The liver sinusoidal endothelial cells (LSEC) and Kupffer cells constitute the most powerful scavenger system in the body. Various waste macromolecules, continuously released from tissues in large quantities as a consequence of normal catabolic processes are cleared by the LSEC. In spite of the fact that pig livers are used in a wide range of experimental settings, the scavenger properties of pig LSEC has not been investigated until now. Therefore, we studied the endocytosis and intracellular transport of ligands for the five categories of endocytic receptors in LSEC.125I-labelled molecules: collagen α-chains, FITC-biotin-hyaluronan, mannan, formaldehyde-treated serum albumin (FSA), and aggregated gamma globulin (AGG) was substantial in cultured LSEC. The endocytosis was mediated via the collagen-, hyaluronan-, mannose-, scavenger-, or IgG Fc-receptors, respectively, as judged by the ability of unlabelled ligands to compete with labelled ligands for uptake. Intracellular transport was studied employing a morphological pulse-chase technique. Ninety minutes following administration of red TRITC-FSA via the jugular vein of pigs to tag LSEC lysosomes, cultures of the cells were established, and pulsed with green FITC-labelled collagen, -mannan, and -FSA. By 10 min, the FITC-ligands was located in small vesicles scattered throughout the cytoplasm, with no co-localization with the red lysosomes. By 2 h, the FITC-ligands co-localized with red lysosomes. When LSEC were pulsed with FITC-AGG and TRITC-FSA together, co-localization of the two ligands was observed following a 10 min chase. By 2 h, only partial co-localization was observed; TRITC-FSA was transported to lysosomes, whereas FITC-AGG only slowly left the endosomes. Enzyme assays showed that LSEC and Kupffer cells contained equal specific activities of hexosaminidase, aryl sulphates, acid phosphatase and acid lipase, whereas the specific activities of α-mannosidase, and glucuronidase were higher in LSEC. All enzymes measured showed considerably higher specific activities in LSEC compared to parenchymal cells.Endocytosis of five Pig LSEC express the five following categories of high capacity endocytic receptors: scavenger-, mannose-, hyaluronan-, collagen-, and IgG Fc-receptors. In the liver, soluble ligands for these five receptors are endocytosed exclusively by LSEC. Furthermore, LSEC contains high specific activity of lysosomal enzymes needed for degradation of endocytosed material. Our observations suggest that pig LSEC have the same clearance activity as earlier described in rat LSEC. Togethes (LSEC) ,8.in vivo and in vitro as avidly as do wild-type mice [The endocytic receptors of rat LSEC can be listed in five categories: (i) collagen α-chain receptor (COLLAR), (ii) hyaluronan receptor (HAR), (iii) IgG Fc receptor, (iv) mannose receptor, and (v) scavenger receptor (SR). With specificity for free α-chains of types I, II, III, IV, V, XI collagens, but not native triple helical collagen, the COLLAR function has so far only been described in LSEC ,10. The ype mice . These rin vivo and in vitro clear a SR ligand [We have previously shown that pig LSEC express a functionally active receptor that both R ligand . Until nR ligand . Apart fWe here show that pig LSEC, but not stellate cells, parenchymal cells (PC) or KC, express the same five categories of endocytosis receptors for soluble and colloidal ligands as reported for rat LSEC. Ligands endocytosed via these receptors are transported along the endocytic pathway and degraded. Assays carried out to measure activity of lysosomal enzymes in cells showed that several enzymes are present at higher specific activities in LSEC than in either KC or PC.8 LSEC. Contaminants in LSEC cultures were epithelial-like cells, stellate cells, KC, and PC. Compared with rat and mice PC, the population of PC from pig are much more heterogeneous in size; consequently, pig LSEC cultures may be contaminated by small PC.The fraction of cells collected after density centrifugation in OptiPrep consisted of LSEC, stellate cells, KC, PC and a minor proportion of unidentified cells, possibly of epithelial nature. In this fraction, LSEC were most numerous, closely followed by stellate cells. Further purification, using either elutriation centrifugation or selective adherence, resulted in 80–95 % purity of LSEC. One cycle of elutriation centrifugation resulted in approximately 10Cultures of KC prepared by selective adherence were prepared by incubating the non-PC fraction, for 15 min, on glass dishes coated with glutaraldehyde-treated BSA This procedure yielded 70–80 % pure KC. The use of tissue culture plastic dishes resulted in a lower purity of KC (40–50%), with a higher proportion of LSEC.125I-labelled AGG, FSA, collagen α-chain (COLLA), mannan and FITC-bHA were incubated with purified cultures of pig LSEC with or without the presence of excess amounts of unlabelled substances known to compete with the radiolabelled ligands for endocytosis in rat LSEC. Control experiments showed that endocytosis was substantial following 2 h incubation at 37°C with only radiolabelled ligands. Expressed as % endocytosis of total added ligand, the results were as follows: AGG, 7.1%; FSA, 42.7%; COLLA, 59.7%; mannan, 38.2%; FITC-bHA, 12.1%  inhibited uptake of trace amounts of 125I-AGG by 73%   and ratsFurthermore, in contrast to liver cell isolation from rat and mice, it is not feasible to remove all PC from the pig NPC suspension by neither isopycnic- or elutriation centrifugation due to the large heterogenity in both density and size of the cells. Heterogenity in density was also observed in LSEC, since the cells were found in all tested OptiPrep-layers, with densities varying from 1.038–1.086 g/ml. Moreover, LSEC were elutriated at all flow rates varying from 20–50 ml/min, indicating heterogeneity in cell size. For mass isolation of LSEC by elutriation centrifugation, we obtained purities between 80–95%, very similar to purities obtained by selective adherence.9 purified LSEC from one pig. The addition of the detergent Pluronic acid F-68 in the elutriation buffer eliminated clotting of cells in the elutriation chamber [Taken together, LSEC purified by selective adherence is faster as long as a limited number of cells are needed. Elutriation centrifugation is more time-consuming, but yields a higher cell number, with up to 1.1 × 10 chamber , therebyin vitro degraded endocytosed IgG-complexed antigen much slower than ligands for the mannose- and scavenger receptor, and that the delay was due to slow departure from early endosomes. We also observed that FITC-AGG delayed the transport of TRITC-FSA to the lysosomes when the ligands were given simultaneously. This is consistent with the observation that AGG reduces the amount of 125I-FSA degraded even if the total amount of endocytosed FSA was not changed. Interestingly, we observed no uptake of FITC-AGG in KC.The ligands COLLA, FITC-bHA, mannan, FSA, and AGG were used to probe the endocytic activity in pig LSEC via the COLLAR, HAR, SR, mannose receptor and IgG Fc receptors, respectively. We found that all these ligands were avidly endocytosed in LSEC. Competition experiments showed that the ligands were taken up in a specific manner, via the five different categories of receptors. Moreover, morphologic pulse-chase experiments using FITC-labelled ligands to study the intracellular transport of endocytosed ligand suggested that all ligands studied, with the exception of AGG, reached lysosomal compartments within a time span of 2 h. At that time, AGG was still found in ring shaped structures which is a typical feature of early and late endosomes ,38. The 125I-FITC-bHA was found as low molecular weight material, demonstrating intracellular degradation (results not shown). We stress that, although practically all cultured LSEC accumulated FITC-labelled ligands in vitro, not all LSEC had taken up TRITC-FSA in vivo. We speculate that the explanation for the heterogeneous uptake in vivo was due to circulatory regulation: not all sinusoids may have allowed entrance of blood at the time of injection, thus preventing LSEC from being exposed to the injected ligand in those sinusoids. This is not an unreasonable explanation, since it is known that hepatic sinusoids may regulate blood flow by a sphincter mechanism [FITC-labelled ligands were seen concentrated in small spherical vesicles scattered over the entire cell body after a 10 min chase. These small vesicles are probably early endosomes reminiscent of small bristle coated vesicles that have been previously observed electron microscopically as vesicles with a diameter of 180 nm, located directly below the cell surface . The vesechanism .If LSEC are an important part of the reticulo-endothelial system in the body they also need a high activity of lysosomal enzymes to degrade waste material endocytosed from the circulation. Therefore, we compared the lysosomal activity in LSEC with the metabolically very active PC and the phagocytic KC. Earlier studies in rat have revealed that KC and LSEC, as compared to PC, contain higher specific lysosomal enzyme activities -43, and in vitro scavenger properties resemble their in vivo properties. Once outside their micro-environment in the liver, rat LSEC dedifferentiate and eventually die after 2–4 days on culture dishes. During