In most recent large efficacy trials of barrier contraceptive methods, a high proportion of participants withdrew before the intended end of follow-up. The objective of this analysis was to explore characteristics of participants who failed to complete seven months of planned participation in a trial of spermicide efficacy.Trial participants were expected to use the assigned spermicide for contraception for 7 months or until pregnancy occurred. In bivariable and multivariable analyses, we assessed the associations between failure to complete the trial and 17 pre-specified baseline characteristics. In addition, among women who participated for at least 6 weeks, we evaluated the relationships between failure to complete, various features of their first 6 weeks of experience with the spermicide, and characteristics of the study centers and population.Of the 1514 participants in this analysis, 635 (42%) failed to complete the study for reasons other than pregnancy. Women were significantly less likely to complete if they were younger or unmarried, had intercourse at least 8 times per month, or were enrolled at a university center or at a center that enrolled fewer than 4 participants per month. Noncompliance with study procedures in the first 6 weeks was also associated with subsequent early withdrawal, but dissatisfaction with the spermicide was not. However, many participants without these risk factors withdrew early.Failure to complete is a major problem in barrier method trials that seriously compromises the interpretation of results. Targeting retention efforts at women at high risk for early withdrawal is not likely to address the problem sufficiently. Retention of participants has been a consistent problem in clinical studies of barrier contraceptive methods. For example, in six large studies of condoms, diaphragms, and spermicides conducted in the past decade, more than 30% of the participants failed for reasons other than pregnancy to complete the intended six months or six menstrual cycles of follow-up -6 Such hIssues regarding the design of barrier method studies have become increasingly important to researchers and public health scientists since the onset of the HIV epidemic because of the urgent need for methods to prevent this disease and other sexually transmitted infections. Numerous new barrier contraceptive methods and microbicides are currently in various stages of development and testing. Devising effective approaches to maximize retention in these studies will be critical.In this analysis, we used data from a large, recently completed randomized trial of the efficacy and safety of five spermicide products to determine whether we could identify specific subgroups of participants who were at particular risk for failure to complete the trial. Our goal was to provide information that might assist in the development of targeted approaches to improve follow-up in future trials.The primary purpose of this randomized trial was to estimate and compare the probability of pregnancy during six months of typical use of five nonoxynol-9 spermicide products. Safety, acceptability, and product use were additional specified outcomes. The trial was conducted at 14 sites in the United States between June 1998 and August 2002. The study was approved by the institutional review boards at each site and at Family Health International. All participants signed written informed consent forms before enrollment.A full description of the trial procedures has been published previously . In brieFollow-up visits were scheduled at 4, 17, and 30 weeks after admission. Each participant was also asked to return to the study site if she wished to discontinue use of the spermicide. At each visit, the participant was interviewed, and a urine pregnancy test was done. At the 4-week and final visits, she completed a seven-page acceptability questionnaire. A pelvic examination was performed at the final visit and at other visits as indicated. Colposcopy substudy participants had a vaginal colposcopy at each follow-up visit. Each participant was asked to do a pregnancy test at home 2, 10, and 23 weeks after admission and to telephone the site with the result. If a participant missed a scheduled contact, study procedures required that staff make at least four attempts to contact her by at least two different modalities  If they could not contact her directly, staff were to try to reach her through an alternate contact person identified by the participant at admission. Compensation for completion of all scheduled visits in the primary study ranged from $120 to $400 at the 14 study sites; at most sites, the amount was divided evenly among the separate visits.In this analysis, we included all randomized participants except for 22 who were discovered to have been pregnant at admission and who therefore contributed no data to the primary analysis. We classified each of the remaining 1514 participants as having completed the study if she considered the spermicide to be her primary contraceptive method for at least 183 days after randomization, or she became pregnant before she stopped relying on it. Otherwise, she was classified as having failed to complete the study. We assigned each participant's last day in the analysis as the earliest of the following dates: the estimated date of fertilization of a pregnancy; the date she was last known to have been relying primarily on the assigned spermicide for contraception; the latest date her pregnancy status could be reliably determined; and 183 days after randomization. These rules were the same as those used in the prior primary pregnancy analyses .We assessed the associations between failure to complete and 17 baseline factors of interest, which were prespecified before the analysis. Among the subset of participants who were in the analysis for at least 6 weeks, we examined the associations between final status category and various factors that characterized their experience during the first 6 weeks in the study. Factors were categorized in part to ensure substantial numbers of participants in each level. Hypotheses about the effects of factors on completion status were tested using chi square tests, Fisher's exact tests, Mantel Haenszel tests. Parameters estimated by multivariable logistic regressions were tested using Wald tests. We included factors in regression models if they were associated with the outcome  in bivariable analyses. None of the included factors were highly correlated. In both bivariable and multivariable analyses, a p-value of <0.05 was considered to indicate a significant association.Of the 1514 participants in this analysis, 635 (42%) failed to complete the study for reasons other than pregnancy. The proportion who withdrew early at each of the 14 study sites ranged from 17% to 83%. Only 3 centers had completion rates ≥65%. Forty nine participants (8% of those who withdrew) were discontinued by the site investigator because of a concern about their safety , staff error, or closure of the trial at the study site  relative risk of failure to complete the trial Table . FactorsOf the 1095 participants who contributed more than 6 weeks to the analysis, those who in their first 6 weeks were not compliant with follow-up visits, coital diary completion, or use of the spermicide during sex were significantly less likely than others to complete the study Table . HoweverIn analyzing data from longitudinal studies, researchers commonly assume that the experience of participants who withdraw early, had they stayed in the study, would have been similar to the experience of those who completed. However, this assumption is generally impossible to confirm and is often implausible. If the assumption is false, the study findings may substantially misrepresent the likelihood of the outcome in the study population. If the degree of misrepresentation is not consistent across study groups, comparisons could be seriously biased. Indeed, some expert epidemiologists have suggested that a trial with losses of greater than 20% of the participants "would be unlikely to successfully withstand challenges to its validity" ,9.Our study, like other recent barrier contraceptive method studies, did not even approach this standard: 42% of our enrolled participants did not complete the trial. Furthermore, the participants who failed to complete were different in key ways from those who did – they reported significantly more frequent coitus at baseline, and they also were more likely to be younger, unmarried, and poorly compliant with study procedures and method use in the first few weeks after admission. All of these characteristics were associated to some extent with elevated risk of pregnancy in our population , which sClearly, increased attention to preventing this problem in future studies is imperative. In performing this analysis, our intention was to explore the potential impact of focusing retention efforts on participants with characteristics that are associated with failure to complete. However, although we did find some factors that were significantly associated with early withdrawal, none was highly predictive; that is, many participants without these factors failed to complete the study, and many with these factors did complete. Therefore, applying special efforts only to the high-risk participants would not likely have been sufficient to raise completion rates to desirable levels. In future trials, aggressive follow-up measures should be instituted universally. Such efforts might include assigning individual "case-workers" to participants, using novel means for communicating with the participants, such as pagers, conducting visits at participants' homes or at other locations convenient for them, providing specific reimbursement for expenses such as travel, parking, and child care, or providing extra incentives for completing follow-up. Researchers should be mindful, however, that one downside to some of these approaches is that they might influence participants' use of the study product or other behaviors related to the study outcome, which is detrimental if the goal of the trial is to estimate effectiveness during "typical use" of the product.In our study, participants who enrolled at study centers where enrollment was slow were at increased risk of failure to complete the study. The reason for this association is unclear. Factors at these centers that hindered enrollment also may have adversely affected participants' interest in remaining in the study. Alternatively, in responding to pressure to hasten recruitment, these centers may have enrolled women who were not good candidates for study completion. This latter possibility emphasizes the need to maintain a careful balance between recruitment and retention goals: rapid recruitment of participants who then drop out of the study is not beneficial to the study as a whole.The amount of reimbursement promised to our participants was strongly associated with final completion status in the bivariable analysis, but this effect was not significant when adjusted for other factors in our multivariable model. Numerous prior studies have shown that modest monetary incentives  increase response rates to surveys or short follow-up studies ,11 Some We were surprised that several of the factors that we expected would be associated with early withdrawal did not show significant associations in this analysis. When we began this analysis, we presumed that one reason for both slow enrollment and poor follow-up rates in barrier method trials is the relatively poor efficacy of these products: women may consider them to be temporary or backup methods and thus may be unwilling to use them as their sole or primary contraceptive for the 6–12 month duration of these studies. However, in our study, participants who strongly wished to avoid pregnancy or who had completed their desired family size were not more likely than others to drop out, nor were participants who expressed concerns about contraceptive efficacy early in the trial. Furthermore, neither early medical problems nor other complaints about the spermicides were predictive of withdrawal. These findings differ from that of a previous randomized trial of spermicides conducted mostly in developing countries. In that trial, participants who initially liked the assigned product very much were more likely than others subsequently to complete the study and to use the product for a longer period of time after admission .In one respect, the poor retention rate in our study and in other barrier method trials is a result of the design of these studies, which typically call for censoring data  when participants stop relying on the assigned contraceptive method. This design prohibits a true intent-to-treat analysis and is consequently a potential source of bias. Clearly, retention would be higher if the trials were designed at the outset to follow all subjects for the full intended duration of follow-up, even if they