) were placed on glass coverslips. After 30 min cells were challenged with a cAMP (100 μM) filled glass capillary and chemotaxis was recorded for 40–45 min either on vidoe tape or images were stored directly on a hard disk. In addition, experiments were done with cells incubated with 2–10 mM EGTA for 30 min to empty Ca2+-storage compartments. Analysis of chemotaxis was done as outlined previously [Chemotactic performance of the amoebae depends on the degree of differentiation, so their shape was checked prior to the chemotaxis assay. 200 μl of cells at 2 × 10eviously .2+ concentration: [Ca2+]iCytosolic free CaPhosphodiesterase: PDE3-isobutyl-1-methylxanthine: IBMXcAMP-dependent protein kinase: PKA2+-ATPase: PMCAPlasma membrane Ca2+] recordings and light scattering experiments. He also determined cAMP levels and designed the study. KBR did chemotaxis experiments at different external conditions. KH carried out [Ca2+]i-measurements. CS did [Ca2+]i-imaging experiments, designed the study and wrote the manuscript. All authors read and approved the manuscript.DFL performed extracellular [Ca


Hyperandrogenic conditions have been associated with a high prevalence of endometrial pathologies related to cell survival. However, the action of androgens on proliferation and apoptosis in endometrial cells is poorly understood. Therefore, the aim of the present study was to evaluate the effect of androstenedione on cell proliferation, cell death and expression of estrogen receptor (ER) isoforms and proteins related to apoptosis in endometrial cells using two in vitro experimental approaches.The endometrial tissue was obtained from 20 eumenorrheic women [28.7 (25 – 35) years] during the early secretory phase. We analyzed cell proliferation (immunohistochemistry of Ki-67 and spectrophotometric assay); apoptosis (DNA fragmentation (TUNEL) and Annexin V-FITC binding); ER-alpha, ER-beta bcl-2 and bax mRNA abundance (RT-PCR) in explants and isolated endometrial epithelial (EEC) and stromal cells (ESC) incubated with androstenedione 1 micro mol/l (A4) or A4 plus hydroxyflutamide 10 micro mol/l (F) for 24 h.In explants, A4 induced an increase of cell proliferation and a decrease on apoptosis in the stromal compartment (p < 0.05). In isolated ESC, proliferation augmented with A4 (p < 0.05), whereas, no significant modifications in the expression of ER-alpha, ER-beta bcl-2 and bax nor in the apoptotic index were observed. In EEC, A4 increase the ER-beta mRNA abundance (p < 0.05) and a decrease of the bcl-2/bax ratio (p < 0.05), without an increase in the apoptotic index. Hydroxyflutamide reverted the effect of androstenedione on the parameters described.These results indicate that androstenedione may modulate cell survival, expression of ER-beta and proteins related to apoptosis, suggesting a potential mechanism that associates the effect of hyperandrogenemia on the endometrial tissue. Polycystic ovary syndrome (PCOS) is a complex endocrine-metabolic disorder, associated to hyperandrogenism, menstrual disturbances and in many cases to insulin resistance ,2. It haEarly reports in isolated endometrial stromal cells (ESC) have shown that androgens can induce decidualization and inhibition of the expression of ER and progesterone receptors ,13. MoreCell proliferation and apoptosis of the endometrium are importantly regulated by the expression of ER , which eOn the other hand, in some tissues including the human endometrium, the control of apoptosis has been associated to proteins related to the Bcl-2 family, like Bcl-2 that promotes cell survival and Bax which is an inducer of apoptosis ,18. EvenPrevious reports have shown that androstenedione is an important androgen detected in the endometrial tissue ,21. TherEndometrial tissue was obtained with pipelle suction curette from the corpus of the uteri of 20 women with regular menstrual cycles, aged 28.7 (25 – 35) years, at the time of bilateral tubal ligation at the San Borja-Arriarán Clinical Hospital, National Health Service, Santiago, Chile. The tissue was obtained during the early secretory phase since the cells obtained from this phase maintain a high degree of proliferation capacity . None of3, 