the first hours in culture only small changes in endocytic capacity of LSEC occur in so far as experiments in our laboratory have shown that pig LSEC cultivated in RPMI for 2 days retain 80% of the endocytic capacity when compared to freshly isolated cells (results not shown). Because of the short cultivation time used in our experiments, the endocytic capacity of the cells would not be influenced by mediators in the medium. But in other experiments with cultures of rat LSEC that were incubated for 18 h or more, it has been shown that inflammatory mediators like tumor necrosis factor-α and interleukin-1β enhance 2–3-fold endocytosis via the SR and mannose receptor, while COLLAR mediated endocytosis remained unaffected [In all our experiments we used freshly isolated cells because we believe that when cells have just been isolated from the intact organ, their affected . Also liaffected . Using iaffected  found a affected ,50 and paffected  which atOur results suggest that pig LSEC are functionally very similar to rat LSEC, at least in terms of clearance activity. It is therefore highly likely that LSEC in pig (as in rat) represent the major site of elimination of an array of soluble waste molecules from the circulation. Several if not all of these waste macromolecules are harmful if allowed to accumulate in the blood. The very active endocytosis in LSEC ensures that these soluble waste macromolecules are never allowed to increase above trace levels in the circulation.125I, and TRITC were from Pierce, Rockford, IL, USA, Institute for Energiteknikk, Norway and ICN Biomedicals Inc., OH, USA. FITC, BSA, 4-methylumbelliferyl-substrates for fluorometric assays of lysosomal enzymes, Triton X-100, mannose, and mannan was from Sigma Chemical Co, St.Louis, MO, USA. Collagenase P was from Boehringer Mannheim, Germany. Fibronectin was kindly donated by Dr. B. Hansen, University of Tromsø, Norway. Human IgG and high molecular weight hyaluronan  were from Pharmacia, Sweden. OptiPrep was from Nycomed, Norway. Human serum albumin was from Octapharma, Ziegelbrucke, Switzerland. Monoclonal mouse anti-human desmin, clone D33, was from DAKO A/S, Denmark. Monoclonal goat anti-mouse IgG, TRITC-conjugate, was from Zymed, CA USA. Two mouse monoclonal antibodies (clones 2G6 and 2B10) against porcine macrophages were kindly provided by Dr. A. Berndt, Institute of Pathology, Friedrich Schiller University, Jena, Germany.1,3,4,6-tetrachloro-3α,6α-diphenylglycoluril (Iodogen), carrier-free NaSus scrofa domesticus, Norwegian strain), weighing 7–8 kg, were fasted for 18 h, drinking water ad libitum, prior to sacrifice. Animals received care according to "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health, NIH publication 86-23 revised 1985.Castrated male piglets (g) to pellet PC, and the resulting supernatant was concentrated by centrifugation (850 g) for 10 min, mixed into a 21% OptiPrep density solution, and centrifuged for 30 min (3300 g). The PC and RBC were pelleted, and a layer consisting of 6 × 108-3.5 × 109 non-PC were recovered. For further purification, 2.0 × 108 non-PC in HBBS solution containing 0.3 % BSA, 0.4 mM EDTA, 200 μg/ml Pluronic acid F-68, and antibiotics were introduced into a standard elutriation chamber of a JE-6B rotor (Beckman Instruments) in a J-21-type Beckman centrifuge at a flow rate of 22 ml/min and a rotor speed of 2500 rpm. The first fraction of 100 ml was enriched in stellate cells as judged by strong staining with anti-desmin antibody. The flow rate was then increased to 35 ml/min, and 150 ml was collected. This fraction contained purified LSEC characterized by their specific accumulation of FITC-labelled formaldehyde-treated serum albumin (FSA). The cells remaining in the chamber were pumped out and discharged after the centrifuge was stopped. These were mainly KC and PC. KC were identified by the immunostaining with two anti-porcine macrophage antibodies. The average numbers of LSEC grown per cm2 were 2.5 × 105 in Falcon dishes (Becton Dickinson, France). An alternative separation technique was also used: the cell suspension of non-PC after the gradient centrifugation was diluted to 4 millions/ml and seeded on glass dishes at a concentration of 5 × 105/cm2 and allowed to attach for 15 min, at 37°C. Prior to use, the glass dishes were washed in 96% ethanol, coated with bovine serum albumin and fixed in 1% glutaraldehyde for 30 min., before being extensively rinsed in distilled water. It was mainly KC that attached well to these dishes. Poorly attached cells that were detached by gentle washing together with non-adherent cells were transferred to fibronectin-coated culture dishes, incubated for another 15 min followed by thorough washing, and supplied with fresh medium to enable attachment and spreading of viable SEC. The purity of these LSEC cultures was between 80–95%.The procedure for isolation of functionally intact LSEC from a single pig liver was as described . Brieflyg). Native triple helical collagen ((Nutacon, Leimunden, The Netherlands) was denatured to single collagen α-chains (COLLA) by incubation at 60°C for 60 min. Biotinylated hyaluronan, bHA, was prepared by incubating HA with Biotin-LC-Hydrazide (Pierce, Rockford, IL, USA) and 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride  in a ratio allowing a maximum of 1 out of 10 carboxyl groups per HA molecule to be labelled [FSA was prepared by treating BSA with 10% formaldehyde in 0.2 M carbonate buffer, pH 10, for 3 days as described . Aggregalabelled .FSA, AGG, collagen, bHA and mannan dissolved in sodium carbonate buffer (0.1 M, pH 9.5) were incubated with FITC or TRITC in a ligand/dye weight ratio of 5:1, at 4°C overnight. To remove unbound dye, the solutions were dialysed against PBS.125I employing Iodogen as the oxidizing agent [6 cpm/μg protein. To radiolabel bHA, FITC was first attached to the biotin, thereby providing an 125I acceptor. For separation of free iodine from radiolabelled FITC-bHA, the reaction solution was dialysed against PBS, giving a final radioactivity of 0,3 × 106 cpm/μg HA.Macromolecular ligands (FSA, mannan, collagen, AGG and FITC-bHA) in PBS were labelled with Nang agent . Radiola125I-ligands (10,000–30,000 cpm per culture) and excess cold ligands. All endocytosis experiments were terminated after an incubation-period of 2 h at 37°C, by transferring the conditioned medium (200 μl), along with 500 μl PBS used for washing of the cells, to tubes containing 500 μl 20% trichloroacetic acid. Following centrifugation of the tubes, the extent of degradation was determined by measuring the radioactivity in the pellets and the supernatants, except for mannan or FITC-bHA where the trichloroacetic acid-precipitation step was omitted due to lack of degradation products being released from the cells. The 125I was attached to the protein core in mannan and to the FITC adduct in FITC-bHA of which both accumulate in the lysosomes, since mammalian cells do not carry degradative hydrolases for these molecules. Cell-associated radioactivity was determined by measuring the amount of 125I released by solubilizing washed cultures in 1% SDS. All experiments were carried out in triplicate.After seeding and cultivation for 2–3 h in 24 well dishes, purified cultures of LSEC were washed and supplied with fresh RPMI 1640 medium containing 1% human serum albumin and trace amounts of one of the six TRITC-FSA (20 mg) in 20 ml physiological saline was administered via the left external jugular vein 90 min prior to liver perfusion. Prior to use, TRITC-FSA was centrifuged at high speed and sterile-filtered in order to remove aggregates which would otherwise be taken up in KC by phagocytosis. Cultures of LSEC were established on fibronectin-coated 14 mm diameter glass coverslips for 3–4 h in serum-free growth medium. The cultures were then washed in PBS, and pulsed in fresh medium with 0.1 mg/ml FITC-ligands for 1 h at 4°C. Because FITC-labelling of proteins remove positive charges, and thus may turn proteins into negatively charged ligands for the SR , FITC-ma2 Falcon culture dishes.Samples of PC were taken from the pellet resulting from the density centrifugation, and solubilized in 0.1 % Triton, whereas KC and LSEC samples were obtained from the solubilizates of cultures seeded on 7.5 cmThe assay conditions of the six enzymes are given in Table -1·gram protein-1 under the conditions stated above.The fluorescence of 4-methylumbelliferone, resulting from the action of the enzymes on the various substrates, was measured in a Shimadzu RF 5000 spectrofluorometer with excitation set at 360 nm and emission at 450 nm. Enzyme activities are given in number of substrate molecules transformed minProtein content in the samples was measured according to Lowry , and BSAThe values are expressed as: mean (standard deviation) unless otherwise noted. We used SPSS 10.0 software package (SPSS, Chicago, IL) for statistical analysis. Analysis of variance (ANOVA) was used to test whether any statistical significance existed between the three cell populations enzyme activity followed by the LSD post hoc comparison test. Probability values of p ≤ 0.05 were considered significant for all tests applied.KHE and GIN designed and carried out the experiments. KHE drafted the manuscript. AR and BS coordinated the study and contributed to the text of the manuscript.