switched contraceptive methods. However, data from participants who are not using the method under study are not necessarily relevant to the efficacy and safety of the method. For the results of these trials to be meaningful, as many subjects as possible must not only complete follow-up but also continue to use the method during the full follow-up period. In our study, almost all the women who gave a reason for withdrawing early either cited problems with the spermicide or indicated that they wished to switch to another method after leaving the study. Our results are consistent with the findings of the 1995 National Survey of Family Growth, which showed that more than 47% of US spermicide users stopped relying on the method within the first 6 months of use . These fOur results suggest that to reduce bias potentially introduced by a large proportion of participants failing to complete the study, future barrier contraceptive method researchers should consider approaches in addition to those directly aimed at tracking and retaining individual participants. For example, both to reduce the burden on participants and to help the study staff maintain focus on follow-up, limiting data collection to critical variables may be appropriate. Complete collection of key data is clearly preferable to inadequate collection of less important data. Reducing the planned duration of follow-up would also certainly reduce withdrawals; although a larger sample size would be needed to provide the desired levels of precision and power, this disadvantage might be overcome if the shorter study were more attractive to potential participants. Given the large proportion of women who stop using the method earlier than 6 months, it is not clear that 6-month pregnancy probabilities are clinically needed anyway. Adding a run-in period to the trial before randomization might be helpful in excluding participants likely to drop out very early after admission, although such an addition might deter enrollment of other women as well, which is also a problem in these trials. Finally, innovative study designs to measure product efficacy should be evaluated. The design proposed by Steiner et al., which compares the one-month pregnancy probability in a relatively small number of women using a contraceptive method to the probability in women using a placebo, offers an alternative to the traditional 6–12 month trial . It showNo authors have any declared interests except the following:Elizabeth Raymond owns stock in Johnson and Johnson.Mitchell Creinin serves as a speaker for Ortho.Alfred Poindexter has had research grants from Columbia Laboratories and serves as speaker for Ortho.EGR helped design the trial, managed the trial, planned this analysis, and drafted the manuscript.PLC and BPL helped design the trial and/or this analysis, performed the analysis, and contributed to the manuscript.JL designed the trial and contributed to the manuscript.Other authors participated in the design of the trial, conducted the trial, and contributed to the manuscript.The pre-publication history for this paper can be accessed here:


S. cerevisiae, C. elegans, E. coli, A. thaliana, D. melanogaster, and H. sapiens. We use genomic sequence information to connect these data and compare global and modular properties of the transcription programs. Linking genes whose expression profiles are similar, we find that for all organisms the connectivity distribution follows a power-law, highly connected genes tend to be essential and conserved, and the expression program is highly modular. We reveal the modular structure by decomposing each set of expression data into coexpressed modules. Functionally related sets of genes are frequently coexpressed in multiple organisms. Yet their relative importance to the transcription program and their regulatory relationships vary among organisms. Our results demonstrate the potential of combining sequence and expression data for improving functional gene annotation and expanding our understanding of how gene expression and diversity evolved.Comparing genomic properties of different organisms is of fundamental importance in the study of biological and evolutionary principles. Although differences among organisms are often attributed to differential gene expression, genome-wide comparative analysis thus far has been based primarily on genomic sequence information. We present a comparative study of large datasets of expression profiles from six evolutionarily distant organisms:  Comparative analysis of sequence and gene expression data from bacteria, yeast, worms, flies, weeds, and humans hints at the potential to extract biological and evolutionary principles We performed a statistical analysis comparing the pairwise correlations between genes in one organism to the correlations between their respective homologues. Indeed, a significant fraction of such correlations were similar see . The strSaccharomyces cerevisiae expression data , while in the other organisms they may be required to accelerate folding during cell growth.The observation that groups of functionally related genes are often coexpressed in multiple organisms prompted us to ask whether also the higher-order regulatory relationships between these groups have been conserved . To addrC. elegans), the standard deviations of the correlation coefficients do not exceed 0.1, even when removing half of the expression profiles. Taken together, these results indicate that, despite the sparseness of the data, our findings reflect real properties of the expression networks and not the specific subset of experimental conditions used.In order to test whether the variations in the regulatory relations among functional groups in different organisms are due to the use of unrelated sets of experimental conditions, we restricted both the human and the yeast expression data to the cell cycle experiments. We found that the correlations between modules did not change qualitatively due to this restriction B and 2C.To compare the higher-order regulatory structures more systematically, we decomposed the expression data of each organism into a set of transcription modules using the iterative signature algorithm (ISA) we proposed recently A and 3B.C. elegans tree exhibits a sharp transition between a regime dominated by a single branch (from which only few less-stable modules branch off) to a part of the tree that rapidly bifurcates into many branches at higher thresholds , most modules have either significantly less or significantly more homologues than expected  of the connectivity k (the number of edges of a particular gene). We find that for all organisms, the connectivity is distributed as a power-law, n(k) ∼ k−γ, with similar exponents γ ≈ 1.1–1.8  that two genes of connectivity k and k′, respectively, are connected with each other . Interestingly, in the three organisms in which large-scale knockout information is available  identified by our modular analysis. The decrease of  modules E. Thus, Comparing genomic properties of different organisms is of fundamental importance in the study of biological and evolutionary principles. Although much of the differences among organisms is attributed to different gene expression, comparative analysis thus far has been based primarily on genomic sequence information. The potential of including functional genomic properties in a comparison analysis was demonstrated in recent studies that compared protein–protein interaction networks of different organisms .In this paper we presented a comparative analysis of large datasets of expression profiles from six evolutionarily distant organisms. We showed that all expression networks share common topological properties, such as a scale-free connectivity distribution and a high degree of modularity. While these common global properties may reflect universal principles underlying the evolution or robustness of these networks, they do not imply similarity in the details of the regulatory programs. Rather, with a few exceptions, the modular components of each transcription program as well as their higher-order organization appear to vary significantly between organisms and are likely to reflect organism-specific requirements.http://barkai-serv.weizmann.ac.il/ComparativeAnalysis/.Nevertheless, coexpression of functionally linked genes is often conserved among several organisms. Based on this finding, we proposed an efficient method that uses coexpression analysis for improving sequence-based functional annotation. An interactive implementation of this algorithm is available at Our analysis was based on the available expression data, which are still sparse for most organisms. It is likely that the modular decompositions we obtained are partial, so additional modules can be identified as more expression data become available. Nevertheless, by analyzing the sensitivity of our results to the number of conditions, we concluded that the composition of the modules themselves is rather robust. Moreover, the higher-order correlations between modules are only slightly affected by the number of conditions.S. cerevisiae . We excluded genes or conditions with more than 90% missing datapoints, resulting in expression matrices of the dimensions shown in Preprocessed expression data from Database  using deDatabase , we onlyFASTA files for amino-acid sequences of coding regions were downloaded from the sources detailed in S. cerevisiae and E. coli) and RNAi experiments (C. elegans) were obtained from the sources indicated in Data for deletion mutants (mG of the genome G) and an associated set of regulating conditions . The defining property of a transcriptional module is self-consistency, which is achieved as follows. First, we assign scores to both genes and conditions that reflect their degree of association with the module. The gene score is the average expression of each gene over the module conditions, weighted by the condition score: g in condition c normalized over genes and conditions, respectively, such that gs, while the module conditions are those conditions in the dataset with the highest scores cs. The ISA identifies transcription modules through iterative refinement of a large number of random gene scores.A transcription modules consist of a set of coregulated genes  in the present analysis, but retained only genes whose score is not less than 70% of the most significant gene  is identical in all expression networks and fixed it to <k> = 0.001. Using the top 0.1% of all possible correlations corresponds to a lower limit on the correlation coefficients between 0.63 for S. cerevisae and 0.85 for D. melanogaster. The results are insensitive to the precise threshold value (see i is k = Σj≠iijA. In order to obtain the connectivity distributions n(k), we used logarithmic binning. The edges of the bins were powers of 2, and we counted the number of genes with ik between two edges and normalized by the bin width. We applied a linear fit to the log values of the bin centers against the normalized counts. We note that the resulting connectivity distributions scale as a power-law for a wide range of thresholds and the exponents only depend weakly on the choice of the threshold. The clustering coefficient of gene i is iC = (Σk>j≠iikAkjAjiA)/[ik(ik −1)/2].Each expression network can be described by a symmetric adjacency matrix http://barkai-serv.weizmann.ac.il/ComparativeAnalysis.Interactive applications for the refinement of sets of homologous genes and the exploration of our modular decompositions of the expression data are available online. We also present details about the highly connected genes in each organism, the pairs of genes that are significantly correlated in two organisms, and the eight modules related to core processes in yeast (and their homologue modules before and after refinement) on our website at Data S1Figure S1 and Figure S2.This note includes (38 KB PDF).Click here for additional data file.Data S2Figure S3, Figure S4, and Figure S5.This note includes (59 KB PDF).Click here for additional data file.Data S3Figure S6.This note includes (11 KB PDF).Click here for additional data file.Data S4(3 KB PDF).Click here for additional data file.Data S5After this work was completed, we succeeded in processing the more than 2,000 human chip experiments deposited at the SMD. Removing genes and conditions with more than 90% missing values resulted in 1,474 expression profiles for 24,795 genes. Our Web tools (“GeneHopping” and “ModuleTree”) allow researchers to use also this updated dataset.(3 KB PDF).Click here for additional data file.Figure S7(137 KB PDF).Click here for additional data file.Figure S8(16 KB PDF).Click here for additional data file.Figure S9(15 KB PDF).Click here for additional data file.Figure S10(33 KB PDF).Click here for additional data file.Figure S11(11 KB PDF).Click here for additional data file.Figure S12(29 KB PDF).Click here for additional data file.Figure S13(19 KB PDF).Click here for additional data file.