25 mmol/L of HEPES aminoacids solution, 100 IU/ml of penicillin, and 5 mg/mL of streptomycin (Sigma). Incubation was performed during 6 h at 37°C in 5% CO2/air in humidified atmosphere in the absence or presence of androstenedione 10-7 to 10-5 M (Sigma) or androstenedione 10-6 M plus hydroxyflutamide 10-5 M (Sigma), the latter added 30 min before androstenedione. After incubation, one piece from both basal and treated tissue explants were frozen in liquid N2 and maintained at -70°C for RT-PCR protocols. Another piece was fixed in 4% buffered formaldehyde for 24 h, embedded in paraffin, and cut into 5 μm thick sections before in situ analysis of apoptosis and immunohistochemistry.Human endometrium was cut into slices (20 to 50 mg wet weight) and incubated in 1 mL of Hank's media supplemented with 2 mmol/L glutamax-I (BRL, Life Technology, Bethesda, MD, USA), insulin-transferrin-selenium (ITS) solution (BRL), 0.1% w/v bovine serum albumin , 26 mmol/L of NaHCO2/air in humidified atmosphere until confluence.The cells were separated and purified according to previously described methods . BrieflyThe glands cells were washed in DMEM and incubated for 1 h (30 min in each side of the culture flask), and then cultured in EEC culture media (DMEM/MCDB-105 (3:1, v/v), 10% charcoal stripped FBS, 2 mmol/L glutamax-I, 0.25 μg/mL ascorbic acid, 0.5 mg% insulin (Sigma), 1 μg% transferrin (Sigma), antibiotic and fungizone), similarly to ESC. In both cell cultures, the media were changed every 3 days. Upon reaching confluence, ESC and EEC were passaged by treatment with 0.5 g/L tripsin-0.2 g/L EDTA solution (GibcoBRL).The purity of cell cultures was greater than 90% for ESC and EEC, evaluated by immunocytochemistry of vimentin and cytokeratin, respectively.5 cells/well or 0.1 × 105 cells/well, respectively. When cells were subconfluent (48 – 72 h of culture), the media were changed to Hank's media and incubated for 24 h. Then, the cells were incubated in fresh Hank's media at 37°C in 5% CO2/air in humidified atmosphere in the absence or presence of androstenedione 10-6 M or androstenedione 10-6 M plus hydroxyflutamide 10-5 M. The latter was added 15 min before androstenedione. The culture was carried out for 24 h to evaluate the early effect of androstenedione on cell survival. The concentration of androstenedione used in the present investigation was established in dose-response experiments and are in agreement with those previously reported [Cells, EEC and ESC, were grown in appropriate medium in 6-well or 96-well plates at 2.5 × 10reported .Sections (4 to 6 μm) of human endometrial tissue obtained from cultured explants were deparaffinized in xylene and hydrated gradually through graded alcohols. Endogenous peroxidase activity was prevented by incubating the samples in 3% hydrogen peroxide for 5 min. The sections were incubated in 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 min. Nonspecific antibody binding was prevented with 2% PBS-BSA for 1 h. Primary antibody of Ki-67  was applied to the samples and incubated overnight at 4°C; the antibody for caspase-3  was incubated for 1 h at 37°C and the antibody for androgen receptor (AR) was incubated overnight at 4°C . The second antibody used in both cases was a biotinylated rabbit antimouse immunoglobulin. The reaction was developed by the streptavidin-peroxidase system and 3,3'diaminobenzidine was used as the chromogen; counterstaining was carried out with hematoxylin. The slides were evaluated in a Nikon optical microscope (Nikon Inc., Melville, NY, USA). The immunohistochemical evaluation was determined as the percentage of positive stained cells. In all cases, at least 500 cells were evaluated by three independent observers.Programmed cell death was detected using TdT-mediated dUTP nick end labeling as previously described (Promega, Madison, WI, USA) . Briefly-6 M alone or androstenedione 10-6 M with hydroxyflutamide 10-5 M, as described above. Twenty μL of a mix of tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl) 2H-tetrazolium; MTS) and an electron coupling reagent  were added to each well and incubated for 2 h at 37°C. The quantity of the soluble formazan product was