The aim of this research paper is to study job satisfaction of physicians and general practitioners at primary health care institutions during the health care reform in Lithuania.Self-administrated anonymous questionnaires were distributed to all physicians and general practitioners (N = 243, response rate – 78.6%), working at Kaunas primary health care level establishments, in October – December 2003.15 men (7.9%) and 176 women (92.1%) participated in the research, among which 133 (69.6%) were GPs and 58 (30.4%) physicians. Respondents claimed to have chosen to become doctors, as other professions were of no interest to them. Total job satisfaction of the respondents was 4.74 point . Besides 75.5% of the respondents said they would not recommend their children to choose a PHC level doctor's profession. The survey also showed that the respondents were most satisfied with the level of autonomy they get at work – 5.28, relationship with colleagues – 5.06, and management quality – 5.04, while compensation (2.09), social status (3.36), and workload (3.93) turned to be causing the highest dissatisfaction among the respondents. The strongest correlation (Spearmen's ratio) was observed between total job satisfaction and such factors as the level of autonomy – 0.566, workload – 0.452, and GP's social status – 0.458.Total job satisfaction of doctors working at primary health care establishments in Lithuania is relatively low, and compensation, social status, and workload are among the key factors that condition PHC doctors' dissatisfaction with their job. In 1989, after regaining its independence, Lithuania inherited an extremely centralised health care system that mainly conditioned ineffective health care management and resource usage. The above situation was an outcome of the former Soviet health care policy, as it did not encourage people or the state to treasure or safeguard its citizens' health [st Century" [The situation has changed a lot though, and the current Lithuanian health policy states that priority shall be given to the development of primary health care ,2. SpeakCentury" . As regaCentury" .As stated in the announcements of the National Board of Health, the situation in the Lithuanian health system is changing; nevertheless, it faces a number of problems. 78.6 % (1,146 out of 1,458) of GP graduates in 1994–2001 were retrained from district physicians and paediatricians [General medicine practice in health care systems of industrialised countries is considered a remarkably well working system that seeks to assess health needs of individuals and to become familiar with their living conditions, taking into consideration their family and social environment . NeverthOne of the ways to look into the existing PHC situation and possible problems is to study job satisfaction of GPs and physicians, as the attitudes people hold toward their jobs are referred to as job satisfaction and this is one of the most widely studied work-related attitudes. Job satisfaction is closely related to the lower level employee turnover and intentions to quit their jobs -9. SinceThe aim of this research is to investigate job satisfaction of physicians and general practitioners in primary health care institutions during the health care reform in Lithuania. In 2003, there were 1,608 GPs and physicians working in Lithuanian PHC establishments . The larResearch tasks:1. To determine motives for choosing a PHC doctor's profession.2. To compare total job satisfaction of Lithuanian PHC physicians' and GPs' as well as their intentions to recommend their children to choose a PHC level doctor's profession.3. To assess factors which determine job satisfaction of PHC physicians, GPs who chose their specialization at higher school, and GPs who were retrained as GPs later in their career.Job satisfaction comprises positive and/or negative attitudes held by individuals in respect to their job . EmployeBronsky and Cook (1994) interviewed 334 Ohayo University graduate physicians . For theIn 1989 American Physical Therapeutics Association (APTA) carried out a survey of hiring and retention aspects . This stOkerlund et all (1994) research revealed that Utah physicians were satisfied with their job . RespondRozier' et al (1998) found out that intrinsic factors, such as ethical practice, improvement of patient health, and perception of satisfaction were more important than big salary and important title . These gBarnes (1998) conducted research of job satisfaction among rehabilitation physicians, occupational therapy specialists, and speech pathologists. Set apart, there are three factors that describe job satisfaction: external context factors and internal context factors, as well as internal content factors . ExternaSpeakman et al (1996) conducted a study of 106 Texas (USA) doctors' job satisfaction . RespondDowell et al (2000) conducted research, the purpose of which was to examine job satisfaction and stress in the work of GPs in New Zealand. Their job satisfaction level was rather high . HoweverResearch was conducted in October – December 2003. Questionnaires were distributed to all Kaunas PHC physicians and GPs (N = 243) in person. The response rate amounted to 78.6 % (191 questionnaire returned).Total job satisfaction of the respondents was measured on a seven-point scale, where 7 stood for highly satisfied and 1 for highly dissatisfied.Intentions to recommend one's children to choose a PHC level doctor's profession were measured on a seven-point scale, where 7 stood for "strongly agree" and 1 for "strongly disagree".The level of satisfaction with different job characteristics was assessed using a job satisfaction questionnaire that embraced questions related to the PHC work specifics. Eight sections of questions were included in order to assess satisfaction with different job characteristics: ability utilisation at work, workload, colleagues, compensation, creativity, autonomy, management, and social status. All statements were measured on a seven-point scale, where 7 stood for highly satisfied and 1 for highly dissatisfied.2 criteria, degrees of freedom number (df) and statistical significance. Relationship between two independent variables was assessed relying on Spearmen's rank correlation, taking into consideration the value of the correlation ratio and statistical significance. Reliability notation: p < 0.05 – statistically significant, p < 0.01 – highly significant.The survey data was processed using SPSS version 11.00 statistical package for data analysis. The statistical data reliability was checked according to χ15 men (7.9%) and 176 women (92.1%) participated in the research, among which 133 (69.6%) GPs and 58 (30.4%) physicians.The analysis of the distribution of the respondents according to their age and specialization  do not intend to recommend their children to choose a PHC level doctor's profession. The assessment of the respondents' general satisfaction with work and their intentions to recommend their children to choose a primary level doctor's profession did not reveal any statistically significant differences between the specialisations (p > 0.05). However, the research revealed a tendency among physicians to be generally more satisfied with their work and more willing to recommend their children to choose a PHC level doctor's profession: total job satisfaction of physicians was 5.03 and intentions to recommend their children to follow their career comprised 2.56, while those of GPs respectively made 4.64 and 2.19.The study of the satisfaction level with different job characteristics shows that respondents were most satisfied with autonomy at work – 5.28, relationship with colleagues – 5.06, and management quality – 5.04 , workload (0.452) and GP's social status (0.458) . Doctors who have worked for 30 – 39 years are most satisfied with their job. Total job satisfaction of GPs and physicians does not have statistically significant difference. Most PHC level doctors (75.5%) do not intend to recommend their children to follow their career.Such characteristics as autonomy at work, social status, and workload have the biggest impact on total job satisfaction. Stamps and Piedmonte's 2000) research shows that the more autonomy activities possess and the less monotony exists at work, and the more employees are satisfied with their work [00 researIn conclusion it can be stated that the nature of a PHC doctor's work and rather low salaries condition relatively low job satisfaction among Lithuanian PHC doctors.The author(s) declare that they have no competing interests.These authors contributed equally to this work.The pre-publication history for this paper can be accessed here:


Phytoestrogens derived from soy foods (or isoflavones) have received prevalent usage due to their 'health benefits' of decreasing: a) age-related diseases, b) hormone-dependent cancers and c) postmenopausal symptoms. However, little is known about the influence of dietary phytoestrogens on regulatory behaviors, such as food and water intake, metabolic hormones and neuroendocrine parameters. This study examined important hormonal and metabolic health issues by testing the hypotheses that dietary soy-derived isoflavones influence: 1) body weight and adipose deposition, 2) food and water intake, 3) metabolic hormones (i.e., leptin, insulin, T3 and glucose levels), 4) brain neuropeptide Y (NPY) levels, 5) heat production [in brown adipose tissue (BAT) quantifying uncoupling protein (UCP-1) mRNA levels] and 6) core body temperature.This was accomplished by conducting longitudinal studies where male Long-Evans rats were exposed (from conception to time of testing or tissue collection) to a diet rich in isoflavones (at 600 micrograms/gram of diet or 600 ppm) vs. a diet low in isoflavones (at approximately 10–15 micrograms/gram of diet or 10–15 ppm). Body, white adipose tissue and food intake were measured in grams and water intake in milliliters. The hormones (leptin, insulin, T3, glucose and NPY) were quantified by radioimmunoassays (RIA). BAT UCP-1 mRNA levels were quantified by PCR and polyacrylamide gel electrophoresis while core body temperatures were recorded by radio telemetry. The data were tested by analysis of variance (ANOVA) (or where appropriate by repeated measures).Body and adipose tissue weights were decreased in Phyto-600 vs. Phyto-free fed rats. Food and water intake was greater in Phyto-600 animals, that displayed higher hypothalamic (NPY) concentrations, but lower plasma leptin and insulin levels, vs. Phyto-free fed males. Higher thyroid levels (and a tendency for higher glucose levels) and increased uncoupling protein (UCP-1) mRNA levels in brown adipose tissue (BAT) were seen in Phyto-600 fed males. However, decreased core body temperature was recorded in these same animals compared to Phyto-free fed animals.This study demonstrates that consumption of a soy-based (isoflavone-rich) diet, significantly alters several parameters involved in maintaining body homeostatic balance, energy expenditure, feeding behavior, hormonal, metabolic and neuroendocrine function in male rats. Some phytochemicals are considered to be endocrine disrupters that mimic or modulate the physiological effects of steroid hormones, especially that of estrogens ,2. Of alPhytoestrogens represent hundreds of molecules possessing non-steroidal, diphenolic structures found in many plants  that have similar chemical and structural properties to those of estrogens -4. ThereOf these three main classifications, human consumption of isoflavones has the largest impact due to its availability and variety in food products containing soy. Furthermore, the