Inconsistent findings have been reported on the occurrence and relevance of creatine kinase (CK) isoenzymes in mammalian liver cells. Part of this confusion might be due to induction of CK expression during metabolic and energetic stress.The specific activities and isoenzyme patterns of CK and adenylate kinase (AdK) were analysed in pathological liver tissue of patients undergoing orthotopic liver transplantation.The brain-type, cytosolic BB-CK isoenzyme was detected in all liver specimens analysed. Conversely, CK activity was strongly increased and a mitochondrial CK (Mi-CK) isoenzyme was detected only in tissue samples of two primary hepatocellular carcinomas (HCCs).The findings do not support significant expression of CK in normal liver and most liver pathologies. Instead, many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. Moreover, the data suggest a possible interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC. These results, if confirmed, could provide important hints at improved therapies and cures for HCC. Creatine kinase (CK) isoenzymes catalyse the reversible transfer of the phosphate group of phosphocreatine (PCr) to ADP, to yield ATP and creatine (Cr). The CK/PCr/Cr system is present primarily in tissues with high and fluctuating energy demands such as brain, heart and skeletal muscle, and serves as a temporal and spatial "energy buffer" that helps to maintain a high intracellular phosphorylation potential in situations of increased metabolic demand .de novo. The liver is the main site of Cr production in the body . After ody see -6). Othe. Othede ody . Finallody  isoenzyme. On the other hand, besides BB-CK which was suggested to be present in endothelial and Kupffer cells, Vaubourdolle et al.  also proIn a number of studies reporting significant levels of CK activity in liver, interference by adenylate kinase (AdK) isoenzymes in the CK activity assays -21 is veBecause of these conflicting data, the goal of the present study was to analyse in detail the CK and AdK activities in pathological liver tissue of patients undergoing orthotopic liver transplantation.The present project was approved by the ethics commission of the University of Innsbruck. In total, 25 liver samples were analysed. Twenty-three samples were obtained from 18 explanted organs of liver transplant recipients, one sample was obtained at autopsy (no. 1), and the last sample was from a normal rat liver. According to pathomorphological criteria, the 25 samples can be divided into 5 groups: (1) Nine samples of cirrhotic liver tissue ; (2) six samples of neoplastic tissue ; (3) three samples of necrotizing liver tissue due to acute or subacute organ rejection ; (4) five samples of macroscopically normal liver parenchymal tissue  surrounding focal liver pathologies ; (5) two samples originating from a normal rat liver (no. 12) and from a patient with steatosis hepatis (no. 1).All steps were performed on ice or at 4°C. Approximately 5 g of liver tissue was homogenized in 45 ml buffer A . The homogenate was subjected to centrifugation for 5 min at 800 g. The pellet was discarded, and the supernatant centrifuged for 4 min at 5,100 g (centrifugation C2). The supernatant of C2 was further clarified by centrifugation for 12 min at 12,300 g, thus yielding the cytosolic fraction. The pellet of C2 was resuspended in 10 ml buffer A, followed by centrifugation for 2 min at 12,300 g (C3). After resuspension of the C3 pellet in 10 ml buffer A and centrifugation for a further 10 min at 12,300 g, the sediment was resuspended in 4 ml buffer A, thus yielding the mitochondrial fraction. One-ml aliquots of the different fractions were immediately frozen in liquid nitrogen and stored at -80°C until analysis.2, 2.1 mM ADP, 2.1 mM NADP, 21 mM N-acetylcysteine, 9 U/ml of hexokinase, and 5.8 U/ml of glucose-6-phosphate dehydrogenase (both from Sigma). Enzymatic activity was measured at 25°C as an increase in NADPH absorbance at 340 nm. For AdK, three separate measurements were made for each sample in the same assay medium. For CK measurements, 5.1 mM AMP was added to the assay medium to inhibit AdK activity. For each sample, three measurements with 10.3 mM PCr and three measurements without PCr  were made, and the CK activity was calculated as the difference of the respective means. All values in this paper represent specific activities per mg of homogenate, cytosolic or mitochondrial protein. Protein amounts were measured according to the method of Bradford -1). Therefore, in the absence of histochemical data, it cannot be concluded with certainty whether the increased levels of CK are due to increased vascularization of the tumour , or to induction of CK expression in the malignant cells.Interestingly, we observed a strong induction of both BB-CK and Mi-CK expression in two samples of primary HCC. Despite CK/AdK activity ratios Induction of CK expression has been observed previously in many types of tumours (see ) and mayA key player in the picture might be the p53 tumour suppressor gene. Mutations in p53 are quite prevalent in HCC, especially in tumours with low cellular differentiation ,43. On tin vivo models  the limited responsiveness of HCC to currently available therapeutic approaches and, thus, (ii) the poor prognosis associated with this disease. Cr analogues (cyclocreatine and β-guanidinopropionic acid) and also Cr itself were previously shown to have antitumour activity, both in cell culture and in  models . Similaepatoma; .The present findings shed light on some old enigmas and open up fascinating avenues for future research. Our findings do not support significant expression of CK in normal liver and most liver pathologies, but rather indicate that many of the previous misconceptions in this field can be explained by interference from AdK isoenzymes. On the other hand, given the need for improved understanding of the molecular pathogenesis of HCC, and for improved therapies and cures, the induction of CK expression in HCC described here calls for a more in-depth analysis of the interplay between p53 mutations, HCC, CK expression, and the growth-inhibitory effects of cyclocreatine in HCC.AdK, adenylate kinase; BB-CK, brain-type cytosolic CK isoenzyme; CK, creatine kinase; Cr, creatine; HCC, hepatocellular carcinoma; Mi-CK, mitochondrial CK; MM-CK, muscle-type cytosolic CK isoenzyme; PCr, phosphocreatine.The authors declare that they have no competing interests.GM and RM covered the medical part of this study. GM, FNG and MW performed the biochemical experiments. MW drafted the manuscript.The pre-publication history for this paper can be accessed here:


Caenorhabditis elegans vulval development, the anchor cell (AC) in the somatic gonad secretes an epidermal growth factor (EGF) to activate the EGF receptor (EGFR) signaling pathway in the adjacent vulval precursor cells (VPCs). The inductive AC signal specifies the vulval fates of the three proximal VPCs P5.p, P6.p, and P7.p. The C. elegans Rhomboid homolog ROM-1 increases the range of EGF, allowing the inductive signal to reach the distal VPCs P3.p, P4.p and P8.p, which are further away from the AC. Surprisingly, ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. This observation led to the discovery of an AC–independent activity of EGF in the VPCs that promotes vulval cell fate specification and depends on ROM-1. Of the two previously reported EGF splice variants, the longer one requires ROM-1 for its activity, while the shorter form acts independently of ROM-1. We present a model in which ROM-1 relays the inductive AC signal from the proximal to the distal VPCs by allowing the secretion of the LIN-3L splice variant. These results indicate that, in spite of their structural diversity, Rhomboid proteins play a conserved role in activating EGFR signaling in C. elegans, Drosophila, and possibly also in mammals.During  ROM-1 increases the range of EGF, which specifies the vulval fate of precursor cells, by functioning in the signal- receiving precursor cells rather than the signal-sending anchor cells Intercellular signaling pathways control many diverse processes, such as cell proliferation, differentiation, survival, migration, shape changes, and responses to the environment. In most instances, the release of the signaling molecules by the signal-sending cells constitutes a rate-limiting step that determines the spatial distribution and temporal duration of the response . On the Drosophila growth factor Spitz, which activates the EGFR in multiple developmental processes  receptor (EGFR) acts in a highly conserved signal transduction pathway that controls various cell fate decisions in metazoans . EGFR lirocesses . Geneticing cell . Drosophroteases . Site-sproteases . The Dro pathway . There a elegans . Rhomboi elegans . Rhomboi elegans  and the ease S2P . On the ease S2P . WhetherC. elegans hermaphrodite vulva serves as a simple model by which to study signal transduction and cell fate determination during organogenesis  in the somatic gonad induces three out of six equivalent vulval precursor cells  in the ventral hypodermis to adopt vulval cell fates , rom-2 (C48B4.2), rom-3 (Y116A8C.14), rom-4 (Y116A8C.16), and rom-5 (Y54E10A.14) , followed by ROM-2 (29% identity) and the more diverged ROM-3 (24% identity), ROM-4 (26% identity), and ROM-5 (29% identity). Mutagenesis experiments with Drosophila Rhomboid-1 have identified a catalytic triad formed by conserved asparagine, serine, and histidine residues that are necessary for the serine protease activity  A. All fihomboids . The trahomboids B. ROM-1 activity . This caactivity B, suggesrom-1, we isolated rom-1 cDNA by RT-PCR. An SL1 trans-spliced leader sequence was identified at the 5′ end of the message that was spliced to the second of the six exons predicted by the C. elegans genome project (http://www.wormbase.org). The remaining intron-exon boundaries were confirmed experimentally and corresponded exactly to the predicted boundaries. The conceptual translation of the 1,071-bp open reading frame (ORF) predicts a protein of 356 amino acids, with very short stretches of hydrophilic amino acids between the seven-pass transmembrane domains, except for a longer loop consisting of 43 amino acids between the first and second transmembrane domains  to transiently knock down their expression  animals with bacteria producing rom-3 dsRNA had no effect on vulval development (unpublished data). Due to the high degree of sequence similarity between rom-3 and rom-4 (69.8% identity), rom-3 RNAi is likely to simultaneously reduce rom-4 function.As a first step to examine the biological function of the pression . Double-rom-1 gene . The rom-1(0) single mutants exhibited no obvious phenotype; they were healthy and fertile. In addition, we obtained the rom-2(ok966) allele from the C. elegans Gene Knockout Consortium. The rom-2(ok966) animals carry a 530-bp deletion that removes the fifth exon, which contains the predicted catalytic center with the essential histidine residue (see rom-2(rf). Consistent with the RNAi experiments, both rom-1(0) and rom-2(rf) single mutants exhibited normal vulval development (rom-1(0) rom-2(rf) double mutants, no defects in vulval development were observed, ruling out a possible redundant function of the two genes  mutation as well as rom-1 RNAi partially suppressed the multivulva (Muv) phenotype caused by overexpression of the LIN-3 EGF growth factor [lin-3(+)] (n1046 gain-of-function (gf) mutation in the let-60 ras gene, which renders vulval development partially independent of upstream signaling (rom-1(0) mutation suppressed the Muv phenotype of hs::mpk-1 animals that overexpress the wild-type MAPK MPK-1 under control of a heat-shock promoter together with Drosophila MEK-2 under control of the interferon-1α promoter  mutation nor rom-2 RNAi affected the Muv phenotype of let-60(gf) animals ]  or by thignaling  (Table 1promoter  (Table 1rom-1(0) mutation did not significantly enhance the vulvaless (Vul) phenotype caused by the lin-3(e1417), lin-2(n397), sem-5(n2019), or let-60(n2021) mutations that reduce the activity of the receptor tyrosine kinase (RTK)/RAS/MAPK pathway (unpublished data). Since these Vul mutants affect the cell fates of only the proximal VPCs , ROM-1 plays no role in the induction of the proximal VPCs by the AC. Thus, ROM-1 enhances the activity of the EGFR/RAS/MAPK pathway to allow the induction of the distal VPCs P3.p, P4.p, and P8.p.The (hs::lin-3extra) (rom-1(0) (lin-15(rf) mutants, all VPCs adopt vulval cell fates independently of the LIN-3 signal, though induction in lin-15(rf) mutants depends on the activity of LET-23 and the other components of the EGFR/RAS/MAPK pathway (rom-1(0) mutation did not suppress the Muv phenotype of lin-15(rf) animals, suggesting that loss of rom-1 function affects the LIN-3-dependent induction of vulval cell fates rather than the LIN-3-independent activity of the EGFR/RAS/MAPK pathway  . Unlike  pathway . The romrom-1 and the Notch and Wnt pathways, since both pathways control vulval cell fate specification in parallel with the RTK/RAS/MAPK pathway (lin-12 notch(gf) animals, no AC is formed, and all VPCs adopt the 2° cell fate (rom-1(0) lin-12(gf) double mutants  mutation  mutation on the egl-17::cfp expression pattern in mid L2 larvae. For this purpose, larvae were synchronized in the mid L1 stage at 13 h of development by letting them hatch in the absence of food, and then development was allowed to proceed by adding food for another 24 h until they reached the mid L2 stage (approximately 37 h of development). Loss of ROM-1 function had no effect on egl-17::cfp expression in the proximal VPCs , but significantly reduced egl-17::cfp expression in the distal VPCs when compared to wild-type animals (egl-17::cfp expression pattern in rom-1(0) animals is consistent with the epistasis data, which showed that loss of rom-1 function affects the induction of only the distal VPCs (see above).To assess how much inductive signal each VPC receives, we examined the expression pattern of the  pathway . In mid and P7.p , and lowand P7.p A and 2B. animals C and 2D.rom-1 reporter by fusing 6.9 kb of the 5′ rom-1 promoter/enhancer region to the green fluorescent protein (gfp) ORF carrying a nuclear localizing signal (zhIs5[rom-1::nls::gfp]). With a translational full-length rom-1::gfp fusion construct, we failed to obtain transgenic lines that consistently expressed ROM-1::GFP. Moreover, a genomic DNA fragment encompassing the entire rom-1 locus failed to produce stable transgenic lines even when injected at relatively low concentrations (1–10 ng/μl), suggesting that elevated levels of ROM-1 are toxic to the animals.To analyze the expression pattern of ROM-1, we generated a transcriptional rom-1::nls::gfp reporter was widely expressed in somatic cells throughout development. Surprisingly, we did not detect any rom-1::nls::gfp expression in the gonadal AC before the L4 stage, while consistent expression was observed in the Pn.p cells and the Pn.a-derived neurons from the L1 stage on. In early L2 zhIs5 larvae, the six VPCs expressed rom-1::nls::gfp at equal levels . After vulval induction, rom-1::nls::gfp was down-regulated in the 1° and 2° descendants of P5.p, P6.p and P7.p, while the 3° descendants of P3.p, P4.p, and P8.p again expressed high levels of rom-1::nls::gfp after they had fused with hyp7 . To test whether rom-1::nls::gfp expression depends on RTK/RAS/MAPK signaling in the VPCs, we introduced the zhIs5 transgene into lin-7(e1413) mutants that exhibit a penetrant Vul phenotype due to reduced LET-23 EGFR activity (lin-7(e1413); zhIs5 animals, the up-regulation of rom-1::nls::gfp occurred less frequently . Thus, the AC signal up-regulates rom-1::nls::gfp expression in the VPCs that adopt vulval cell fates.The transcriptional l levels A and 3B.al fates C and 3D.ith hyp7 E and 3F.r fusion G and 3H r fusion . To test1 and Z4 . Uniformactivity . In lin-rom-1(+) function on vulval induction in gonad-ablated animals. If ROM-1 acts exclusively in the AC, then the rom-1(0) mutation should not affect vulval induction in gonad-ablated animals. On the other hand, if ROM-1 acts in cells other than the AC, then the rom-1(0) mutation should suppress vulval induction even in the absence of the AC. Since the inductive AC signal is absolutely required to initiate vulval development (let-60(gf) or hs::mpk-1 animals that exhibit a hyperactive EGFR/RAS/MAPK signaling pathway causing AC-independent vulval induction (lin-3(+) animals because, as reported previously by lin-3 under control of its own promoter, some vulval differentiation could still be observed in the absence of the AC, pointing at an additional source of LIN-3 from the transgene in cells outside of the gonad (rom-1 function in gonad-ablated lin-3(+), let-60(gf), or hs::mpk-1 animals caused a strong further reduction in vulval induction (lin-15(rf) animals that exhibit lin-3 independent vulval differentiation was not affected by the rom-1(0) mutation .To examine whether ROM-1 acts in cells other than the AC (which is part of the somatic gonad), we tested the effect of loss of elopment , we perfnduction . In addihe gonad , row 2. egl-17::cfp expression pattern after removal of the AC. In gonad-ablated animals, residual egl-17::cfp expression was observed in all VPCs  loss-of-function mutation [lin-3(0)] was much stronger than the decrease observed in gonad-ablated lin-3(+); hs::mpk-1 animals (lin-3(0) mutation was suppressed by the hs::mpk-1 transgene). Vulval induction in lin-3(0); hs::mpk-1 animals was not affected by gonad ablation since the lin-3(0) allele eliminated lin-3 function in the AC  reduction-of-function mutation almost completely abolished the expression of the egl-17::cfp marker (egl-17::cfp expression in the VPCs. Loss of rom-1 function in a lin-3(0); hs::mpk-1 background caused no further decrease in vulval induction, suggesting that ROM-1 does not affect vulval development in the absence of LIN-3  ; let-60(gf) and rom-1(0); hs::mpk-1 double mutants to levels comparable to those found in let-60(gf) and hs::mpk-1 single mutants (lin-31 promoter (lin-31::cre) that was used as a negative control had no effect on vulval induction ; let-60(gf) animals (rom-1 in the AC under control of the AC-specific enhancer (ACEL) (ACEL::rom-1), which is located in the third intron of the lin-3 locus  Muv phenotype by rom-1(0) (rom-1 function.The absence of detectable ::rom-1) . The linnduction . Consist-3 locus . In contlin-3 dsRNA in the Pn.p cells in order to down-regulate by RNAi any possible lin-3 expression in the VPCs (lin-3 cDNA fragment under control of the same Pn.p cell-specific lin-31 promoter used above (lin-31::lin-3i) was introduced into wild-type animals . In wild-type L4 larvae, the 1° descendants of P6.p in the vulF toroid ring , secrete LIN-3 to specify the ventral uterine (uv1) cell fate in the somatic gonad . The lin-31::lin-3i transgene almost completely suppressed the Muv phenotype caused by the lin-31::lin-3S transgene, while the lin-31::cre transgene that was used as negative control had no effect  and hs::mpk-1 animals  animals lacking a gonad  single mutants exhibited wild-type levels of vulval induction, the egl-38(rf) mutation reduced the Muv phenotype of hs::mpk-1 animals to a similar degree as the rom-1(0) mutation or the lin-31::lin-3i transgene  produce LIN-3 to amplify the inductive signal.lin-3 locus encodes two splice variants termed LIN-3S (short) and LIN-3L (long) that are generated by the differential choice of the splice donor of exon 6  transgenic lines generated by injection of a relatively low (1 ng/μl) or high (100 ng/μl) concentration of the lin-3S minigene exhibited a strong Muv phenotype (with induction indices ranging from 4.1 to 5.6). In contrast, the lin-3L construct caused a Muv phenotype only when injected at a high concentration. . For the lin-3XL construct, we obtained variable results; some lines exhibited a weak Muv and others no or even a Vul phenotype (unpublished data). Since we failed to observe a consistent phenotype with this minigene construct, we did not further pursue the analysis of the lin-3XL minigene.The f exon 6 B  background. The lin-3S and lin-3L transgenes both rescued the larval lethality of lin-3(0) mutants, yielding adult Muv animals (lin-3(0); lin-3S and lin-3(0); lin-3L animals were sterile. Loss of rom-1 function did not affect the viability or the Muv phenotype of lin-3(0); lin-3S animals  mutants was reduced by loss of rom-1 function (rom-1(0), lin-3(0); lin-3L animals that escaped the larval lethality exhibited a weaker Muv phenotype than lin-3(0); lin-3L animals, suggesting that the function of LIN-3L during vulval induction partially depends on ROM-1 activity . The lin-31::lin-3S and lin-31::lin-3L transgenes both caused a strong Muv phenotype in the presence and absence of the AC  mutants an unprocessed, membrane-bound form of LIN-3 that is retained on the plasma membrane of the AC induces the 1° fate in the adjacent VPC P6.p through juxtacrine signaling . It is possible that the reporter constructs used were lacking some of the regulatory sequences necessary to drive strong lin-3 expression in the VPC lineage. Other potential sources of LIN-3 may be the posterior ectoderm or the excretory system in the head. However, it seems unlikely that LIN-3 secreted from cells at the anterior or posterior end of the animal influences vulval induction, since we did not observe a bias favoring the induction of anterior or posterior VPCs in the absence of the AC.Three lines of evidence indicate that ROM-1 functions in the signal-receiving VPCs rather than the signal-sending AC. First, a omboid-1  suggest he gonad  and by tal cells , had essL4 stage , and occrom-1::nls::gfp are highest in the proximal VPCs  that adopt 1° and 2° vulval cell fates, suggesting that the proximal VPCs are competent to secrete LIN-3 in response to the inductive AC signal. LIN-3 from the proximal VPCs may facilitate the induction of the more distally located VPCs by paracrine signaling  mutation, including the ovulation defects (unpublished data). This may explain why loss of rom-1 function causes neither the larval lethality nor the sterility observed in lin-3 mutants. Furthermore, our data indicate that LIN-3L function in the VPCs almost completely depends on ROM-1 activity. It seems improbable that the 15 amino acid insertion in LIN-3L could change the substrate specificity toward the ROM-1 protease. A more likely explanation for the inherent difference in the dependence of the LIN-3 splice variants on ROM-1 is suggested by the experiments performed with Drosophila Spitz  as well as the newly identified longer variant (LIN-3XL) differ by 15 and 41 amino acid insertions in the juxtamembrane region just prior to the predicted Rhomboid cleavage site at the start of the transmembrane domain . Our expla Spitz . When exla Spitz , tissue-tebrates . Differetebrates . The tisCaenorhabditis elegans  (dpy-19(e1259), lin-12(n137gf), rom-1(zh18) (this study), rom-2(ok966) (C. elegans Gene Knockout Consortium), and unc-119(e2498); LGIV: let-60(n1046) (lin-3(n1049), unc-5(e53), unc-44(362), lin-45(sy96), unc-24(e138), mec-3(e1338), dpy-20(e1282), egl-38(n578), and mec-3(n3197); LGX: sem-5(n2019) and lin-15(n309); extrachromosomal and integrated arrays: zhEx22, zhE66, zhEx72, zhEx68, zhEx69,zhEx73, zhEx78, zhEx81, zhE88, zhEx89, syIs12[hs::lin-3extra] ], huIs7 , and pTG96  were used as a cotransformation markers ] was integrated in the genome animals following γ-irradiation with 3,000 Rad to generate the array zhIs5 and backcrossed six times before analysis. Double and triple mutants were constructed using standard genetic methods. Where cis-linked markers were used they are indicated in the table legends.Standard methods were used for