measured at 450-nm absorbance in an ELISA reader (Sigma) and expressed as optical density units (OD).CellTiter 96 AQueous Cell Proliferation Assay (Promega) was used to perform cell proliferation, following manufacturer's instructions. Briefly, EEC or ESC were plated in 96-well until sub-confluence; then, the cells were cultured with androstendione 10-6 M alone or androstenedione 10-6 M with hydroxyflutamide 10-5 M, as described above. The cells were dissociated by 0.25 g/L trypsin-0.1 g/L EDTA treatment, gently re-suspended in cold binding buffer to approximately 1 × 106 cells, and incubated with Annexin V-FITC, as indicated by the manufacturer. The cells were counter-stained with propidium iodide, analyzed and counted by two independent observers in at least 1000 cells in each experimental condition using a fluorescent and optical microscope (Nikon, UFX-NX model. Nikon Inc., Melville, NY, USA). The results are expressed as percentage of apoptotic Annexin V-FITC positive cells respect to total cells counted.Apoptosis was performed using Annexin V-FITC Apoptosis Detection Kit (Oncogene Reasearch Products, Boston, MA, USA). Briefly, after EEC and ESC achieved sub-confluence in 6-well plates, the cells were incubated with androstenedione 10Total RNA was isolated using TRIzol Reagent (GibcoBRL) from endometrial tissue and ESC culture according to the manufacturer's instructions. Total RNA was then reverse transcribed, and cDNA was subjected to polymerase chain reaction (PCR) using specific primers for ERα and ERβ, bcl-2 a2, 0.15 mM of dNTP, 0.63 U of Taq DNA polymerase (Fermentas Hannover, MD, USA), and 0.4 μM of each primer; for bcl-2 and bax was 3 mM MgCl2, 0.25 mM of dNTP, 0.63 U of Taq DNA polymerase, and 0.4 μM of each primer. The PCR amplification was carried out in the Thermocycler, model PTC-100 (MJ Research Inc, Watertown, MA), as previously reported [Semiquantitative RT-PCRs were achieved in the exponential linear zone amplification for each gene studied. Two μg of total RNA were used for reverse transcription in a total volume of 20 μl using the Revertaid H Minus M-Mulv Reverse (Fermentas Hannover, MD, USA). The PCR condition for ERα and ERβ was 2 mM MgClreported .The PCR products were electrophoretically resolved on 1% agarose gel and stained with ethidium bromide. The bands were evaluated using an image analyser , and normalized relative to β-actin PCR product and expressed as arbitrary units (AU).To confirm the specificity of RT-PCR products, the fragments were purified with CONCERT Rapid PCR Purification System (GibcoBRL) and sequenced using an ABI PRISM310 automated Sequencer .The results are expressed as the percentage of changes obtained in the treated respect to the basal condition. Comparisons between groups were performed by ANOVA following Dunnett test. The significance level was set at 5%. Results are expressed as mean ± standard error of the mean (SEM).-7 M – 10-5 M), the percentage of positive cells increased significantly only in the presence of androstenedione 10-6 M and on the stromal compartment  . Moreover, in EEC showed a positive staining in the cytoplasm. The nucleolar antigen Ki-67 was detected in the nucleus of EEC and ESC. After six hours of incubation with androstenedione at different concentrations , Figures  and 1E. Basal values of cell proliferation were 0.39 ± 0.09 OD and 0.64 ± 0.16 OD for EEC and ESC, respectively, whereas, the percentage of annexin V positive cells was 16.3 ± 4.6% for EEC and 12.7 ± 4.3% for ESC. The addition of androstenedione significantly increased cell proliferation in ESC cultures . Table In isolated cells, the basal mRNA abundance for bcl-2 in EEC was 0.47 ± 0.07 AU and for bax 0.39 ± 0.25 AU and in ESC, basal expression of bcl-2 mRNA was 0.87 ± 0.16 AU and 1.09 ± 0.10 AU for bax. Androstenedione induced a decrease of 58% of bcl-2 mRNA expression (p < 0.05) and a 30% increase of bax mRNA (p < 0.05) in EEC . In contrast, androstenedione treatment induced a decrease in ERβ mRNA abundance . However, the ratio between the level of ER isoforms did not change. No significant modifications were observed when hydroxyflutamide was added to the cultures.On the other