phytoestrogens principally derived from soy foods have received prevalent usage due to their 'health benefits' of decreasing: a) age-related diseases (cardiovascular & osteoporosis), b) hormone-dependent cancers (e.g. breast & prostate) and c) postmenopausal symptoms -6. HowevThe purpose of this study was to examine, in a comprehensive manner, important hormonal and metabolic health issues by testing the hypotheses that dietary soy-derived phytoestrogens influence: 1) body weight and adipose deposition, 2) food and water intake, 3) metabolic hormones (i.e., leptin, insulin, T3 and glucose levels), 4) brain neuropeptide Y (NPY) levels, 5) heat production [in brown adipose tissue (BAT) quantifying uncoupling protein (UCP-1) mRNA levels] and 6) core body temperature. This was accomplished by conducting longitudinal studies where male Long-Evans rats were exposed (from conception to time of testing or tissue collection) to a diet rich in phytoestrogens vs. a diet low in phytoestrogens.Long-Evans male and female rats [10 per sex at 50 days old] were purchased from Charles River Laboratories (Wilmington, MA, USA) for breeding. These animals were caged individually and housed in the Brigham Young University Bio-Ag vivarium and maintained on an 11-hour dark 13-hour light schedule (lights on 0600–1900). The animals and methods of this study were approved by the institute of animal care and use committee (IACUC) at Brigham Young University (BYU).Upon arrival all animals were allowed ad libitum access to either a commercially available diet with high phytoestrogen levels (Harlan Teklad Rodent Diet 8604, Madison, WI, USA) containing 600 micrograms of phytoestrogens/gram of diet [or specifically this diet is high in isoflavones, 600 parts per million or ppm]; referred to hereafter as the Phyto-600 diet, or a custom phytoestrogen-free diet; referred to hereafter as the Phyto-free diet, obtained from Ziegler Bros. (Gardner, PA, USA) and water . In the Body weights and food intake were measured on a Mettler 1200 balance [in grams (g) ± 1 g; St. Louis, MO, USA], white and brown adipose tissue and prostate weights were measured on a Sartorious balance [in milligrams (mg) ± 1 mg; Brinkman Inst. Co., Westbury, NY, USA]. Water intake was measured in drinking tubes [in milliliters (ml) ± 1 ml]. White adipose tissue (WAT) was dissected inferior to the kidneys and superior to the testes in the abdominoplevic cavity  and then weighed in grams ± 0.01 g. Brown adipose tissue was dissected from between the scapular blades (inter-scapular region) and weighed in milligrams (mg) ± 1 mg.Serum leptin and insulin levels were determined by kits purchased from Linco Res. Inc. (St. Charles, MO, USA) [from arterial blood samples of 33 and 55 day-old male animals and venous blood samples collected from 75 day-old rats. This was due to exhausting the arterial supplies from the available blood samples for other assays and thus venous blood was assayed at 75 days of age]. Serum thyroid (T3) levels were assayed by a kit purchased from Diagnostic Systems Labs. Inc. (Webster, TX, USA) and glucose levels were detected by a kit (#510) purchased from Sigma Chem. Co. (St. Louis, MO, USA).Subsequent to blood collection (above), after the animals were sacrificed, brains were removed rapidly, frozen on dry ice and then stored at -80°C until microdissection. Coronal slices 300 μm thick were sectioned on a microtome cryostat. The paraventricular nucleus, arcuate nucleus and median eminence regions of the hypothalamus were microdissected by punch technique and homogenized in 100 μl of 0.1 M HCl. Tissue protein was determined by the Lowry method . The NPYBody temperature was monitored by radio telemetry by implanting a very small electronic chip [under the skin above the left thoracic cavity near the heart] that measured and transmitted core body temperature (± 0.1°C) to a notebook-sensor monitor (BioMedic Data Systems Inc., Seaford, DE, USA) within 2 seconds and repeated measurements were made throughout the day and/or the duration of the experiments.32P-dCTP (0.1–0.15 μl), and Jumpstart Taq polymerase (Sigma Chem. Co., St. Louis, MO, USA) (1 U). Each tube was then subjected to the following protocol: 95°C for 5 min, 20 cycles of 94°C for 30 sec, 60°C for 30 sec, 72°C for 45 sec, followed by 72°C for a final 10 min. interval. With this profile, the UCP-1 and 18S fragments were amplified within the linear range (20 cycles for UCP-1). The primers for UCP-1 were GTGAAGGTCAGAATGCAAGC (sense) and AGGGCCCCCTTCATGAGGTC (antisense), the resultant fragment was 197 bp. The sequence of the UCP-1 fragment was verified by the DNA Sequencing Center at BYU. The PCR products were then subjected to non-denaturing polyacrylamide gel electrophoresis and the gels were exposed to autoradiographic film. Optical density (O.D.) of each band was determined using the NIH imaging system (Version 1.61). For each sample the O.D. ratio UCP:18S was determined. Each RT-PCR protocol was repeated and O.D. ratio values averaged over at least two runs.Uncoupling protein (UCP-1) mRNA levels were determined in brown adipose tissue (BAT) collected from the interscapular region of each male rat. The BATs were homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted. Two micrograms (2 μg) of total RNA were reverse transcribed (RT) for 60 min at 42°C using Superscript II (Invitrogen, Carlsbad, CA, USA) (200 U). Each 20 μl reaction contained 0.1 M DTT (2 μl), 10 mM dNTP mix (1 μl), 10X PCR buffer (2 μl), random decamers (0.4 μl), and RNaseOUT (Invitrogen) (40 U). A duplex PCR reaction was then performed on each RT product, with 18S rRNA serving as the internal control. Each 50 μl reaction contained the RT product (2 μl), UCP-1 primers (2 μl), 18S primers [2 μl of 3:7 ratio of 18S primer to18S competimer (Ambion, Austin, TX, USA)], 10X PCR buffer (5 μl), 10 mM dNTP mix (0.625 μl), All data are presented as the mean ± SEM with p < 0.05 deemed significant. The data were tested by analysis of variance (ANOVA) (or where appropriate by repeated measures), followed by pairwise comparisons  to detect significant differences between the diet treatment groups (p < 0.05).When food and water intake was measured in young adult animals, surprisingly the Phyto-600 fed males displayed slight but significantly higher food Figure  and wateThe effects of dietary phytoestrogens on body weights in pre-, early adult and young adult age male rats are shown in Figure White adipose tissue (WAT) weights were not measured in 33 day-old animals, since relatively little fat deposition was observed in the abdominopelvic cavity  at this age. However, at 55 and 75-days of age, males fed the Phyto-free diet displayed significantly higher white adipose tissue weights (approximately 50% greater) compared to Phyto-600 values Figure .In 33, 55 and 75 day-old male rats, circulating leptin and insulin levels were within the normal ranges , however, at each age males fed the Phyto-free diet displayed significantly higher leptin Figure  and insuFrom the animals collected on 33, 55 and 75 days of age not enough serum was left after other assays were performed to quantify glucose levels on these days. However, circulating glucose levels were assayed in non-fasting 65, 80 or 110 day-old animals, Phyto-600 fed males displayed slightly higher values (that were not significantly different) compared to Phyto-free fed males [age 65 days old- Phyto-600 = 113.5 (± 4.4) vs. Phyto free = 93.5 (± 9.0) mg/dl, n = 8 per group; age 80 days old- Phyto-600 = 137.2 (± 3.8) vs. Phyto-free = 122.5 (± 3.9) mg/dl, n = 10 per group; age 110 days old- Phyto-600 = 123.5 (± 5.0) vs. Phyto-free = 113.8 (± 3.6) mg/dl, n = 5 per group, (mean ± SEM), data not shown graphically].Since leptin plays an important role in regulating brain NPY levels that in turn influences food/water intake, NPY levels were determined in three hypothalamic regions [i.e., the periventricular nucleus (PVN), median eminence (ME) and the arcuate nucleus (ARC)] in 75 day-old males exposed to the diet treatments. In the PVN and ARC (but not the ME) NPY levels were significantly higher (by approximately 40 %) in Phyto-600 fed males vs. the Phyto-free male values  levels were determined from venous blood samples. Phyto-600 fed males displayed significantly higher T3 levels compared to Phyto-free fed values [age 65 days old- Phyto-600 = 2.4 ± 0.2 vs. Phyto-free = 1.5 ± 0.4 pg/ml (± SEM), n = 8 per diet treatment; age 110 days old- Phyto-600 = 1.9 ± 0.4 vs. Phyto-free = 0.8 ± 0.3 pg/ml, n = 5 per group, (mean ± SEM) data not shown graphically].The effects of dietary phytoestrogens on uncoupling protein-1 (UCP-1) mRNA levels in brown adipose tissue (BAT) from 75 day-old animals is shown in Figure Finally, when core body temperatures were recorded during a 24-hour interval, Phyto-free fed males displayed, in general, slight but significantly higher values compared to Phyto-600 animals during the dark phase of the light/dark cycle when rodents are most active Figure . HoweverEstrogen is known to play a dual role in regulating body weight, food intake and adipose tissue deposition. On the one hand, estrogens decrease food intake, increase locomotor activity and hence decrease body weight ,11. HoweIt was previously observed in our laboratory that dietary phytoestrogens significantly alter food and water intake ,25,26. TThe present studies also suggest a mechanism that may underlie the diet-induced effects on NPY . A number of previous studies have demonstrated a reciprocal relationship between circulating insulin and leptin titers and NPY concentrations in PVN. Thus, experimentally-induced reductions in either insulin  or leptiWhile it is clear that thyroid hormone levels are influenced by estrogens where increases are seen in T3 and T4, presumably by increasing in the production of thyroid binding globulin in the liver , the pubUncoupling proteins (UCP-1 through UCP-5) are expressed in various tissues from many different species  that play important  role(s) in the regulation of energy expenditure, or thermogenesis ,41. UncoThis study demonstrates that consumption of a widely used commercially available soy-based rodent diet, (i.e., the Phyto-600 diet rich in isoflavones), alters several hormonal, metabolic and neuroendocrine parameters involved in maintaining body homeostatic balance, energy expenditure and feeding behavior in male rats. Further research is warranted in examining the important aspects of the neuroendocrine and metabolic influences of dietary phytoestrogens via the consumption of soy in humans and laboratory animals. This is especially true when diet is usually not considered as an influencing factor in the experimental design -50.neuropeptide Y (NPY), white adipose tissue (WAT), brown adipose tissue (BAT), uncoupling proteins (UCP), phytoestrogen-rich diet (Phyto-600), phytoestrogen free diet (Phyto-free), periventricular nucleus (PVN), median eminence (ME), arcuate nucleus (ARC), norepinephrine (NE), thyroid (T3, T4), National Institutes of Health (NIH), reverse transcriptase-polymerase chain reaction (RT-PCR), Food and Drug Administration (FDA), optical density (OD), luteinizing hormone (LH), follicle stimulating hormone (FSH), Brigham Young University (BYU), estrogen receptor (ER), high performance liquid chromatography (HPLC)The author(s) declare that they have no competing interests.JPP, TDL, LB and GR contributed equally to this paper in various aspects of this study. KDRS carried out the isoflavone quantifications, WRC conducted the NPY analyses and EDL conceived of the study, designed, coordinated and drafted the manuscript along with the other authors.