maintaining and manipulating  elegans . The C. -1(mu38) ; LGIII: 0(n1046) , lin-3] , gaIs36[-Dmek-2] , and arI17::cfp] . Unless 17::cfp] .The cons markers . The extrom-1::nls::gfp reporter construct (pRH2) was generated by ligating a HindIII-NheI–restricted 6,998-bp genomic fragment spanning the entire 5′ upstream region of F26F4.3 isolated by PCR amplification with the primers OAD49 (5′-GGAAGCTTGCATGCCCAACGAAATCGATA-3′) and OAD59 (5′-GGGCTAGCCATGTTGTGGAGAAGGAGAAC-3′) into the HindIII-XbaI site of pPD96.04. The rom-1::gfp translational reporter construct (pAD31) was generated by PCR amplification of a 3,146-bp genomic fragment containing 1,849 bp of 5′ sequences and the entire rom-1 ORF using the primers OAD47 (5′-GACTCTAGAGTTGTCAAAAGGTCACGGG-3′) and OAD51 (5′-ATCCTCTAGAGTTGAGCAATTTTCGTTGTTCCAC-3′') followed by XbaI restriction and ligation to XbaI-digested vector pPD95.75. The upstream promoter region of this construct was further extended by replacing a 420-bp PstI fragment with a 2,099-bp PstI genomic fragment corresponding to positions –1,432 and –3,531 relative to the predicted translation start codon of F26F4.3. The lin-31::rom-1 construct (pAD16) was generated by ligating a 1,601-bp SalI-NotI fragment spanning the entire rom-1 coding sequence amplified with the primers OAD44 (5′-TTTTGGTCGACCTCCTTCTCCACAAC-3′) and OAD45 (5′-TTTGGCGGCCGCCTATGAGCAATTTTCG-3′) into the SalI-NotI site of the pB253 vector  showed strong and specific GFP expression in the AC beginning in the mid L2 stage as reported  and OAD32 (5′-CGTATCTGCAGAATCCAACTCGATATTAATTAC-3′) using first-strand cDNA synthesized from mixed-stage total RNA as template. The PCR-amplified products were size-fractionated by agarose gel electrophoresis, cloned into the pGEMT vector (Promega), and sequenced to identify clones encoding individual splice variants. To obtain full-length lin-3 cDNA construct (pAD27), a 1,996-bp XhoI fragment from the EST clone yk1053b07 (confirmed to encode full-length lin-3XL cDNA by DNA sequencing) was first subcloned into the XhoI site of a modified pBluescriptSK  vector (pAD23) in which the PstI site had been destroyed by restriction with EcoRV and SmaI and religation of the resulting blunt ends. To generate full-length lin-3S and lin-3L cDNA constructs , 1,065-bp and 1,110-bp PstI fragments specific for each splice variant isolated from the partial cDNA clones described above were used to replace the 1,188-bp PstI fragment in the full-length lin-3XL cDNA construct (pAD27). The lin-31::lin-3S and lin-31::lin-3L constructs were generated by cloning the 1,133-bp and 1,178-bp XhoI cDNA fragments of the S and L splice variants into the SalI site of pB253  were generated by cloning a 6.1-kb genomic fragment spanning the entire ORF of lin-3 and 574 bp of 5′ and 236 bp of 3′ sequences amplified with the primers OAH137 (5′-CCAGAAAGTTCATGTGAATCAT-3′) and OAH138 (5′-TCACAGGAACTGAGAGGGAGAGTG-3′) into the pGEMT vector. From this construct, a 6,206-bp ApaI-SacI fragment was subcloned into pAD23 to obtain pAH62. The minigenes encoding each of the splice variants were obtained by replacing the 2,728-bp lin-3 genomic PstI fragment with 1,065-, 1,110-, and 1,188-bp PstI fragments isolated from cDNAs of the different splice variants. To construct the lin-31::lin-3 hairpin plasmid (pAD35), a 964-bp NdeI-HindIII lin-3S cDNA fragment from pAD25 was cloned into NdeI-HindIII–digested pAD27 using the recA–E. coli SURE strain as host to obtain pAD32. The resulting 1,918-bp lin-3 hairpin fragment was excised with XhoI from pAD32 and subcloned in E. coli SURE into the SalI site of the pB253 vector to obtain pAD35.The transcriptional 3 vector . To genethe ACEL , was clopromoter . Transgereported . The Kpnof pB253 . The linrom-1 function was generated by in vitro transcription using a 350-bp rom-1 cDNA fragment corresponding to the nucleotides –17 to 725 relative to the predicted start codon of the ORF inserted into pGEM-T (Stratagene) as template. Transcripts were prepared using T7 and Sp6 RNA polymerase and annealed prior to injection as described  deletion mutant was isolated from an ethyl methanesulfonate–mutagenized library consisting of approximately 106 haploid genomes as previously described . The mutant strain was backcrossed six times against N2 before further experiments were done.The escribed . DNA pooVulval induction was scored by examining worms at the L4 stage under Nomarski optics as described . The numhttp://www.ncbi.nlm.nih.gov/) accession numbers of the Rhomboid genes discussed in this paper are C.e. ROM-1 (AAA91218), C.e. ROM-2 (CAA82377), C.e. ROM-3 (CAB55154), C.e. ROM-4 (CAB55122), C.e. ROM-5 (AAF60768), D.m. Rho-1 (CAA36692), D.m. Rho-2 (AAK06752), D.m. Rho-3 (AAK06753), D.m. Rho-4 (AAK06754), D.m. Rho-6 (NP_523557), H.s. Rho-1 (CAA76629).The GenBank (


This study describes the 24-h changes in plasma prolactin levels, and dopamine (DA), serotonin (5HT), gamma-aminobutyric acid (GABA) and taurine concentration in median eminence and adenohypophysis of newborn male rabbits.Animals were kept under controlled light-dark cycles , housed in individual metal cages, and fed ad libitum with free access to tap water. On day 1 after parturition, litter size was standardized to 8–9 to assure similar lactation conditions during the experiment. Groups of 6–7 suckling male rabbits were killed by decapitation on day 11 of life at six different time points during a 24-h period.Plasma prolactin levels changed significantly throughout the day, showing a peak at the beginning of the active phase (at 01:00 h) and a second maximum during the first part of the resting phase (at 13:00 h). Median eminence DA concentration also changed significantly during the day, peaking at the same time intervals as plasma prolactin. A single maximum (at 13:00 h) was found for adenohypophysial DA concentration. Individual adenohypophysial DA concentrations correlated significantly with their respective plasma prolactin levels. A maximum in median eminence 5HT concentration occurred at 21:00 h whereas adenohypophysial 5HT peaked at 13:00 h. Median eminence 5HT concentration and circulating prolactin correlated inversely. In the median eminence, GABA concentration attained maximal values at 21:00 h, whereas it reached a maximum at 13:00 h in the pituitary gland. Median eminence GABA concentration correlated inversely with circulating prolactin. In the median eminence, taurine values varied in a bimodal way showing two maxima, at the second half of the rest span and of the activity phase, respectively. In the adenohypophysis, minimal taurine levels coincided with the major plasma prolactin peak (at 01:00 h). Circulating prolactin and adenohypophysial taurine levels correlated inversely.The correlations among the changes in the neurotransmitters analyzed and circulating prolactin levels explain the circadian secretory pattern of the hormone in newborn male rabbits. The mechanisms that regulate prolactin secretion are complex . Two majIt is well known that basal secretion of prolactin varies throughout the day, describing a characteristic pattern with maximal values close to the light-dark transition ,8. Such The rat is very immature at birth, so that newborn and suckling rats are very sensitive to manipulations that can affect adulthood -18. CircThe rabbit is probably the best-studied laboratory animal in the wild, due to its abundance, size and importance as an agricultural pest ,24. WildIn contrast to the large amount of information available on circadian rhythms in adult mammals, studies on circadian phenomena in neonates are few ,27. For This study was performed using 24 multiparous, lactating Californian × New Zealand White crossbreed doe rabbits. Animals were housed in research facilities of the Animal Production Department. They were maintained under controlled light-dark cycles , housed in individual metal cages, fed at libitum using a commercial pellet diet  with free access to tap water. On day 1 after parturition, litter size was standardized to 8–9 by adding or removing kits to assure similar lactation conditions during the experiment. This study was performed according to the CEE Council Directive  for the care of experimental animals. Groups of 6–7 suckling male rabbits were killed by decapitation on day 11 of life at six different time points throughout a 24-hour cycle. The brains were quickly removed, and the median eminence and the anterior pituitary were taken out. Anterior pituitaries were weighed and homogenized in chilled (0–1°C) 2 M acetic acid. After centrifugation , the samples were either analyzed for DA and 5HT or boiled for 10 min and further centrifuged at 14000 rpm for 20 min to measure GABA and taurine.L-RP-1. Hormone was labeled with 125I by the chloroamine-T method [Staphylococcus aureus  was used to precipitate the bound fraction [Plasma prolactin levels were measured by a specific homologous RIA method  using AFT method . The volfraction . All sam2/H+ reference electrode were: conditioning electrode: -0.4 V; preoxidation electrode: +0.10; working electrode: +0.35 V. Indoleamine and catecholamine concentration was calculated from the chromatographic peak heights by using external standards and was expressed as pg/μg protein. The linearity of the detector response for DA and 5HT was tested within the concentration ranges found in median eminence and adenohypophysial supernatants.DA and 5HT concentration was measured by high pressure liquid chromatography (HPLC) using electrochemical detection , as described elsewhere . A C-18 Amino acids were isolated and analyzed by HPLC with fluorescence detection after precolumn derivatization with O-phthalaldehyde (OPA) as described elsewhere . An aliqStatistical analysis of results was performed by a one-way analysis of variance (ANOVA) followed by post-hoc Tukey-Kramer's multiple comparisons tests. Curve estimation in regression analysis was made by using SPSS software, version 10.1 . P values lower than 0.05 were considered evidence for statistical significance.Figure Figures 2 = 0.16, b0 = -123.7 and b1= 18.1 .Median eminence DA concentration changed in a bimodal way as a function of time of day, showing two maxima, coinciding with those of plasma prolactin at the active and resting phase of the diurnal cycle .As shown in Figure 2= 0.21, b0 = 25.7 and b1 = -0.22, F = 6.6, p < 0.01).Figure 2= 0.42, b0 = 11.6 and b1 = -0.11, F = 17.4, p < 0.0001).Figure The present study, performed in neonatal male rabbit pups sacrificed at 6 different time intervals during a 24-h cycle, describes for the first time significant changes in plasma prolactin levels throughout the day. In concomitant measurements of median eminence and adenohypophysial concentration of DA, 5HT, GABA and taurine, a clear daily pattern was found in almost every case. Contrasting with neonatal rats that did not display any circadian pattern of plasma prolactin , a dailyIn adult rabbits, daily patterns of prolactin secretion depend on light/dark phases . The preThe activity of several nuclei of rabbit hypothalamus increases with age and with experience of anticipatory arousal . HoweverAmong other possible neuromodulators of prolactin secretion, the arcuate nucleus receives a dense serotonergic innervation consisting of a population of brainstem neurons arising mainly from the midbrain raphe nuclei  and fromTaurine has also been implicated in the regulation of prolactin release ,13,35,36A relatively dense innervation of GABA terminals exists in the external layer of the median eminence , and theGABA acting on specific receptors in the anterior pituitary has been reported to suppress prolactin secretion ,40, althIn suckling male rabbits plasma prolactin and median eminence and anterior pituitary concentration of several neuromodulators change on a daily basis. The existence of significant correlations among several of the neurotransmitters analyzed and plasma prolactin levels may explain the circadian secretory pattern of prolactin at this age in suckling rabbits. Collectively, the present results differ from the reported absence of circadian rhythmicity of prolactin and median eminence and adenohypophysial neuromodulators in rats at a comparable age.The author(s) declare that they have no competing interests.MPA and PC carried out the experiment and the immunoassays and the analysis of catecholamines, indoleamines and amino acids. DPC and AIE designed the experiments. Also, DPC performed the statistical analysis. PR took care of the experimental animals. AIE supervised its technical implementation and drafted the manuscript. All authors read and approved the final manuscript.