hand, basal expression of ERα mRNA was 0.31 ± 0.05 AU and 0.39 ± 0.08 AU for ERβ in EEC; in ESC was 0.82 ± 0.23 AU for ERα and 0.86 ± 0.28 AU for ERβ.Androstenedione tended to modify mRNA abundance of ERα in EEC and ESC, a 30% and 25% diminution was obtained, respectively (p = 0.07). In EEC, gene expression of ERβ increased 48% with androstenedione (0.39 ± 0.08 AU vs 0.56 ± 0.18, p < 0.05) . Therefore, these in vitro models are important approaches to understand the potential role of androstenedione in the PCOS endometria. It is well established that women with PCOS exhibit a high prevalence of hyperplasia and endometrial cancer, which is associated with disturbances in the regulation of cell survival -6. AccorIn summary, our results indicate that androstenedione may modulate cell survival, the expression of β-ER isoforms and proteins related to apoptosis. These observations are closely related to the control of endometrial cell function and may help to understand the possible effect of the hyperandrogemia on endometrial tissue.MM conceived and designed the study, carried out the experimental protocols, and drafted the manuscript. SQ, MC and KB performed the RT-PCR and scored of immunohistochemistry, MA carried out the score of immunohistochemistry studies. CJ reviewed and supported in the drafting the manuscript and MV conceived the study as principal investigator and participated in drafting the manuscript.


Most animals (including humans) show a high level of bilateral symmetry: on the surface, the right side of our body resembles the left. A closer and deeper look, however, reveals an underlying asymmetry. The heart, for example, is on the left side in most humans, and the liver on the right. This left-right asymmetry develops early on in the embryo, and research in the past few years has revealed some of the molecular and cellular mechanisms that establish the left-right axis, which conveys positional information to cells in the growing embryo. We know that the formation of the axis relies on “crosstalk” between cells, which involves long-range signaling molecules (or ligands) and cell-surface receptors on cells that receive the signal.The molecules involved in the formation of the left-right axis during embryogenesis, along with their functions, are conserved among vertebrates. They include members of the Transforming Growth Factor beta (TGF-ß) family—such as the agonists (or ligands) Nodal, Vg1/GDF, and activin, and the antagonist (a molecule that interferes with agonist/ligands) Lefty—on the signaling side and members of the EGF-CFC family—such as the activin receptor and its coreceptors—on the receiving side. The EGF-CFC proteins play important roles in early vertebrate embryogenesis; mutations in these genes in the zebrafish (and mouse) result in a range of developmental defects, including problems in left-right axis specification. While ligand-stimulation of the activin receptor by Nodal and Vg1/GDF requires the EGF-CFC coreceptors, activin can activate the activin pathway without a coreceptor. Lefty—being an antagonist—can block activation of the activin receptor, though it is not clear how.Through genetic and biochemical studies in zebrafish and frog embryos, Simon Cheng, Alex Schier, and colleagues have now clarified a piece of this very complex signaling puzzle by demonstrating that Lefty inhibits a subset of TGF-ß signals—Nodal and Vg1/GDF but not activin—by blocking EGF-CFC coreceptors. They went on to show that a short, specific region of the signal molecules—accounting for less than 4% of the entire protein—determines whether the signals activate the activin receptor in an EGF-CFC coreceptor-dependent or -independent fashion and therefore governs susceptibility to Lefty. These findings suggest that subtle sequence differences between related signals can dramatically influence their function.Gene families are thought to arise from gene duplications, and the studies described here illustrate how members of the same gene families can gain diverse roles by specific interactions with coreceptors and antagonists. Additional studies will be necessary to reveal the structural basis for the observed diversity.