The ciliary neurotrophic factor (CNTF) receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor α component (CNTFR). This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s) is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC) associated either with the soluble receptor subunit cytokine-like factor-1 (CLF) or the soluble form of the binding receptor itself (sCNTFR).clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR.Here we show that, during development, This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development. CLC (cardiotrophin-like cytokine) shares homology with CNTF (ciliary neurotrophic factor) and CT-1 (cardiotrophin-1) and requires co-expression with either CLF (cytokine-like factor-1) or the soluble form of the CNTFR to be secreted . The CLCclc has only been studied in adult mouse tissues [clc is very low when compared with that of clf or cntfr. The highest level of clc expression was observed in the muzzle, a very heterogeneous region containing different positive tissues, as described below. Clc expression is also observed in lung, kidney, brain and skeletal muscles such as the tongue or limb muscles.Since the expression of  tissues , we firsin situ hybridization experiments to determine the pattern of expression of clc in the environment of developing motoneurons and compare it with the expression of both clf and cntfr. Motoneuron death occurs between E14.5 and E18.5 in mice lacking in the ability to produce a functional CNTF receptor complex [clc mRNA expression levels at E16.5. Clc is expressed in muscles along the whole rostro-caudal axis, at the brachial level  from E16.5 or E18.5 mouse tissues according to the manufacturer's instructions. Complementary cDNA was synthesised from 2 μg of RNA by random hexamer priming using MMLV reverse transcriptase (Promega). Quantitative PCR was performed using a capillary real-time LightCycler (Roche Diagnostics), and the data analysed using "Fit Point Method" (Roche Diagnostics). For comparison of gene expression levels, all quantifications were normalized to endogenous cntfr clone was as previously described [clf [clc probes corresponded to the isolated cDNAs.Plasmid cDNA clones were linearized and transcribed with T7 or T3 polymerase using digoxigenin (Dig) or fluorescein (Fluo)labeling reagents (Roche Diagnostics). Probes were used at a concentration of 500 ng/ml. The escribed  and the bed [clf  and clc In situ hybridization was performed as described previously [eviously  on 20 μmper se, express clc and clf, double in situ hybridization / immunohistochemistry was carried out as described [nlacZ mice, which express the nlacZ reporter gene under the control of a muscle-specific myosin light chain promoter. After in situ hybridization, slides were rinsed in PBT (PBS, 0.1% Triton), and sections were successively incubated for 1 h with blocking solution containing 2% BSA, 2% heat-inactivated donkey serum in PBT and then overnight at 4°C with rabbit anti-β-galactosidasel (1/1000, Cappel). After three washes in PBT, slides were incubated 1 h at RT with a biotin donkey anti-mouse secondary antibody. Slides were then washed in PBS, and TBS (50 mM Tris-HCl, 0.15 M NaCl, pH 7.6), and incubated for 30 min at RT in ABC streptavidin/HRP in TBS. Staining was revealed with DAB (D4293, Sigma) in the presence of H2O2.In order to confirm that muscle fibers, escribed  on sectiin situ hybridization was performed as described previously [Double eviously . BrieflyThe author(s) declare that they have no competing interests.clc and clf probes before publication. OL participated in the experimental design and coordination of the research. All authors read and approved the final manuscript.BB performed in situ hybridizations whereas DD and HG performed RT-PCR analyses. GE and JFG provided the


G protein-coupled receptors (GPCRs) transduce signals from extracellular space into the cell, through their interaction with G proteins, which act as switches forming hetero-trimers composed of different subunits (α,β,γ). The α subunit of the G protein is responsible for the recognition of a given GPCR. Whereas specialised resources for GPCRs, and other groups of receptors, are already available, currently, there is no publicly available database focusing on G Proteins and containing information about their coupling specificity with their respective receptors.gpDB is a publicly accessible G proteins/GPCRs relational database. Including species homologs, the database contains detailed information for 418 G protein monomers (272 Gα, 87 Gβ and 59 Gγ) and 2782 GPCRs sequences belonging to families with known coupling to G proteins. The GPCRs and the G proteins are classified according to a hierarchy of different classes, families and sub-families, based on extensive literature searchs. The main innovation besides the classification of both G proteins and GPCRs is the relational model of the database, describing the known coupling specificity of the GPCRs to their respective α subunit of G proteins, a unique feature not available in any other database. There is full sequence information with cross-references to publicly available databases, references to the literature concerning the coupling specificity and the dimerization of GPCRs and the user may submit advanced queries for text search. Furthermore, we provide a pattern search tool, an interface for running BLAST against the database and interconnectivity with PRED-TMR, PRED-GPCR and TMRPres2D.The database will be very useful, for both experimentalists and bioinformaticians, for the study of G protein/GPCR interactions and for future development of predictive algorithms. It is available for academics, via a web browser at the URL:  G protein-coupled receptors (GPCRs), form one of the major groups of receptors in eukaryotes; they possess seven transmembrane α-helical domains, as confirmed by analysis of the crystal structure of Rhodopsin . The stuAgonist binding to GPCRs leads to association of the hetero-trimeric G protein with the receptor, GDP-GTP exchange in the G protein α subunit followed by dissociation of the G protein into α-GTP and βγ complexes. The dissociated subunits can activate or inhibit several effector proteins such as adenylyl cyclase 1–9, PLCβ 1–4, tyrosine kinases, phosphodiesterases, phosphoinositide 3-kinase, GPCR kinases, ion channels, and molecules of the mitogen-activated protein kinase pathway, resulting in a variety of cellular functions , and alsAs mentioned above, G proteins, form hetero-trimers composed of Gα, Gβ and Gγ subunits. G protein α subunits, possess an intrinsic GTPase activity, which enables them to act as time switches: Hydrolysis of the bound GTP to GDP promotes the re-association of the α subunit with the βγ dimer and renders the G protein in an inactive form -14. G prToday, there exist general-purpose databases gathering information for receptors , and othIn order to construct the database, initial sequence information was retrieved from the publicly available databases: PIR , SWISS-PThe data has been organized on the basis of a relational model and is stored in a PostgreSQL database system. The user has supervisory access through our Apache web-server. The database is managed by interferential software, written in Java, which tends to settle any web-server's query. The main innovation of the database, resides on its relational scheme Figure . It is wC. elegans) and all plants and fungi do not have such a detailed classification. Gβ and Gγ are subdivided into 6 and 13 different types, respectively. GPCRs are usually classified into several classes, according to the sequence similarity shared by the members of each class. Here, we have to mention that in this classification scheme, the classes are usually termed families, but we chose as before [Each database entry contains the following fields: gpDB name, gpDB id, UniProt accession number, Protein description and classification, sequence, species, organism common name, taxonomy, links to other databases (such as PDB, InterPro, Prints, Prosite, Pfam, GPCRDB, MIM or Smart) and coupling preference (if existent). Information on coupling preference is accompanied by links to PUBMED, corresponding to original articles reporting the interaction. There is also a field showing the reported effector molecules on which G Proteins act, and GPCRs accessory proteins, also accompanied by links to original articles. As we already noted, G proteins are classified into three classes (Gα, Gβ, Gγ). Gα class is further subdivided into four families (Gi/o, Gq/11, Gs, G12/13) and each family is subdivided into different subfamilies and types. This classification is mainly based on proteins present in vertebrates and in the vast majority of invertebrates, while some invertebrates (s before ,35 to res before ,23,36 anWe further classified GPCRs into 64 different families and each family is further subdivided into different subfamilies, based mainly on TIPS classification scheme that takes into account the native ligand(s) that binds to a particular GPCR. Currently, information on coupling specificity is available only for GPCRs, belonging to the classes A, B, C, D, E and Frizzled/Smoothened, thus only GPCRs belonging to these classes are deposited in the database. A sample entry of the database is shown in Figure The application possesses a user-friendly environment, through which, the user may retrieve the necessary information, find available resources and cross-references and perform additional tasks such as running predictive algorithms, performing alignments, etc. In the main page of gpDB the user may find links for the following tools: Navigation, Text Search, BLAST Search, Pattern Search. There is also an extensive user's manual page, describing in detail the available tools Figure . In summThrough the navigation tool, the user has the ability to browse the database following the hierarchy Figure . The navAlternatively, following the link of G PROTEINS, the user may browse through: G PROTEIN CLASSES, G PROTEIN FAMILIES, G PROTEIN SUB-FAMILIES, G PROTEIN TYPES and finally to individual G proteins. At each point, the user may navigate up or down the hierarchy tree. Finally, the user may obtain a detailed view of a particular GPCR or G protein (See Entry description).In the Text Search area, the user can search for any text in the fields of his/her preference. The user can enter any word in one or more of the available boxes under the name: 'Protein Name', 'Species', 'Description', 'Gene Name' and 'Cross-References'. Advanced queries can be performed using parentheses, and logical operators such as AND, OR, NOT, AND NOT as described in the documentation. Expressions in separate search fields are combined with the AND operator, so every entry of the result set will satisfy the expressions of all the search fields the user has chosen. The user has the option to choose whether the query will be performed against the GPCRs or the G proteins included in the database.With the BLAST search tool , the useUsing the Pattern Search tool (a home made tool), the user may perform searches for finding specific patterns in protein sequences of the database. The user, once again, has the option to choose whether to perform the Pattern search against the GPCR sequences or the G proteins sequences. The input of the Pattern Search tool could be either a standard regular expression pattern, or a pattern following the PROSITE  syntax. DRY. [AGS].{3, 6}And intracellular loop of the Gi/o coupled GPCRs, has the simple interpretation, that we must have the consecutive residues Aspartate, Arginine Tyrosine (DRY), followed by any single residue(.), followed by one only of the following residues Alanine (A), Glycine (G) or Serine (S), followed by 3 to 6 residues of any type, ending up to an Alanine (A). We have collected, the 40 most discriminative patterns for each one of the three classes of coupling specificity, reported in [), and the user has the option, to use them in order to perform searches against the database.taken from the work of Moller and co-workers , that waorted in  (found aThe output of the Pattern search application consists of a list of the sequences matching the particular pattern. Following the appropriate links the user may retrieve the detailed view of the target sequence(s).Furthermore, from the detailed view of an entry the user has the option to perform some additional tasks. These include, running PRED-TMR , PRED-GPThe database that we present here has some innovative and unique features not available in any other publicly accessible resource. The relational scheme, on which the database is organised, is especially designed to capture the coupling preferences of G proteins to GPCRs according to the reported data in the scientific literature. General sequence databases, such as Uniprot, do not include fields showing the coupling preference of GPCRs, but rather contain such information (if they do) in the free-text field of FUNCTION. Other specialised databases already exist focusing only in specific groups of receptors. For example GPCRDB , is the The database provides a starting point for the development of algorithms predicting the coupling specificity of GPCRs to G proteins, an issue addressed already in the past by some teams ,41, but The information, which the database comprises of, is essentially information regarding protein-protein interactions, which in turn, may be utilised in various ways. Currently, the most informative publicly available general purpose resource, concerning protein-protein interaction data, is the Database of Interacting Proteins . ProteinWe present here a relational database, gpDB, summarizing the existing publicly available information regarding G proteins and their interactions with GPCRs. This database fills a gap in the already available resources, regarding GPCRs, and maintains an excellent functionality and interconnectivity with the publicly available databases and web-tools. The database is unique, since no other such database already exists, and will be useful for both molecular biologists conducting experiments, but also for bioinformaticians that manage large amount of data, building algorithms and performing functional classification of proteins in the genomic context.. Non-academics should contact Prof S. J. Hamodrakas at shamodr@cc.uoa.gr to obtain a license. All comments, suggestions, corrections and additions, should be sent to biodb@biol.uoa.gr.The gpDB is freely available for academic users at ALE carried out the data collection and annotation, classified the G proteins and drafted the manuscript, PGB designed the relational scheme of the database, classified GPCRs and drafted the manuscript, ICS implemented the SQL database and the web-server pages and SJH coordinated and supervised the whole project. All authors read and approved the final manuscript.