We assessed the applicability of the T&C test as an accurate and convenient means to screen for dementia in primary care and community settings.The study group comprised 59 patients and 405 community participants, all of who were aged 65 years and over. The time component of the T&C test evaluated the ability of a subject to comprehend clock hands that indicated a time of 11:10, while the change component of the T&C test evaluated the ability of a subject to make 1,000 Won from a group of coins with smaller denominations .The T&C test had a sensitivity and specificity of 73.0 and 90.9%, respectively, and positive and negative predictive values of 93.1, and 66.7%, respectively. The test-retest and interobserver agreement rates were both 95% (κ = 0.91) . The association between the T&C test and K-MMSE test was modest, while significant . The T&C test scores were not influenced by educational status.We conclude that the T&C test is useful as supplemental testing of important domains  to traditional measures such as the MMSE. However, because T&C test is simple, rapid, and easy to use, it can be applied conveniently to elderly subjects by non-specialist personnel who receive training. Dementia, an acquired persistent impairment of cognitive functioning is an increasingly common problem in Korea, and is associated with increased morbidity and mortality, functional loss, caregiver burden, and institutionalization . NeverthScreening for dementia has been recommended to increase detection of dementia. The Mini-Mental State Examination (MMSE) is a brief screening test that quantitatively assesses the cognitive status of elderly people ,5. AlthoWe have therefore introduced a T&C test, and assessed the applicability of the T&C test as a convenient and accurate means to detect early stage dementia in primary care and community settings.One study group initially comprised 60 participants who either visited or were admitted to a hospital that was located in an urban area, namely Gwangju city (referred to forthwith as area A) between November and December 2001. Of these 60 participants, 37 were diagnosed with Alzheimer's disease or vascular dementia, 11 had mental illness such as schizophrenia or depression, 7 had organic brain disease such as cerebral apoplexy or Parkinson's disease, and 5 were alcoholics.Another study group initially comprised 412 participants who were recruited from all residents of Jangseong-county, Jeonnam province, South Korea, aged 65 and over in 2002(referred to forthwith as area B). The area consists of 11 towns and had an estimated population 54,528 of whom about 16.5% were aged 65 and over. The subjects were selected from each stratum (town) using cluster sampling. All subjects in whom vision or hearing was impaired were excluded from the study. We selected 59 (of 60) and 405 (of 412) participants from area A and B, respectively. All participants gave informed consent, and when participants with dementia cannot provide informed consent, caregivers were asked to provide it.The 'time' component of the T&C test evaluated the ability of a subject to comprehend that the hands of a clock indicated 11:10. The diameter of the clock was 15 cm, and the distance between the clock and the participant was 25–35 cm . When the subject responded incorrectly on the first attempt, the interviewer posed the same question, i.e., a second attempt was permitted. The time (in seconds) that it took for the participant to respond correctly was recorded. A time limit of 60 s for a response was imposed.In the 'change' component of the T&C test, the participants were required to make 1,000 Won from a group of coins of several smaller denominations, namely one 500, seven 100, and seven 50 Won coins, that were placed on a table in front of the participant. The participants were given 120 s to complete the task, and an additional 120 s was granted if the subject failed to complete the task at the first attempt. The time (in seconds) that it took for the participant to complete the task was recorded by the interviewer.The participants who completed both of the aforementioned tasks of the T&C test successfully were determined to be negative for suspected dementia, whereas participants who failed to complete either or both of the tasks were determined to be positive for suspected dementia. The T&C test was performed prior to the other cognitive tests by an interviewer who was unaware of the results of the other tests.In the participants from area A, the T&C test was conducted by a nurse, while a physician who were blinded to the result of the T&C test performed a clinical examination and neuropsychiatric inventory. The diagnostic criteria for dementia were based on those of the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition) . To deteThe participants from area B were interviewed by interviewers who had undergone sufficient training to be able to conduct the K-MMSE and T&C test.All participants were interviewed for data on social and demographic factors such as address, age, sex, educational status, and the number of family members living together.To assess the validity of the T&C test as a method to screen for dementia in the participants from area A, we compared the results of the test to a reference standard (diagnosed by a physician), and evaluated sensitivity, specificity, and positive and negative predictive values. To assess the test-retest reliability, the one type of interviewer  conducted the same test twice at an interval of 24 h (n = 22 participants). To assess the interobserver reliability, two different types of interviewer  conducted the same test, at an interval of 24 h (n = 22 participants).To assess the applicability of the T&C test as a method to screen for dementia in the participants from area B, the association between the T&C and K-MMSE test scores was evaluated.The sensitivity, specificity, and positive and negative predictive values and 95% Confidence interval of the T&C test were analyzed. After classifying the participants from area B into a group in which dementia was suspected, and another group in which dementia was not suspected based on the results from the T&C test, a Student's t-test was used to compare the total K-MMSE score with the scores for each of the components of the K-MMSE test, and Spearman's rank correlation was used to analyze the correlation between the T&C and K-MMSE test scores. SPSS for Windows  and Stata Software 6.0  were used to analyze data and statistics.For the participants from area A, the average age was 73.2 ± 7.9 years, 55.9% of the group was female, the average number of years of education was 4.2 ± 5.4, and 62.7% lived alone. For the participants from area B, the average age was 73.1 ± 6.1 years, 58.5% of the group was female, the average number of years of education was 3.1 ± 4.0, and 29.0% lived alone  .There was a significant difference between the total K-MMSE score and the scores for each of the components of the K-MMSE test between participants that were classified as positive and those that were classified as negative for dementia according to the T&C test Table . The assThe results of a logistic regression in which the T&C test as a dichotomous and dependent variable was performed with education status adjusting for age and sex revealed no association between them. The odds ratio for T&C test associated with educational status is 0.877 (95% CI = 0.766–1.004).For the time task in the T&C test, 75.8% of the participants produced a correct response on the first attempt after 6.3 ± 6.7 s, and 45 participants (11.1%) produced a correct response on the second attempt. For the change task in the T&C test, 81.2% of the participants produced a correct response on the first attempt after 12.7 ± 14.2 s, and 43 participants (10.6%) produced a correct response on the second attempt. 34 participants(8.4%) were tested twice for both the time and change task. None of the subjects refused to respond during the tests.Interracial variability in both the etiology of dementia and the accuracy of cognitive testing suggests that there is an urgent need to develop racially appropriate methods of cognitive assessment. The rate of vascular dementia due to cerebrovascular disease is much higher in Koreans than in other races; this is due to insufficient prevention, diagnosis, and treatment of hypertension, diabetes, and hyperlipidemia in Korea. Vascular dementia, unlike Alzheimer's disease, can be prevented by appropriate treatment and prevention for the risk factors of cerebrovascular diseases, can be treated to improve symptoms and inhibit progression of the disease.In the present study, the original T&C test of Inouye et al.  was modiWhen compared to the results of Kawamato , in whicIn elderly patients, the assessment of cognitive function is affected by psychological factors and by the circumstances under which the tests are conducted. In our measurement of the reliability of the T&C test, the test-retest and interobserver agreement rates were both remarkably high . In a study by Inouye et al.  the testIn the present study, no association was observed between T&C test scores and educational status. This result may be explained by the fact that interpreting the hands of a clock and calculation using change are behaviors that are common to all people during daily life, irrespective of race and education. In addition, unlike the language-focused MMSE, the questions in the T&C test cannot be misinterpreted or misunderstood, and are effective for assessing calculation ability and attention. Clock test (similar to T&C test) is less likely to be confounded by educational attainment , and meaThis study has several important limitations. First, while the hospital patients underwent an evaluation of their medical history as well as neurological and physical examinations before a diagnosis of dementia was made, the elderly community sample were evaluated using only the K-MMSE as a reference. Second, the hospital patients would appear to be a rather unusual sample in that there was a high prevalence of psychiatric morbidity in the non-demented subjects. Third, dementia in the present study was not classified as vascular or Alzheimer's-type, nor did we consider the severity of symptoms. Finally, the T&C test has limitations in assessment of a wide range of deficits associated with dementia. However, because T&C test is simple, rapid, and easy to use, the T&C test may pose particular advantages in primary care and community settings where frequent assessment of cognitive functioning is required. The T&C test adds supplemental testing of important domains  to traditional measures such as MMSE. In addition, because the T&C test is less influenced by educational status, it may be particularly useful in populations with diverse educational and cultural backgrounds.We conclude that the T&C test is useful as a supplemental testing of important domains  to traditional measures such as the MMSE, because sensitivity of T&C test is not great and the association between the T&C test and MMSE is modest. However, because T&C test is simple, rapid, and easy to use, it can be applied conveniently to elderly subjects by non-specialist personnel who receive training. In addition, the T&C test is less influenced by educational statusThis work was supported by grants from Chonnam National University Hospital (CUHRI-U-200241).The author(s) declare that they have no competing interests.JAR conceived of the study, collected data and drafted the manuscript. EKC participated in its design, performed data collection, and reviewed the manuscript. MHS participated in data analysis and reviewed the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