Drosophila have demonstrated the value of enhancer "trapping" using an invertebrate to address this functional genomics problem.Among functional elements of a metazoan gene, enhancers are particularly difficult to find and annotate. Pioneering experiments in Sleeping Beauty transposon-based transgenesis cassette to establish an enhancer trapping technique for use in a vertebrate model system, zebrafish Danio rerio. We established 9 lines of zebrafish with distinct tissue- or organ-specific GFP expression patterns from 90 founders that produced GFP-expressing progeny. We have molecularly characterized these lines and show that in each line, a specific GFP expression pattern is due to a single transposition event. Many of the insertions are into introns of zebrafish genes predicted in the current genome assembly. We have identified both previously characterized as well as novel expression patterns from this screen. For example, the ET7 line harbors a transposon insertion near the mkp3 locus and expresses GFP in the midbrain-hindbrain boundary, forebrain and the ventricle, matching a subset of the known FGF8-dependent mkp3 expression domain. The ET2 line, in contrast, expresses GFP specifically in caudal primary motoneurons due to an insertion into the poly(ADP-ribose) glycohydrolase (PARG) locus. This surprising expression pattern was confirmed using in situ hybridization techniques for the endogenous PARG mRNA, indicating the enhancer trap has replicated this unexpected and highly localized PARG expression with good fidelity. Finally, we show that it is possible to excise a Sleeping Beauty transposon from a genomic location in the zebrafish germline.We modulated a This genomics tool offers the opportunity for large-scale biological approaches combining both expression and genomic-level sequence analysis using as a template an entire vertebrate genome. Caenorhabditis elegans and Drosophila melanogaster, and only six times as many as Saccharomyces cerevisiae [in silico analysis of the regulatory sequences even more complex. To further complicate the matter, enhancer sequences diverge in evolution, co-evolving with their respective transcription factors, and often do not work across large evolutionary distances – worm to fly, for example [Human, mouse and rat genomes likely have less than 40 000 genes each -4. This revisiae -7. The irevisiae . Further example . This maDrosophila. The first fly enhancer trap vectors were based on the P element transposon and often used the transposase's own promoter fused to the beta-galactosidase reporter gene for enhancer detection [LacZ reporter in cells corresponding to the expression patterns of nearby genes, validating the approach [engrailed, fushi tarazu and Hsp70 were successfully developed for enhancer trapping in the fruitfly [LacZ fusion protein [hobo and piggyBac with insertion site preferences distinct from those of the P element have been used in Drosophila [Drosophila genes (reviewed by [Enhancer detection ("trapping") using insertion site context vectors was popularized as a genomics tool in etection -12. Seveapproach . In othefruitfly -16. Furtfruitfly , green ffruitfly , and eve protein  as reporosophila ,21. The iewed by ) and allDrosophila enhancer trap transposons is not significantly higher than the average 15% rate obtained with regular P elements, most insertions do not result in a mutation [The ability to excise from a genomic location has been instrumental to the utility of P element based vectors. For mutation-causing insertions, reversion of the mutant phenotype by P element excision proves that a given insertion causes the mutation. Since the mutagenicity of mutation . In thesmutation .Drosophila prompted application of this approach in the mouse [Drosophila, the lacZ reporter was shown to be expressed in part of the target gene's expression domain [The success of enhancer trapping in he mouse -27. As wn domain . Despiten domain ).Drosophila can be largely attributed to the advantages of this experimental system over mouse. In Drosophila, large numbers of transgenic organisms can be readily generated and screened for gene expression patterns. It is far less practical in the mouse. This is partly due to the availability of efficient and precise transgene delivery tools in the fruitfly: the native P element, hobo and piggyBac transposons. In contrast, early mouse experiments were carried out by non-facilitated DNA transgenesis. This approach is less efficient and prone to induce deletions and other genome rearrangements in the recipient locus, as noted in the first published mouse enhancer trap locus [Drosophila genome also contributed to the success of enhancer trapping, making the path from an enhancer trap insertion to the identification of the affected gene straightforward, especially once the Drosophila genome was sequenced.We believe the success of enhancer trapping in ap locus ,30. The Danio rerio is a vertebrate model system that provides many of the advantages found in invertebrates. A few hundred transparent, externally developing embryos can be obtained from a single pair of fish per week. The zebrafish genome is about two-fold smaller than the mouse genome, and its sequencing and annotation are nearing completion. Finally, transposon tools for efficient and precise transgene delivery into the zebrafish genome are available. We focused our research on the Sleeping Beauty (SB) transposon system [Sleeping Beauty transposon system offers advantages in expression as well as ease of construction and testing of diverse vectors that can be done using basic molecular biology tools. Furthermore, the SB system offers the possibility of transposase-induced excision out of the genome to induce local deletions or to revert possible mutant phenotypes.The zebrafish n system ,32. Whiln system ,34, the Sleeping Beauty transposon system is a viable experimental approach using as template a vertebrate genome.In this report, we investigated the potential of the SB transposon system for enhancer detection in zebrafish. Our results indicate that zebrafish enhancer trap lines with diverse GFP expression patterns can be readily generated using the SB system. Most of the obtained lines harbor a single transposon insertion event, facilitating the rapid identification of transposon insertion sites responsible for specific GFP expression patterns. We show that two enhancer trap lines exhibit GFP expression patterns matching the expression patterns of the target genes, and that both expected and novel gene expression patterns can be identified using this genomics tool. We conclude that enhancer trapping using the Sleeping Beauty transposons are flanked by relatively large inverted repeats. These repeats might function as silencer elements and not allow for transcriptional regulation across them. Second, the promoter we used  and transgenic (F1) zebrafish [S1EF1α-GM2 or γ Cry1/pT2/S1EF1α-GM2 were scored for any GFP fluorescence and for eye-specific GFP fluorescence at 3 dpf  increase in injected embryos showing any GFP fluorescence. In contrast, the increase in eye-specific GFP expression was ten-fold  may notn vector . We tookpromoter  to the pebrafish . EmbryosS1EF1α is expressed too strongly in transgenic animals. Since most successful enhancer traps in Drosophila and mice were based on truncated or weak promoters, we decided to attenuate the S1EF1α promoter by removing sequences upstream of Bst1107I (S2EF1α) and EcoRI (S3EF1α) restriction enzyme sites . In contrast, when pT2/S2EF1α-GM2 was used, most of the GFP-positive fish exhibited fairly strong, ubiquitous expression. Closer analysis indicated that many of these "ubiquitous" expression patterns were rather unique, with GFP expression often noted to be particularly strong in some tissues, consistent with a tissue-specific expression pattern superimposed on a ubiquitous expression pattern (data not shown). In most cases, GFP expression segregated as a single integration event, indicating that a tissue-specific expression pattern was not likely being masked by a ubiquitous expression pattern from a different insertion event. A similar phenomenon has been observed with Drosophila enhancer traps [S2EF1α-GM2 pilot screen yielded three kinds of GFP expression in its progeny. Some were ubiquitously GFP positive, some showed a hatching gland-specific expression profile, and some exhibited both. When three F1 fish with both ubiquitous and hatching gland specific expression were raised and outcrossed, the two expression patterns exhibited independent segregation: 24% of the embryos were GFP negative, 26% expressed GFP ubiquitously, 25% had hatching gland-specific GFP expression, and 25% had both hatching gland-specific and ubiquitous expression (n = 245). Independent segregation indicates that the two transposon insertions causing the two expression patterns are unlinked. Two independent integration events were confirmed by Southern hybridization and inverse PCR (data not shown). The hatching gland-specific GFP expressing embryos were used to establish our first enhancer trap line, ET1  wereS2EF1α-GM2 and SB10 transposase mRNA mix. 2102 embryos survived to day 3 for scoring, of which 848 were mosaic GFP positive and were selected to be raised. 330 survived to adulthood and were screened for germline transmission of GFP expression, primarily by sibling incrossing. This approach provided a lower estimate of the transgenesis and expression rate because it does not distinguish instances were both parents are transgenic. In this screen, at least 80 of the founder fish produced GFP-expressing progeny resulting in a minimum estimate of a 24% transgenesis rate. The actual transgenesis rate is closer to 30% because most of the fish were screened by incross, and if a pair produced GFP-expressing progeny, only one parent was counted as a transmitter. Eight of the GFP-expressing fish displayed distinct GFP expression patterns . The expression is very pronounced between 20 and 40 hours post-fertilization (hpf), when it marks the hatching gland Figure . ExpressWe analyzed the ET2 line in detail because of the highly specific expression of GFP in these fish. GFP expression was first observed at the 16 somite stage, when 2 bilateral cells in the spinal cord of the 10 anterior somites become GFP positive Figure . At lateSouthern analysis indicated that there is a single transposon insertion in this line, and it is linked to GFP expression Figure . InverseAplysia [S2EF1α-GM2 enhancer trap recapitulates the expression pattern of the endogenous PARG gene, we conducted whole mount in situ hybridization on ET2 outcross embryos to compare PARG and GFP reporter expression ation is a protein modification that is extensively studied at the biochemical level and is associated with changes in DNA replication, recombination, repair and transcription , for a rAplysia . Most orAplysia . PARG acAplysia -53, but n Figure . In situWe characterized insertion events in other enhancer trap lines. GFP positive F1 or F2 fish were outcrossed, and embryos were sorted into GFP positive and GFP negative pools. Genomic DNA was prepared from each pool, and Southern analysis was conducted to assess transposon copy number and linkage to the GFP expression pattern. In all lines except ET1 (see above), a single GFP expression-linked transposon insertion event was detected by Southern hybridization. We then conducted inverse PCR analysis on the DNA from GFP positive embryos to identify the insertion locus. For verification, DNA from embryos from an independent outcross was prepared and PCR was run with primers flanking the insertion site to link GFP expression and transposon insertion at a particular locus. Verified enhancer trap loci are presented in Table mkp3 gene within 30 kb of the insertion site. Our attempts to amplify the predicted novel candidate gene from maternal and post-somitogenesis zebrafish cDNA libraries using 2 different primer pairs failed, while mkp3 cDNA was readily amplified in parallel PCR reactions (data not shown). This suggests that the novel target gene may be a false prediction by Genescan. The mkp3 gene encodes a dual specificity phosphatase which was cloned as a member of fgf8 synexpression group and is a negative feedback regulator of FGF8 signaling. mkp3 is expressed in the midbrain-hindbrain boundary, forebrain, tailbud, branchial arches, developing ear, pectoral fin buds and other tissues [mkp3 mRNA expression pattern in 23 hour embryo  and polyA (3' exon) and other related constructs. We then constructed two further truncations to the S1EF1α promoter in the transgenesis cassette [.In this paper, we describe the first use of enhancer trapping, or enhancer detection, as an experimental approach in zebrafish. We show that cassette  and founDrosophila enhancer trap vectors and was speculated to stem from the transcriptional complexity of neural tissue [Many of the obtained enhancer trap lines express GFP in the nervous system. This was previously observed with both mouse and l tissue ,28. SeveThe ET2 line harbors a transposon insertion into the zebrafish gene for poly(ADP-ribose) glycohydrolase (PARG). We demonstrate that both PARG and GFP in ET2 line are expressed in caudal primary motoneurons of 23 hour old embryos. Thus, GFP expression in the ET2 line mimics that of an endogenous gene (PARG), indicating that transgene expression is under control of an endogenous enhancer. A very intriguing question is what the actual trapped enhancer sequence is, how far away from the genomic enhancer the trap can insert and still detect it, and weather artificial enhancer trap approach Figure  can be umkp3 locus. GFP expression in that line closely resembles part of the mkp3 expression domain, suggesting that the enhancer trap transposon in that line is under control of a subset of mkp3 enhancer elements.The ET7 line has a transposon insertion into a predicted novel gene 30 kb downstream of the zebrafish hgg1 gene is specific to the polster and hatching gland depends on nodal signaling and is absent in one-eyed-pinhead mutants [one-eyed-pinhead mutation by morpholino injection in ET1 homozygotes and observed a complete loss of hatching gland-specific GFP expression (data not shown). While the ET1 line expresses GFP in an organ that can be readily observed using regular light microscopy techniques, other lines visualize tissues that are not nearly as easily morphologically accessible. In particular, the ET2 line visualizes the position of primary motoneuron cell bodies and axonal trajectory. Morpholinos against known genes or new members of the zebrafish secretome [Zebrafish enhancer trap lines will be valuable in future developmental genetics studies, be it classical mutagenesis or morpholino "knockdown" screening . GFP exp mutants . We phenecretome  can be sSleeping Beauty transposon from a plasmid results in local deletions with fairly high frequency which is dependent on the cell type or tissue used [Sleeping Beauty transposons significantly increases in cells with a compromised DNA repair pathway [Sleeping Beauty transposon from a genomic location in zebrafish germ line is accompanied by a deletion of flanking genomic DNA, and it should be possible to compromise the embryo's DNA repair machinery to induce such deletions at a high frequency.A further utility offered by the transposon system is the possibility to revert a mutant phenotype or to generate localized deletions by transposon excision . We succsue used . Further pathway ,63. It rSleeping Beauty transposons is an easily scalable and efficient experimental technique in zebrafish. Obtaining fish with different GFP expression patterns is not the rate limiting step in this process. Preliminary molecular analysis of the insertion site is also straightforward using inverse PCR techniques. Identification of candidate genes should benefit from the progress in zebrafish genome sequencing and annotation. The main bottleneck step is the detailed biological analysis of GFP and the corresponding candidate gene expression profile.Our experiments indicate that enhancer detection using Drosophila, the generation of transposase-expressing lines of flies made enhancer detection and P-element mutagenesis in general a mainstream approach. Even without a similar gain in efficiency, transposase expressing fish lines would make enhancer trapping as well as related gene- and poly(A)-trap methodologies even more accessible for high-throughput functional analysis of the vertebrate genome.In S1EF1α-GFP (pDB358) was previously published [S1EF1α-GFP (pDB375), a BamHI-HindIII fragment from Cry1-GFP3 [X. laevis γ-Cry1 promoter was cloned into the Ecl136II site of pDB358. To produce pT2/S2EF1α-GFP (pDB371), a part of the EF1α promoter was deleted from pDB358 by ligation of the Bst1107I-NcoI and NheI-NcoI fragments of pDB358. Similarly, the EcoRI-NcoI and NheI-NcoI fragments of pDB358 were ligated to produce pT2/S3EF1α-GFP (pDB372).pT2/ublished . To makery1-GFP3  containiFor inverse PCR experiments, zebrafish genomic DNA was digested and ligated as described . 1 and 2SpeI and transcribed with T7 RNA polymerase (Promega) and DIG labeling kit (Roche). GFP probe was made by amplifying GFP with 46 base pairs of EF1α promoter from pT2/S1EF1α-GM2 using primers EF1a-F2 and GFP-R0, and cloning it into Topo TA cloning kit resulting in pSS100. pSS100 was linearized with SpeI and transcribed with T7 RNA polymerase using DIG labeling kit. To make mkp3 antisense probe, mkp3 cDNA was amplified from maternal cDNA library with primers Mkp3-F1 and Mkp3-R1 and cloned into Topo TA cloning kit to produce pDB528. The plasmid was linearized with SpeI and transcribed with T7 polymerase using DIG labeling kit.A partial sequence for the zebrafish poly(ADP-ribose) glycohydrolase cDNA was amplified using primers neuroC1-F1 and neuroC1-R1 and cloned using a Topo TA cloning kit (Invitrogen) to make pDB376. To make antisense RNA probe, pDB376 was digested with Embryos injected with SB10 transposase mRNA and transposon DNA mix were raised as described ,64. In pHomozygous ET1 embryos were injected with SB10 transposase mRNA, raised and screened for loss of hatching gland specific GFP expression, or for a change in the GFP expression pattern. Twenty six fish were screened (R0, for Remobilization), and 2 gave GFP negative embryos, with an additional 2 giving ubiquitously GFP positive embryos, suggesting that germline remobilization events may have occurred in as many as 15% of transposase injected embryos. Ubiquitous GFP positive embryos (one in each of the two R0) did not survive. Of the two R0's that gave GFP negative embryos, one gave mosaic hatching gland expression in the next generation. PCR with transposon specific and flanking primers did not show any changes in the locus. The second R0 produced 19 embryos that were GFP negative from the total of 671 embryos obtainted. An R1 adult was outcrossed, embryo DNA was prepared, and PCR with primers HG1-1 and HG1-2 was conducted. The resulting PCR fragment was cloned using PCR 4 Topo cloning kit (Invitrogen). Plasmids were sequenced using M13 Forward primer, and one clone with a transposon footprint was identified. To confirm that it was not due to PCR contamination, a second clutch of embryos was obtained, the procedure was repeated, and the same footprint was obtained (data not shown).in situ analysis. DB designed and built the transposons used in this study and was responsible for drafting the manuscript for publication. SE conceived and supervised the study and edited the manuscript. All authors read and approved the final manuscript.The experiments described in this paper were planned, conducted and analyzed as a joint effort between the authors. In particular, DB, AD, SH and ZW contributed to fish screening, line establishment and to scientific descriptions of these lines, DB and SS to molecular analysis, AD and DB to GFP expression and


Mometasone furoate (MF) is a highly potent glucocorticoid used topically to treat inflammation in the lung, nose and on the skin. However, so far no information has been published on the human glucocorticoid receptor activity of the metabolites or degradation products of MF. We have now determined the relative receptor binding affinities of the known metabolite 6β-OH MF and the degradation product 9,11-epoxy MF to understand their possible contribution to undesirable systemic side effects. In competition experiments with human lung glucocorticoid receptors we have determined the relative receptor affinities (RRA) of these substances with reference to dexamethasone (RRA = 100). We have discovered that 6β-OH MF and 9,11-epoxy MF display RRAs of 206 ± 15 and 220 ± 22, respectively. This level of activity is similar to that of the clinically used inhaled corticosteroid flunisolide (RRA 180 ± 11). Furthermore we observed that 9,11-epoxy MF is a chemically reactive metabolite. In recovery experiments with human plasma and lung tissue we found a time dependent decrease in extractability of the compound. Hence, we provide data that might contribute to the understanding of the pharmacokinetics as well as the clinical effects of MF. Mometasone furoate (MF) is a highly potent topical glucocorticoid for the treatment of asthma , allergiBesides the efficacy of a corticosteroid, safety issues have to be taken into consideration. For topically applied glucocorticoids, the high local anti-inflammatory activity should be paralleled by a low systemic exposure. Therefore, a prolonged redistribution from lung tissue into systemic circulation and a rapid and complete hepatic metabolism of the compounds to inactive derivatives are favorable. For MF, a very low systemic bioavailability of less than 1 % has been reported . HoweverThere had been, however, not much information on the extent and site of metabolite formation in humans. Recent studies now provided some of the required information ,13,15,16Despite the recent discovery of the major metabolite 6β-OH MF and the abundant degradation product 9,11-epoxy MF it is still not clear whether these compounds retain any significant binding affinity to the human glucocorticoid receptor. In the present study we addressed this open question and we present some evidence that the degradation product might bind tightly, most possibly covalently, to protein structures in human lung tissue and plasma.3H]-Dexamethasone was obtained from Amersham (Freiburg, Germany). All other chemicals were obtained from Sigma-Aldrich-Chemie (Taufkirchen, Germany) or E. Merck (Darmstadt, Germany).Mometasone furoate (MF), 6-hydroxy mometasone furoate (6-OH MF), mometasone and 9,11-epoxy mometasone furoate (9,11-epoxy MF) were generous gifts from GlaxoSmithKline (Greenford, England). [Human lung tissue resection material was obtained from patients with bronchial carcinomas who gave informed consent. Cancer-free tissue was used for the experiments. None of the patients was treated with glucocorticoids for the last 4 weeks prior to surgery. Tissue samples were shock frozen in liquid nitrogen after resection and stored at -70°C until usage. To collect sufficient material for the experiments, tissue samples of three or more patients were pooled. Lung cytosol for receptor competition experiments was prepared as detailed in . Plasma 3H] labelled dexamethasone by various concentrations of 6-OH MF, mometasone and 9,11-epoxy MF was determined.The competition experiments were performed according to the procedure described earlier . The disMF or 9,11-epoxy MF, respectively, were added to human plasma, lung tissue suspension (0.5 g / 20 ml) or buffer (0.2 M phosphate buffer, pH 7.4) yielding an initial concentration of 0.3 μg/ml. Only glass lab ware was used for these experiments to exclude any non-specific binding effects of the highly lipophilic compounds to plastic material. Samples were incubated at 37°C in a shaking water bath. At designated time intervals samples of 1.0 ml were removed, subjected to a fluid extraction with diethylether and analyzed by HPLC.18 column (150 × 4.6 mm I.D., 5 μm particle size, Waters, MA).Samples were prepared and analyzed as described previously . The HPLWe determined the relative receptor affinities (RRAs) of 6β-OH MF, 9,11-epoxy MF and mometasone base by competition assays with reference to dexamethasone (RRA = 100). Both, the metabolite 6β-OH MF and the degradation product 9,11-epoxy MF displayed residual receptor binding affinities about twice as high as dexamethasone  metabolite 6β-OH MF and its degradation product 9,11-epoxy MF to understand their possible contribution to undesirable systemic side effects. For the first time we provide data that both compounds are significantly active at the human glucocorticoid receptor with binding affinities twice as high as dexamethasone and similar to that of the clinically used inhaled corticosteroids flunisolide and triamcinolone acetonide . Furtherin vivo formation of these compounds was still lacking. Studies of Teng et al. identified 6β-OH MF and 9,11-epoxy MF as candidate compounds that can indeed be formed in vivo either by hepatic metabolism or by simple degradation of MF [Glucocorticoids currently used for topical application in asthma therapy all share the safety relevant property of extensive metabolism and formation of inactive metabolites. For MF, however, data was sparse so far. Though putative metabolites and degradation products with binding affinity to the rat glucocorticoid receptor have been previously suggested , it was on of MF ,16. We don of MF .Usually hydroxylation at the 6β position results in inactivation of the corticosteroid. The 6-OH metabolite of various glucocorticoids displays little or no residual binding affinity to the receptor (e.g.) ,18. Thiset al. [The MF degradation product 9,11-epoxy MF also displays a significant receptor binding affinity with an RRA of about 200. This RRA is within the range that could be expected from the studies of Isogai et al. . Since 9et al. , it can Besides the significant residual receptor binding affinity of 9,11-epoxy MF we discovered that this compound undergoes follow-up reactions. After incubation with plasma clearly less of 9,11-epoxy MF compared to the parent compound MF was recovered by extraction with an organic solvent. This extraction procedure usually reliably retrieves all non-covalently bound substance from the incubation mixture. In human lung tissue, it was even more obvious that 9,11-epoxy MF was recovered completely from buffer, but not from the tissue suspension. About 17 % of 9,11-epoxy MF was "lost" after three hours of incubation. This observation cannot be explained by simple non-specific tissue binding since the tissue adsorption reaches equilibrium very quickly after about 20 min  seems toIn contrast to other inhaled corticosteroids MF generates an active metabolite, 6β-OH MF, in the liver. The degradation product 9,11-epoxy MF, which is formed in human lung tissue and plasma, exhibits significant receptor affinity as well. Additionally, we found that 9,11-epoxy MF undergoes follow-up reactions. Our data contribute to the understanding of how the claimed low bioavailability of MF parent compound after inhalation might still be accompanied by HPA axis suppression. Thus, our findings are consistent with both pharmacokinetic and clinical data. We strongly suggest a clinical trial that determines both efficacy and safety in parallel as well as all known metabolites and degradation products after application of MF.AV carried out all experiments and the data analysis and participated in the design of the study. PH conceived of and designed the study and wrote the manuscript. All authors read and approved the final manuscript.