The project should lead to large-scale models of molecular and cellular processes involved in neuronal signaling. A prerequisite is the proper storage of knowledge coming from the literature.DopaNet DopaNet Molecular Pages are highly structured descriptions of quantitative parameters related to a specific molecular complex involved in neuronal signal processing. A Molecular Page is built by maintainers who are experts in the field, and responsible for the quality of the page content. Each piece of data is identified by a specific ontology code, annotated  and linked to the relevant bibliography. The Molecular Pages are stored as XML files, and processed through the DopaNet Web Service, which provides functionalities to edit the Molecular Pages, to cross-link the Pages and generate the public display, and to search them.DopaNet Molecular Pages are one of the core resources of the DopaNet project but should be of widespread utility in the field of Systems Neurobiology. Genes to Cognition consortium . Anot. AnotGen Gilman ,4), whic, whicGenSimilarly, we started at the end of 2001 the mesotelencephalic dopamine consortium . The inA first step, prior to the design of large-scale dedicated experiments, consists in data mining the current literature in molecular and cellular neurobiology for existing quantitative knowledge. The resulting data has to be properly stored and annotated.α and βγ subunits. The information collected deals with the structure of the complex, its anatomical distribution within DopaNet target cells, and its functional properties. Each page is under the responsibility of its maintainer(s), who decide which data is to be included or not, and acknowledge the input of the various contributors. All the data included in a Molecular Page is annotated , and linked to bibliographic references. In addition, each single data stored in the databases of DopaNet is attached to one or several terms of the DopaNet Neuronal Ontology. This ontology will therefore act as a glue, relating the various pieces of data one to the other.A DopaNet Molecular Page is a collection of annotated numerical data relative to a "molecular complex" present in one or several DopaNet target cells. The "molecular complex" is taken here in the sense of the DopaNet Neuronal Ontology (see below), as a "stable assembly of molecules", a "molecule" being described as a "set of atoms linked together by covalent bounds". As a consequence, we can have a Molecular Page storing data relative to a molecular complex made up of components, that are themselves described in other Molecular Pages. An anticipated example is an heterotrimeric G protein and its 2(beta2)3 nAChR. Each term can be the child of several others. Therefore the complete picture is not a genealogical tree, but rather a network or relationships.An ontology is defined here in its information science meaning, as a hierarchical structuring of knowledge. In our case, it is a relational vocabulary, that is a set of terms linked together, aiming to describe a neuron. Each term has a definition and a unique identifier. Terms are related by "is a" inheritances, which represent sub-classing, and "part of" inheritances which represent deepening knowledge. For instance, the nicotinic receptor subunit alpha6 "is a" nicotinic receptor subunit, and is "part of" the There are several biological ontologies, the most famous (and complete) being Gene Ontology ,10). Num. Num10])A Molecular Page is made up of a header followed by several lists, each list containing a sequence of identical elements. There are currently twelve main lists, described below. Several other lists of items are used to described the page data at a finer level. According to the molecular complex described in the page, some of the lists can be empty.The Molecular Page header contains the name of the molecular complex described in the page, an abbreviation, the unique ontology code used to identify the page, the dates of creation and last modification of the page, and the page status. The possible status are:The Molecular Page has been submitted by the maintainers and is ready for public release.A new version of the Molecular Page, not yet ready for public release.A new Molecular Page in construction, that has never been submitted for public release.α4)2(β2)3 is described in the Page describing the complex "_2(beta2)_3 nAChR" . The related Ontology term can be found at For instance, the nicotinic acetylcholine receptor _2(beta2)_3 nAChR"                         status="stable">                         The main lists that compose a Molecular Page are:Maintainers are the only people authorized to directly modify the Molecular Pages. They are responsible for the quality and the completeness of the data included in the Page. However, maintainers are not assumed to systematically gather the information all by themselves. They are encouraged to contact experts to help them. Helpful people should be acknowledged as contributors.Contributors are all the people who bring new information about a Molecular Page, or correct an existing piece of information. Contributors can be seen as the equivalent of authors of an article. Except maintainers (who are contributors by definition), they cannot directly modify a Molecular Page. They have to contact a maintainer instead. Note that the database administration team can directly modify the Molecular Pages to comply with the guidelines.A Molecular Page describes a molecular complex. This complex is made up of components (at least one). The listOfComponents describes those components, their stoichiometry, and lists useful related resources. Each component is annotated by its ontology code.α4 and the subunit β2.The complex "_2(beta2)_3 nAChR" [DA:0000027] is made up of two components, the subunit <listOfComponents> <component DopaNetontology="DA:0000188"  name="alpha4 nicotinic receptor subunit"                     stoichiometry="2">                     <listOfResources>   <resource identifier="ACHa4hosa"     name="Ligand-Gated Ion Channel database"                     references="1"                   "> url="                    <taxon>Homo sapiens</taxon>             </resource>     </listOfResources>     </component>  </listOfComponents> The function of a molecular complex is most often modulated by permutations between various states . Accordingly, most of the quantitative data are actually relevant only for one state or a subset of states. Those states should therefore be listed, described and annotated. The quantitative data described in the "functional" lists (see below) will refer both to the states of the molecular complex itself, listed here, and the list of states of other relevant Molecular Pages.The complex "_2(beta2)_3 nAChR" may exist under (at least) three different states: "basal", "active", and "desensitized".<listOfStates>  <state identifier="basal" name="basal">         <description>                In the basal state, the ionic pore is closed. This state displays a weak affinity for agonists such as acetylcholine or nicotine.                 </description>                 </state>       </listOfStates>  A list of properties that depends solely on the molecular complex itself, and not on its relationships with other entities, such as ligands or substrates. Example of such properties are molecular weight or Stoke radius.<listOfGenericProperties>  <property name="MW" stateMolecule="basal">        <taxon>Homo sapiens</taxon>            <listOfValues>                <value mean="310971" unit="Dalton">                        <comment>without covalent modifications.</comment>                             </value>                        </listOfValues>                 </property>        </listOfGenericProperties>  listOfExtracellular shall be necessary at some point.The distribution of the molecular complex and its components is described within the relevant DopaNet target cells: cortical glutamatergic pyramidal neuron, mesencephalic dopaminergic neuron, striatal cholinergic interneuron, striatal enkephalinergic/GABAergic medium spiny neuron, striatal substance p/GABAergic medium spiny neuron. It is likely that a Each cell is divided into compartments, where the distribution of transcripts and molecules can be described. The approach used to explore the distribution is specified, since both the accuracy and the quantitativeness of the observations strongly depends on the method chosen. As for all the following data, the species where the study has been conducted is also mandatory.α4 is present in 100% of neurons and β2 is probably also present in 100% of neurons.One entry in the complex "_2(beta2)_3 nAChR" is the fact that in the cell soma of the rat mesencephalic dopaminergic neuron, single cell RT-PCR experiments showed that <listOfCells>  <cell cellName="mesencephalic dopaminergic neuron" DopaNetontology=" DA:0000702">    <listOfCompartments>         <compartment DopaNetontology="DA:0000137" name="cell soma">           <listOfTranscripts>               <transcript method="single cell RT-PCR" references="17">                     <taxon>Rattus norvegicus</taxon>                         <description>                            a4 is present in 100% of neurons. b2 is probably also present in 100% of neurons.                               </description>                           </transcript>                     </listOfTranscripts>               </compartment>          </listOfCompartments>      </cell>  </listOfCells>The ligands of a molecular complex are molecules or ions that bind to it. The size of the ligand relative to the molecular complex is irrelevant. Within the Molecular Page of "transforming growth factor receptor type I", one ligand is "transforming growth factor betal". Conversely, in the Molecular Page of "transforming growth factor beta1", one ligand is "transforming growth factor receptor type I"! See table kon, koff or Km can be stored in a controlled manner, in order to be easily retrieved later. Whenever possible, the quantitative values are related to the states of the molecular complexes involved, not only the state of the molecular complex subject of the Molecular Page but also the state of the ligand. This remark holds for the substrates, the translocators and the modulated substances as well (see below). See table Functional parameters such as The desensitized "_2(beta2)_3 nAChR" of the rat binds acetylcholine with a Ki versus the epibatidine of 8.6 ± 1.98 nM.<listOfLigands>   <ligand DopaNetontology="DA:0000184"       name="acetylcholine"                    origin="endogenous">                    <listOfProperties>          <property name="Ki_epibatidine"               references="10 18"                          stateMolecule="desensitized">                             <taxon>Rattus norvegicus</taxon>                <listOfValues>                 <value mean="8.6" sd="1.98" unit="nanomole per litre"/>                      </listOfValues>                 </property>             </listOfProperties>         </ligand>       </listOfLigands> Km, kcat or Vmax.All substances modified as a result of an interaction with the molecular complex. The parameters stored here are for instance The translocators are substances that go from one subcellular compartment to another, the translocation being mediated by the molecular complex. Typical parameters are conductance or relative permeability.The active complex "_2(beta2)_3 nAChR" of the rat translocates cations with a conductance of 13.3 ± 1.5 pS.<listOfTranslocators>  <translocator DopaNetontology="DA:0000264"            name="cation"                         origin="endogenous">                              <listOfProperties>          <property name="conductance" references="14" stateMolecule="active">            <taxon>Rattus norvegicus</taxon>                   <listOfValues>                 <value mean="13.3" sd="1.5" unit="picosiemens"/>                        </listOfValues>                  </property>            </listOfProperties>          </translocator>       </listOfTranslocators>  modulated entries are to be avoided as much as possible, since they generally reflect a set of binding and/or enzymatic events.In many case, one knows about the effect of a molecular complex on a substance, without knowing the detailed mechanism of action. The Possible conversions between the states described in the listOfStates, such as a conformational transition, or a covalent modification.The complex "_2(beta2)_3 nAChR" undergoes conformational transitions between the basal and active states. <listOfTransitions>  <transition state 1="basal" state2="active">        <comment>                       In the absence of ligand, the equilibrium is strongly displaced toward the basal state. Agonists, such as acetylcholine and nicotine, stabilise the active state and shift the equilibrium. The transition from basal to active corresponds to an opening of the ionic pore.                  </comment>             </transition>       </listOfTransitions>  The list of bibliographic resources used to gather and annotate the data. Each piece of data included in the Molecular Page should be linked to those bibliographic items by internal references.Molecular Pages are saved as XML files , and theMolecular Pages XML files are stored within two different repositories, depending on the status of the Page. One repository contains only the stable Pages ready for public release , while the other repository contains also the unstable and the forthcoming Pages .In addition to the two XML repositories, there is also a third HTML repository, containing the human-readable HTML versions of the stable Pages, automatically generated from their XML counterparts using XSL Transformations  with theDopaNet Web Service has been designed and implemented, which provide functionalities to:As described above, Molecular Pages are continuously modified and updated by the maintainers, with the help of the contributors. In order to automatize, safe-guard and simplify as much as possible the work required by a maintainer to create and edit a Molecular Page, an application called the 1. authenticate Page maintainers2. browse pages by maintainers3. grant exclusive Page editing rights to a maintainer4. create and edit a Page via a rich user interface5. save or submit the edited Page, setting the "stable/unstable" statusThe DopaNet Web Service is made of both server-side and client-side components, all written in Java, and communicating via either the SOAP  or the HIn addition to support the remote creation and editing of Molecular Pages, the DopaNet Web Service provide also functionalities to:1. register new DopaNet contributors2. update existing DopaNet contributor information3. browse Molecular Pages by status4. search Molecular PagesSearching of Molecular Pages is implemented by using the API provided by the Apache Xindice  native XAlthough in their early stage of development, DopaNet Molecular Pages provide a unique source of structured, annotated quantitative data about the molecules involved in neuronal signaling. They will feed both the experimental biologist and the theoretician with the best available estimates for all kind of knowledge, whether biochemical, anatomical or functional. This will allow them to design better experiments or formal models, and to benchmark their results. As a side-effect triggered by the mandatory annotations, DopaNet Molecular Pages will also a bibliographic resource, each page being the equivalent of a small review of the literature.Gene Ontology is now a fully grown project, and is being widely used in several biological domains. Nevertheless, in its present form, Gene Ontology was not found suitable to be directly used by the DopaNet project. We hope to collaborate with Gene Ontology maintainers in the future. In particular, effort will be made to complete Gene Ontology in the area of Neurobiology. However, DopaNet Neuronal Ontology will never actually be a subset of Gene Ontology. Indeed, the purpose of the latter is to classify the gene products – and one of its most useful application so far has been the annotation of sequence database entries. The purpose of DopaNet Ontology is broader in term of knowledge, and not limited to the classification of gene products. At the same time it is focused onto a specific system, and therefore of interest for a narrower audience.molecular function, biological process, and cellular component. Only the latter is at the moment relevant to DopaNet purposes, that is the Molecular Pages. However, it is anticipated that the biological process vocabulary will be needed in the near future, for instance to annotate electrophysiological data. DopaNet cellular component vocabulary is larger than Gene Ontology one, since it contains the different kinds of neuronal cells  In additology, .A cellular component may be for instance an anatomical structure, e.g. "dendrite" or "synaptic vesicle" but also a cell or a protein. Note that a "molecule" is defined in the Neuronal Ontology as a set of atoms covalently linked. A molecule cannot contains other molecules. Hence, a protein made up of several subunits, or a polypeptide and a co-enzyme are not "molecules", but "molecular complexes". Although our ontology is built for DopaNet purposes, it can be viewed as a more general "Neuronal Ontology". Therefore, we incorporate terms related to components present (or events taking place) in any neuron, not necessarily DopaNet target cells. In particular, such additions are advised if they clarify some hierarchical relationship.As described above, a "molecular complex" in DopaNet Neuronal Ontology contains one or several components, also present in the "molecule" branch. It could be considered redundant that all monomeric proteins are represented by two terms, as a "molecule" part of a "molecular complex". However, the meanings of the two branches are different. The "molecule" can be seen as an ideal entity, while the latter would rather represent an actual physical object of the cell. Moreover, the hierarchical structures of the two branches are different. In addition, a lot of proteins have only recently been discovered as functional complexes (e.g. the polymeric G-protein coupled receptors), and more are to be discovered. Finally, the systematic dissociation between the functional molecular complex and its components is handy when it comes to write the Molecular Pages.The Alliance for Cellular Signaling was a pioneer in designing Molecule Page. Contrary to DopaNet Molecular Pages, their focus is truly a "molecule" rather than a "molecular complex". For instance, an heteropolymeric receptor will not be represented by a Molecule Page, but rather by a collection of Pages, one per subunit.Kd In addition, pieces of quantitative knowledge, like binding or enzymatic reactions, should be provided in standardized format such as the Systems Biology Markup Language [DopaNet Molecular Pages are highly structured. While this could appear as an obvious choice, it actually comes with a double burden. First, the edition interface has to be sufficiently complex to reflect the underlying structure. This complexity certainly acts as a repellent for the biologist who wish to build a Molecular Page. Second, the high quality required, in particular concerning the annotations, leads to the rejection of a significant portion of the published knowledge. However, we think that a piece of data that cannot be properly annotated is of limited use for the community. For instance, a large amount of pharmacological properties is published without the species. Since those properties vary from one species to the other, one cannot easily re-use the value provided. Similarly, a numerical piece of knowledge cannot be used without caution if one does not know the method used to collect it, and the variability attached to it. Currently, the access to the data is only possible through the web interface. Moreover, although the user is able to search the content of the Molecular Pages using various criteria, the result is always presented as one or several Molecular Pages. However, the DopaNet Web Service should be enriched on a steady pace, and specific pieces of data should be served soon. One can envision interfaces providing precise and meaningful responses to queries like "All Kd for the ligand X of all molecules that bind it", under the form of a list of Language .Ka, Kd, Ki, Kp, IC50, but all those quantities can only be analyzed in regards of the knowledge about the various states of the complex, and its conformational transitions. The distributed annotation can cause concerns related to peer-validation and quality control. With the help of Nature Publishing Group, a peer review process has been set-up by the Alliance for Cellular Signaling to survey the edition of its Molecule Pages. Such an infrastructure is currently out of reach of DopaNet. However, we ensure that the maintainers are always recognized experts in the fields, or, for members of the EBI group, work in close relation. In addition, we included as much as possible guidance through the constraints imposed by the Page editing environment. That way, any Molecular Page complies with at least a minimal set of quality rules. Such an approach has already been successful in other areas. One of the most striking example is the Debian operating system project ([The Molecular Pages are maintained in a distributed fashion, with one or several experts in charge of each complex. Such an approach is mandatory for two reasons. Firstly, the knowledge accumulated by the project will soon become much too large to be handled by one individual, or even one team. Secondly, the level of detail and accuracy sought by the resource is such that only experts can fruitfully mine the adequate literature for relevant information. To extract the simple affinity of a receptor for a ligand can be a daunting prospect. Not only that affinity can be expressed by various parameters with different meaning, project , that mOn the contrary of the Molecular Pages, the Neuronal Ontology is currently developed only by the EBI team. Everyone can contribute by sending their suggestions, but for the sake of coherence the final building is centralized.DopaNet Molecular Pages allow to store annotated numerical data about molecular complexes involved in neuronal signaling. Although the Pages are one of the core resources of the DopaNet project, and therefore their focus on the mesotelencephalic dopamine system, the repository should be of widespread utility in the field of Systems Neurobiology. This is also the case of The DopaNet Neuronal Ontology. The resource is in its early stage of development and will benefit much from the feedback of users.All data contained in the DopaNet Molecular Pages may be copied and redistributed freely, under terms derived from the MIT license ..More information about the DopaNet project can be found at the URL DopaNet ontology is available at the URL DopaNet Molecular Pages are available at the URLNLN designed the database DTDs and schemas, wrote the XSL and acted as the final editorial authority on Molecule Pages. MD implemented all the edition and validation software, as well as the user interface, including the servers.