Coronary artery calcium score incrementally improves coronary risk prediction beyond that provided by conventional risk factors. Limited information is available regarding rates of progression of coronary calcification in women, particularly those with baseline scores above zero. Further, determinants of progression of coronary artery calcification in women are not well understood. This study prospectively evaluated rates and determinants of progression of coronary artery calcium score in a group of healthy postmenopausal women.We determined coronary calcium score by computed tomography and recorded demographic, lifestyle and health characteristics of 914 postmenopausal women, a subset of those enrolled in the Women's Health Initiative Observational Study. The 305 women with calcium score ≥10 Agatston units at baseline were invited for repeat scan. This analysis includes the 94 women who underwent second scans.2, and baseline calcium score was 162 ± 220 Agatston units. Mean interval between scans was 3.3 ± 0.7 years. A wide range of changes in coronary calcium score was observed, from -53 to +452 Agatston units/year. Women with lower scores at baseline had smaller annual increases in absolute calcium score. Coronary calcium scores increased 11, 31 and 79 Agatston units/year among women with baseline calcium score in the lowest, middle and highest tertiles. In multivariate analysis, age was not an independent predictor of absolute change in coronary calcium score. Hydroxymethylglutaryl coenzyme A reductase inhibitor (statin) use at baseline was a negative predictor (p = 0.015), whereas baseline calcium score was a strong, positive predictor (p < 0.0001) of progression of coronary calcification.Mean age of study participants was 65 ± 9 years (mean ± SD), body mass index was 26.1 ± 6.1 kg/mAmong postmenopausal women with coronary calcium score ≥ 10 Agatston units, rates of change of coronary calcium score varied widely. In multivariate analysis, statin use was a negative independent determinant, whereas baseline calcium score was a strong positive predictor of annual change in coronary calcium score. Coronary calcium, assessed by computed tomography, strongly and independently predicts coronary risk -3. Age iThe rate of progression of coronary calcification appears to further stratify risk ,7, but rCalcium scores differ in men and women , but proIn this study, we prospectively assessed the rate of progression of coronary calcification in an ethnically diverse group of healthy women with coronary calcium scores of at least 10 Agatston units at baseline, and identified independent predictors of progression.Study participants were a subset of women enrolled in the Women's Health Initiative Observational Study  at the GThe entire Observational Study cohort comprises 93,676 women at 40 clinical sites. For this ancillary study, participants at the George Washington and Howard/Medstar clinics (n = 4435) were invited for computed tomography. Baseline scans were performed on the 914 women who responded to the invitation. Of these, 528 had no coronary calcium detected and 81 had calcium scores of 1 – 9 Agatston units. The remaining 305 women with calcium score ≥10 Agatston units were mailed a letter inviting them to have a second scan; African-American women received two mailings because of a historically lower response rate. This analysis includes the 94 women with serial scans, which were performed a mean of 3.3 ± 0.7 years after the baseline study.Participants provided data on a wide range of health variables including dietary habits, medical history and anthropometric measures. Questionnaire measures assessed self-reported hypertension, diabetes mellitus , current smoking, high cholesterol requiring pills, postmenopausal hormone therapy, and family history of premature coronary disease . Statin use at baseline was assessed by medication inventory.Dietary fat consumption was assessed using a food frequency questionnaire based on instruments used in the Women's Health Trial . NutrienPhysical activity was assessed by questions on a frequency and duration scale of four walking speeds and three other types of activity classified by intensity (strenuous, moderate or light). For thiPlasma lipids were only measured in a 1% random subsample of Observational Study participants, so were not included as variables in these analyses.2 field of view (pixel size, 0.586 mm). Images were analyzed by the Agatson method [Images were acquired using an Imatron C – 150 scanner. Thirty contiguous 3-mm slides (100 ms/slice) were acquired during a single breathhold beginning 1 cm caudad to the carina. Each level was triggered by ECG in end-diastole . Images were obtained with a 30-cmn method .Descriptive statistics such as frequencies, percentages, means and standard deviations (SD) were used to describe the study population and to explore the relationships between coronary calcium score and several explanatory variables. Group comparisons were made by t test, chi square and, where appropriate, the Mantel-Hanzel chi square test. In Table SAS System for Windows v8.02.Determinants of annual change in calcium score were evaluated in a multiple linear regression model which included age, baseline calcium score, and statin use at baseline as independent variables Table . In a seBaseline demographic and health characteristics of the 94 women with serial scans are shown Table . Among tTable Annual change in calcium score and age-adjusted change in calcium score are shown by tertile of calcium score at baseline Table . Women wCoronary risk factors are shown by tertile of annual change in calcium score Table . Body maIn multiple linear regression analysis, age was not independently associated with annual change in calcium score Table . Statin In this ethnically diverse group of Women's Health Initiative observational study participants, the rate of progression of coronary calcification was 33%/year among women with calcium scores between 10 and 190 Agatston units at baseline, and 14%/year for women with higher calcium scores. The rate of change in calcium score ranged widely in individual women, from -53 to +452 Agatston units/year. In multivariate analysis, statin use was negatively associated with progression of coronary calcification, whereas baseline calcium score was a strong and independent positive predictor of progression.One strength of this analysis is the inclusion of a wide range of variables affecting atherosclerotic risk, including body mass index, physical activity, dietary fat consumption, postmenopausal hormone and statin use in addition to conventional risk factors. Another strength is the relatively long interval between scans, 3.3 years (mean), enhancing accuracy of the estimated rate of progression. This study includes only women, a group traditionally underrepresented in studies of coronary computed tomography ,8,9,11, The rate of progression of coronary calcification observed in this analysis is within the range reported by others ,9,11. SiEfforts to identify independent predictors of progression of coronary calcification have been limited, particularly in women. A retrospective study of 55 high-risk men identified baseline calcium score and Lp(a) as independent predictors of progression in a multivariate model which did not include statin use . A prospIn contrast, smoking was found to be an independent determinant of progression in a larger study of 311 men and 184 women, which also identified baseline calcium score as a potent predictor of progression .th percentile has been reported as 59 Agatston units or 42 volumetric units, and the 90th percentile as 202 Agatston units or 163 volumetric units [Another limitation of this study was use of Agatston score, rather than calcium volume score. The latter provides better reproducibility , but wasic units . We cannth percentile scores for 50–54 year old men and women are 99 and 3, respectively [th percentile scores are 247 and 49, respectively. If baseline score is the major determinant of progression, a gender difference in rates of progression would be expected on this basis alone. In fact, male gender has been reported as an independent determinant of progression [Calcium scores differ in men and women ; for exaOur observations raise several issues with regard to the potential use of coronary calcium score progression as either a clinical tool or an outcome for atherosclerotic intervention trials. First, the variance for this variable is high, both in our study and in others ,9,12, liIn an ethnically diverse cohort of postmenopausal women with coronary calcium score ≥10 Agatston units, rates of progression or coronary calcification vary widely. In multivariate analysis, statin use was inversely associated with progression, whereas baseline calcium score was a strong positive predictor of progression.The author(s) declare that they have no competing interests.JH study concept, design, data analysis, initial and final manuscript preparation. AK data analysis. AP and JB data acquisition and management, manuscript revision. LLA-C and BVH manuscript revision. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here: