Ras induces expression of p19Arf, which, in turn, activates p53 and growth arrest. Here, we used a multistage model of squamous cell carcinoma development to investigate the functional interactions between Ras, p19Arf, and p53 during tumor progression in the mouse. Skin tumors were induced in wild-type, Arfp19-deficient, and p53-deficient mice using the DMBA/TPA two-step protocol. Activating mutations in Hras were detected in all papillomas and carcinomas examined, regardless of genotype. Relative to wild-type mice, the growth rate of papillomas was greater in Arf-p19deficient mice, and reduced in p53-deficient mice. Malignant conversion of papillomas to squamous cell carcinomas, as well as metastasis to lymph nodes and lungs, was markedly accelerated in both Arf-p19  and p53-deficient mice. Thus, p19Arf inhibits the growth rate of tumors in a p53-independent manner. Through its regulation of p53, p19Arf also suppresses malignant conversion and metastasis. p53 expression was upregulated in papillomas from wild-type but not  Arfp19-null mice, and p53 mutations were more frequently seen in wild-type than in  Arfp19-null carcinomas. This indicates that selection for p53 mutations is a direct result of signaling from the initiating oncogenic lesion, Hras, acting through p19Arf.Ectopic expression of oncogenes such as  A squamous cell carcinoma model shows Ras mutation not only initiates tumor development but, through Arf and p53, directly influences the subsequent evolutionary trajectory of the tumors Ras, and two tumor suppressors, Arf,p19  and p53, using a multistage epithelial tumor model.Tumor development and metastasis is a multistep process of somatic cell evolution that includes uncontrolled proliferation, impaired apoptosis, loss of differentiation, immortalization, neovascularization, invasion, and metastatic spread . This evRas is among the most frequently mutated oncogenes in human cancer, with approximately 30% of tumors carrying an activating mutation in one of three family members, Hras, Kras, or Nras  seen in human tumors ; p = 0.010) and  Arf+/−p19 mice had an average of 2.60 more papillomas ; p = 0.045) in weeks 18–30 after DMBA administration. Average papilloma size was also greater in both  Arf−/−p19 and  Arf+/−p19 mice compared to wild-type mice ( Arf−/−p19 mice were greater than 8 mm in diameter versus 14% (38/267) from wild-type mice (p < 0.0001). Papillomas from  Arfp19-deficient mice measured up to 16 mm in diameter while very few papillomas on wild-type mice measured more than 9 mm. Thus,  Arfp19 deficiency resulted in faster growing papillomas, indicating a role for  Arfp19 in regulating the early stages of benign tumor growth.Both in vitro and in vivo studies have demonstrated that p19ppressor . However5 wk see . Papilloype mice A. Relatiype mice B. This eHras are found in more than 95% of DMBA/TPA-induced skin tumors ,  Arf+/−p19 (4/4), and  Arf−/−p19 (5/5) mice contained the identical A→T transversion at codon 61 of Hras, resulting in an amino acid change from glutamine to leucine and a constitutively activated Ras protein ; p = 0.005) and 3.11 times higher for  Arf+/−p19 mice ; p = 0.073) compared to wild-type mice. The reduced carcinoma latency and increased conversion frequency in the  Arfp19-null mice implicate loss of p19Arf as a critical rate-limiting step in malignant SCC progression.The rate of malignant conversion of papillomas to carcinomas is greatly increased in the absence of p53 function . To deteer 22 wk C. By 28  Arf+/−p19 and  Arf−/−p19 mice also showed a range of grades but a significant number (9/12) were characterized as spindle cell carcinomas. These were characterized by packed and spindle-shaped cells with elongated pleiomorphic nuclei and abundant abnormal mitotic figures. These cells grew in a homogenous pattern with very little evidence of the cellular organization typical of low-grade tumors. These tumors showed focal areas of squamous differentiation, indicating that they were derived from squamous epithelium.Histologic analysis revealed that the carcinomas from control mice ranged in grade from well-differentiated to poorly differentiated SCCs. Carcinomas from  Arfp19 deficiency also increased dissemination and establishment of metastatic SCCs. Carcinoma-bearing p19Arf-deficient mice frequently presented with enlarged lymph nodes, and in several cases tumors were noted on the lungs.  should be seen in the remaining wild-type  Arfp19 allele in carcinomas. Alternatively,  Arfp19 could be recessive, in which case LOH or reduction to a homozygous null state would be expected. The fate of the wild-type allele of  Arfp19 in tumors from heterozygous mice was assessed by semiquantitative PCR analysis of genomic DNA. Three of 15 (20%) papillomas examined showed evidence of loss of the wild-type  Arfp19 allele, compared to ten of 15 (67%) carcinomas (p = 0.0027)  A, indicamal skin B. IncreaArf and p53 were upregulated in papillomas, that p53 expression was reduced in  Arp19f-null papillomas, and that loss of p19Arf had a similar effect on tumor progression as that of loss of p53, provide strong in vivo support of the model whereby p19Arf regulates p53 in response to mutational activation of Hras. However, enhanced tumor growth in  Arfp19-null mice, in contrast to reduced tumor growth in p53-null mice ; p = 0.0001) 10–16 wk after the DMBA administration, while  Arfp19-null mice averaged 2.68 more papillomas ; p = 0.015) 18–40 wk after the DMBA administration  (unpublished data). Thus, p53-regulated apoptosis does not appear to play a major role in SCC development, at least at the papilloma stage.To determine whether tumor growth in mice lacking p19ng index C. The p1Arf and p53 cooperate during tumor progression, papilloma to carcinoma conversion was evaluated in Arf−/−, p53−/−,p19 and Arf−/−p53−/−p19 littermates. Tumor progression was accelerated in all  Arf-p19 and p53-deficient genotypes compared to wild-type littermates p53  A, indicaints see B. This c+/−p53 mice there is strong selective pressure to lose the wild-type allele during conversion to malignancy p19 were assessed for LOH of p53 by semiquantitative PCR analysis of genomic DNA. Two out of seven papillomas from  Arf+/+p53+/−p19 mice show loss of the remaining p53 allele, while all papillomas examined from  Arf+/−p53+/−p19 (n = 8) and  Arf−/−p53+/−p19 (n = 8) mice show retention of wild-type p53  from Arf+/−p53+/−,p19  but only one of 12 (8%) from  Arf−/−p53+/−p19 (p = 0.036) showed loss of p53. Thus, deletion of p19Arf disrupts the activation of p53 and thereby reduces selection for mutations in p53 during malignant progression.In lignancy . As p19Atype p53 B. Seven Hras and the tumor suppressors Arfp19 and p53. Somatic mutation of Ras is an early and frequent event in this model of tumor development. Against this backdrop, p19Arf has at least two distinct tumor suppressor properties, which act at different stages of tumor development and which show a range of gene dosage effects. Loss of one or both  Arfp19 alleles leads to accelerated growth of benign tumors, indicating  Arfp19 is partially haploinsufficient for suppression of this early growth phenotype. Although p19Arf regulates p53 at this stage, suppression of tumor growth per se by p19Arf does not appear to be mediated through p53. p19Arf also inhibits the benign to malignant transition and subsequent tumor cell dissemination and metastasis, and this effect of p19Arf is, in contrast, mediated through p53. LOH of Arfp19  occurs preferentially in malignant tumors, indicating complete loss of p19Arf is favored during progression. Thus, p19Arf inhibits several stages in Ras-driven tumor progression. Furthermore, Ras is connected to both p19Arf and p53 through a signaling pathway, indicating that selection for mutations in the p19Arf / p53 pathway are a direct consequence of the initial Ras mutation.Using a multistage model of tumor progression, we have examined the functional interactions between the oncogene Hras mutations are found at high frequency in papillomas from wild-type and both p19Arf- and p53-deficient mice indicates that squamous epithelial cells harboring Hras mutations have a strong selective advantage, with or without the presence of p19Arf or p53. This permits analysis of the effects of Ras on defined genetic backgrounds in the natural setting of tumor cell evolution. The expression levels of both p19Arf and p53 were increased in wild-type papillomas but not in  Arf−/−p19 papillomas, indicating that p19Arf regulates p53 in response to activated Ras in vivo. However, other signals to induce p53, such as those stemming from DNA damage, remain intact in the absence of p19Arf, as shown by the rapid increase in p53 in irradiated  Arfp19-null tumors. Thus, of the many stimuli that have been shown to activate p53 using a variety of experimental systems  or exogenous stimuli . However, it remains possible that these other modes of p53 activation might predominate in other tumor types or in other circumstances.The observation that  systems , the RasArf and p53 were induced in papillomas, loss of p19Arf or p53 had opposite effects on early tumor growth. p19Arf deficiency resulted in increased tumor cell proliferation and tumor growth while p53-deficient mice had reduced tumor cell proliferation, tumor numbers, and tumor size. The observation that tumors harboring mutant Ras grew faster in the presence of p53 than in the absence of p53 differs from in vitro studies, in which ectopically expressed Ras induces p53-dependent growth arrest or senescence  were provided by Martine Roussel and Charles Sherr , and carcinogenesis studies were performed on the F3 littermates of this cross. 20 mice of each genotype,  Arf−/−, p19 Arf+/−,p19 and  Arf+/+p19 were treated. The backs of 8-wk-old male and female mice were shaved and treated with a single application of DMBA  followed a week later by twice weekly applications of TPA (200 μl of 10−4 M solution in acetone) for 15 wk. The number and size of papillomas on each mouse were recorded every 2 wk. Mice were sacrificed if moribund or following detection of carcinomas. Tumors were frozen for DNA extraction and/or fixed in formalin to be processed and stained with hematoxylin/eosin (H&E) for histological examination.p19es Sherr . To incrp53 (7 backcross to NIH) were crossed to  Arfp19-deficient (F4 backcross to NIH) mice to generate   Arf+/− p53+/−p19 mice. These mice were intercrossed to generate all nine possible  Arf /p53p19 genotypes. Some 20–30 mice of each genotype were subjected to the same DMBA/TPA protocol and monitored as described above.Mice deficient for p53   and p53 .In order to remove abundant keratin present in papillomas and carcinomas, nuclear extracts were prepared as described  with modHras was amplified with standard PCR , with 5′-GACTCCTACCGGAAACAGGT-3′ and 5′-CTGTACTGATGGATGTCCTC-3′ primers. We used the internal primer 5′-TGGTCATTGATGGGGAGACA-3′ to sequence exon 2, using PE Biosystems  Dye-Terminator and Big-Dye cycle sequencing.Genomic DNA was prepared from tumor and normal tissue by QIAamp DNA Mini Kit . A 400-bp PCR fragment containing exon 2 of Sections of normal skin, papillomas, carcinomas, and other organs were removed and fixed in 10% normal buffered formalin for 4 h. After fixation, tissues were processed and then embedded in paraffin. From the tissues, 4-μm sections were cut and stained for either p53  or pan-keratin  using high-temperature antigen retrieval in 10 mM citrate buffer (pH 6), or for BrdU  after treating with 2N HCl followed by 0.1% trypsin. After staining with the primary antibody, the sections were stained with a biotin-conjugated secondary  followed by StreptABComplex/HRP (Dako). Slides were developed with DAB/NiCl and counterstained with methyl green. Control sections with no primary antibody were run concurrently. Other sections were cut and stained using a standard H&E method. Proliferation index was determined by counting the number of BrdU-stained cells per 40× field. The apoptotic index was determined by counting the H&E slides for the number of apoptotic figures per 40× field. All counts were done on a Nikon  Labophot-2 microscope.Arfp19 wild-type allele in Arf+p19/− tumors, wild-type and knockout alleles were amplified by PCR separately then combined for electrophoresis. Primers 5′-AGTACAGCAGCGGGAGCATGG-3′ (Arf1), 5′-TTGAGGAGGACCGTGAAGCCG-3′ (Arf2), and 5′-ACCACACTGCTCGACATTGGG-3′ (ArfN) were used to amplify wild-type (Arf1 and Arf2) and knockout alleles (Arf2 and ArfN) from 100 ng of genomic DNA using 68 °C for annealing and extension at 90 s for 30 cycles. Equal amounts of each PCR product were then combined for electrophoresis on a 2% TAE agarose gel. Wild-type and knockout alleles of p53+/− tumors were amplified in a separate reaction as described . To measure LOH of the escribed  for 30 cArfp19 (or Arf /p53p19) genotypes on the development of papillomas, longitudinal profiles of papilloma counts were analyzed using the generalized estimating equation (GEE) approach (Arfp19 (or Arf /p53p19) genotypes. Estimates of relative odds were adjusted for sex effects. Fisher's exact test was used for comparing two proportions such as comparing LOH proportions between papillomas and carcinomas. All statistical tests were two-sided.In order to assess the impact of approach . GEE is approach  was used


Low adherence is a key factor in explaining impaired effectiveness and efficiency in the pharmacological treatment of hypertension. However, little is known about which factors determine low adherence in actual practice.The purpose of this study is to examine whether low social participation is associated with low adherence with antihypertensive medication, and if this association is modified by the municipality of residence.1288 users of antihypertensive medication were identified from The Health Survey in Scania 2000, Sweden. The outcome was low adherence with antihypertensives during the last two weeks. Multilevel logistic regression with participants at the first level and municipalities at the second level was used for analyses of the data.Low social participation was associated with low adherence with antihypertensives during the last two weeks , independently of low educational level. However, after additional adjustment for poor self-rated health and poor psychological health, the association between low social participation and low adherence with antihypertensives during the last two weeks remained but was not conclusive . Furthermore, the association between low social participation and low adherence with antihypertensives during the last two weeks varied among municipalities in Scania .Low social participation seems to be associated with low adherence with antihypertensives during the last two weeks, and this association may be modified by the municipality of residence. Future studies aimed at investigating health-related behaviours in general and low adherence with medication in particular might benefit if they consider area of residence. The effectiveness ,2 and efLow adherence is one important cause of uncontrolled hypertension ,10. Yet,Social participation is an important concept for understanding the influence of social factors on individual health and behaviour, and can be viewed as a feature of individual social networks . Good soMultilevel analysis handles both the micro-scale of people and the macro-scale of context simultaneously within one model . This anThe first aim of this study is to examine whether low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level and health status . The second aim is to analyse whether the hypothesised association between low social participation and low adherence with antihypertensives during the last two weeks varies between municipalities in Scania.The Health Survey in Scania 2000 (HSS-2000) was a postal self-administered questionnaire sent out to a random sample of 23 437 individuals born from 1919 to 1981 living in Scania. The purpose of the HSS-2000 was to obtain information about health conditions and different types of health hazards among the inhabitants of Scania . The proThe Ethical Committee at the Medical Faculty of Lund University approved the study proposal of The HSS-2000, and all of the participants received written information about the survey.Use of antihypertensives was based on an affirmative answer to the question "Have you during the last year used medicine, which was bought at the pharmacy...?" and indicating "Medication for high blood pressure"Low adherence with antihypertensives during the last two weeks (dichotomised) was based on the question "Have you used (this) medicine during the last year, but not during the last 2 weeks?" Those participants who answered yes were considered to have low adherence.Age was categorised in five groups: <35 (reference), 35–44, 45–54, 55–64 and ≥ 65 years.Low social participation (dichotomised) was assessed after the respondent stated involvement in three or fewer activities (lowest quartile) of 13 formal or informal activities , which the respondent might have participated in during the previous 12 months [2 months .Low educational level (dichotomised) was defined as having nine years of education or less.Poor self-rated health (dichotomised) was defined as a value of ≤ 3 on an ordinal self-rated health scale ranging from 1 ("Very bad") to 7 ("Very good") [y good") .Poor psychological health (dichotomised) was determined by giving three or more affirmative answers to the 12 items composing the Standardised General Health Questionnaire (GHQ-12) [(GHQ-12) .Because of the hierarchy in the data, with individuals nested in municipalities, we used multilevel logistic regression  with indi was created to study the influence of low social participation on low adherence with antihypertensives during the last two weeks, adjusting for age and sex. The second model ii was extended to also include low educational level, because low educational level could be a confounder in the association between low social participation and low adherence with antihypertensives during the last two weeks. The third model iii additionally contained poor self-rated health and poor psychological health. Impaired health may affect both low social participation and low adherence with antihypertensives.The first model The results are shown as odds ratios (OR) with 95% confidence intervals (CI)We calculated the second level variance  regarding prevalence of low adherence with antihypertensives during the last two weeks , and the second level variance regarding the association between low social participation and low adherence with antihypertensives during the last two weeks . We also calculated the covariance between intercept and slope residuals. The covariance gives information about whether the association between low social participation and low adherence with antihypertensives during the last two weeks depends on the prevalence of low adherence in the different municipalities .Parameters were estimated using the Restricted Iterative Generalized Least Squares (RIGLS) and penalised quasilikelihood (PQL). Extra-binomial variation was explored systematically in all models and we found no evidence for under- or over-dispersion. The MLwiN, Version 1.1 software package  was usedLow adherence with antihypertensives during the last two weeks was found among 11% (145/1 288) of the participants and 49% (635/1 288) were classified as having low social participation. The participants mean age was 63 years. Those participants classified as having low social participation more often reported low adherence with antihypertensives during the last two weeks, low educational level, poor self-rated health and poor psychological health than those who were not classified as having low social participation   and in the association between low social participation and low adherence with antihypertensives during the last two weeks (slope variance) Table . The negOur results suggest that low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level. In other words, the association between low social participation and low adherence withstood adjustment for socio-economic position  in our analyses. Social participation might therefore be considered as a real construct, and not only a proxy for socio-economic position. However, the association between low social participation and low adherence with antihypertensives during the last two weeks was weakened after additional adjustment for poor self-rated health and poor psychological health. Furthermore, the association between low social participation and low adherence with antihypertensives during the last two weeks may vary between municipalities in Scania.The weakening of the association between low social participation and low adherence with antihypertensives during the last two weeks after we adjusted for poor self-rated health and poor psychological health may be an expression for confounding. Impaired health may negatively affect both social participation and adherence with antihypertensives. On the other hand, the observed reduction of the association may instead be telling us that physical and mental health are in the pathway between low social participation and low adherence with antihypertensives during the last two weeks . Social Our present finding that the association between low social participation and low adherence with antihypertensives during the last two weeks may vary between municipalities in Scania gives empirical support to the existence of cross-level interactions  associated with health-related behaviours, such as low adherence with medication. These behaviours may be a result of the interaction between a person and his or her area of residence . In a prThe present study shows that multilevel regression analysis can be used for investigation of geographical disparities in health and health-related behaviour , without analysing any specific area characteristic . In multThe rather low participation rate (59%) may increase the risk of selection bias and reduce the ability to generalise the results and compare them to other populations. Nevertheless, the participation rate for participants aged 51–80 was about 65% and the participants using antihypertensives had a mean age of 63 years.The low adherence question we used was stated in a non-threatening manner, which might facilitate for participants to give an honest response and not underreport low adherence . Self-rePeople with low social participation and low adherence with antihypertensives during the last two weeks may have been less inclined to respond to the HSS-2000 questionnaire. This possible selection bias could lead to an underestimation of the association between low social participation and low adherence with antihypertensives during the last two weeks.Our results suggest that low social participation is associated with low adherence with antihypertensives during the last two weeks, independently of low educational level. In addition, the association between low social participation and low adherence with antihypertensives during the last two weeks seems to vary between the municipalities in Scania, which gives empirical support to the existence of cross-level interactions  associated with health-related behaviours, such as low adherence with medication. We have recently showed that factors related to the area of residence influence the individual blood pressure level, especially in people using antihypertensive medication , which iFuture studies aimed at investigating health-related behaviours in general and low adherence with medication in particular might benefit if they consider that area of residence may modify associations between individual variables.The author(s) declare that they have no competing interests.JM and KJ developed the original idea, participated in the design of the study, performed the statistical analyses and drafted the manuscript. LR and JS participated in the design of the study and revised the manuscript. TL participated in the design of the study, helped to collect the data and revised the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/5/17/prepub


Identifying regions of the human genome that have been targets of natural selection will provide important insights into human evolutionary history and may facilitate the identification of complex disease genes. Although the signature that natural selection imparts on DNA sequence variation is difficult to disentangle from the effects of neutral processes such as population demographic history, selective and demographic forces can be distinguished by analyzing multiple loci dispersed throughout the genome. We studied the molecular evolution of 132 genes by comprehensively resequencing them in 24 African-Americans and 23 European-Americans. We developed a rigorous computational approach for taking into account multiple hypothesis tests and demographic history and found that while many apparent selective events can instead be explained by demography, there is also strong evidence for positive or balancing selection at eight genes in the European-American population, but none in the African-American population. Our results suggest that the migration of modern humans out of Africa into new environments was accompanied by genetic adaptations to emergent selective forces. In addition, a region containing four contiguous genes on Chromosome 7 showed striking evidence of a recent selective sweep in European-Americans. More generally, our results have important implications for mapping genes underlying complex human diseases. An analysis of 132 human genes suggests that the migration of modern humans out of Africa into new environments was accompanied by genetic adaptations to emergent selective forces Despite intense study and interest, a detailed understanding of the evolutionary and demographic forces that have shaped extant patterns of human genomic variation remains elusive. An important goal in studies of DNA sequence variation is to identify loci that have been targets of natural selection and thus contribute to differences in fitness between individuals in a population. Identifying regions of the human genome that have been subject to natural selection will provide important insights into recent human history , the funThe neutral theory of molecular evolution , which pOne way out of this conundrum is to recognize that population demographic history affects patterns of variation at all loci in a genome in a similar manner, whereas natural selection acts upon specific loci . Thus, bHere, we describe an extensive analysis of the molecular evolution of 132 genes that were comprehensively resequenced in 24 African-Americans and 23 European-Americans. In total, over 2.5 Mb of baseline reference DNA was sequenced, spanning 20 autosomal chromosomes and the X chromosome. The sampling of a large number of loci dispersed throughout the genome has allowed us to clarify the relative contributions of demography and selection to patterns of genetic variation at individual genes. Specifically, we developed a rigorous computational approach for taking into account multiple hypothesis tests and demographic history, and we found that while many apparent selective events can instead be explained by demography, there is also strong evidence for positive or balancing selection at eight genes in the European-derived population. In addition, we describe a striking example of a previously unreported recent selective sweep in European-Americans that spans four contiguous genes on Chromosome 7. More generally, our data provide insight into the demographic histories of African-American and European-American populations and have important implications for genetic association studies of complex diseases, as several of the genes showing evidence of selection have been implicated in susceptibility to complex human diseases.p < 0.05) in one or more tests of the allele frequency distribution .We resequenced 132 genes primarily involved in inflammation, blood clotting, and blood pressure regulation and discovered a total of 12,890 SNPs . We firsribution . Thus, tThe direction of Tajima's D, Fu and Li's D*, and Fu and Li's F* is potentially informative about the evolutionary and demographic forces that a population has experienced. For example, negative values reflect an excess of rare polymorphisms in a population, which is consistent with either positive selection or an increase in population size. Positive values indicate an excess of intermediate-frequency alleles in a population and can result from either balancing selection or population bottlenecks. In the European-American sample, we observed eleven significantly positive and five significantly negative values for one or more of these three test statistics . In the The observations of both significantly positive and significantly negative values of Tajima's D, Fu and Li's D*, and Fu and Li's F*, combined with the largely nonoverlapping set of significant genes, could reflect selective pressures unique to one population , different demographic histories, spurious results, or most likely some complex combination of all of these factors. Although these results are intriguing, their interpretation is confounded by two issues: (1) We have not corrected for multiple hypothesis tests, and (2) rejection of the standard neutral model can result from either selective or demographic forces. In the subsequent sections, we develop approaches to address these issues with the dual goals of identifying genes that possess strong evidence of natural selection and of inferring population demographic history.p values (–8) distribution  for one or more tests of the allele frequency distribution  and S2. Although neutrality tests of the allele frequency distribution reveal many significant deviations, it is impossible to unambiguously interpret these data as evidence for natural selection, because the null model used to assess significance makes unrealistic assumptions about population demographic history. In principle, it is possible to distinguish between demography and selection, because demography affects all loci in the genome, whereas selection acts upon specific loci. Thus, by sampling a large number of loci dispersed throughout the genome, we can begin to construct a more realistic null hypothesis by which to evaluate the evidence for or against selection .To this end, we used the empirical data to explore four different demographic models A, which We reestimated the significance of Tajima's D, Fu and Li's D*, Fu and Li's F*, and Fay and Wu's H in each population for each of the four demographic models using the best-fit parameter values. All simulations included recombination and correction for multiple tests using the FDR method (with a FDR of 5%) as described above. Population history can clearly have a profound effect on tests of natural selection , and givEPHB6, TRPV6, TRPV5, and KEL;EPHB6, TRPV6, TRPV5, and/or KEL possess specific alleles that have conferred local adaptation to a unique environmental pressure in European-derived populations. Consistent with this hypothesis, we observed strong levels of population subdivision , which An alternative explanation for why we observed fewer significant results in African-Americans than in European-Americans is that African-Americans are an admixed population , and theABO in African-Americans  of accelerated evolution in either the human (TRPV6 and EPHB6) or chimp (DCN) lineage. In addition, Recently, EPHB6, TRPV6, TRPV5, and KEL. To our knowledge, this is the largest footprint of selection that has been described in the human genome, and likely reflects the combination of strong and recent selective pressures and reduced recombination in this region . Based on our current data it is impossible to identify which gene (or perhaps genes) has been the target (or targets) of selection. However, TRPV6 is a particularly interesting candidate, as it possesses three nonsynonymous amino acid substitutions  that are each nearly fixed for the derived allele in European-Americans, show significant frequency differences between European-Americans and African-Americans, and are located in the most significant regions of both Tajima's D and reduced nucleotide diversity  to determine the derived allele for Fay and Wu's H test. These data were generated under the auspices of the SeattleSNPs Program for Genomic Applications, which resequences candidate genes involved in inflammatory processes in humans. In general, we resequenced the complete genomic region for each gene, including introns and approximately 2 kb 5′ of the gene and 1 kb 3′ of the gene using Big-Dye terminator chemistry on an ABI 3700 or ABI 3730XL . For several exceptionally large genes, such as F13A1, less than complete coverage was obtained (see http://pga.gs.washington.edu/).Human DNAs were obtained from the Coriell Institute . We analyzed DNA from 24 African-Americans from the Human Variation Panel, African-American Panel of 50 (HD50AA) and DNA from 23 European-Americans derived from various CEPH pedigrees. We also sequenced each gene in a common chimpanzee . In order to correct for multiple tests, we repeated the coalescent simulations as described above, but included recombination. Following 4 coalescent realizations, we sampled the recombination rate from a Gamma distribution whose expectation equals the average genome-wide recombination rate of 10–8/generation .We initially assessed the significance of these statistics by comparing the observed values to 10ulations , conditineration . The posST as described in We quantified the allele frequency differences between the European- and African-American samples by the statistic FTRPV6 is the target of selection and the selected haplotype is defined by the C157R, M378V, and M681T polymorphisms. If mutations are Poisson-distributed, the expected number of segregating sites in a genealogy is E[S] = μE[T], where S, μ, and T denote segregating sites, neutral mutation rate of the locus, and total branch length of the genealogy, respectively. Assuming a star-shaped genealogy, E[T] = n × t, where n is the number of selected haplotypes. Thus, the time back to the selective sweep, t, can be estimated by S/(nμ). For TRPV6 in European-Americans, n = 45 , S = 11, and μ = 2.5 × 10–5.We estimated the time since the selective sweep for the Chromosome 7q region in European-Americans by analyzing the amount of nucleotide diversity that has accumulated on the selected haplotype as described in Wfor each population. The bottleneck model is specified by the parameters F (the inbreeding coefficient) and t (the time in years measured from the present) at which the bottleneck occurred. Values of F and t considered were F =  and t = . The exponential expansion model is determined by the parameters α (the growth rate/generation) and t . Values considered for α and t were: α =  and t = . The population structure under an island model is specified by the population migration rate between demes, M = 4om,N where oN and m are the effective subpopulation size and fraction of migrants in each subpopulation per generation, respectively. Values of M considered were M = . The structure model assuming population splitting with no subsequent migration is determined by the parameter t (the time in years since the populations diverged). Values of t considered were t = . In all simulations we assumed an effective population size of 10,000 and a generation time of 25 y in order to facilitate comparisons to a previous study .Click here for additional data file.Table S2(534 KB DOC).Click here for additional data file.Table S3(87 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/LocusLink/) for the genes discussed in this paper are ABO (28), ACE2 (59272), APOH (350), BDKRB2 (624), BF (629), C2 (717), CCR2 (1231), CD36 (948), CEBPB (1051), CRF (10882), CRP (1401), CSF2 (1437), CSF3 (1440), CSF3R (1441), CYP4A11 (1579), CYP4F2 (8529), DCN (1634), EPHB6 (2051), F10 (2159), F11 (2160), F12 (2161), F13A1 (2162), F2 (2147), F2R (2149), F2RL1 (2150), F2RL2 (2151), F2RL3 (9002), F3 (2152), F5 (2153), F7 (2155), F9 (2158), FGA (2243), FGB (2244), FGG (2266), FGL2 (10875), FSBP (10646), GP1BA (2811), ICAM1 (3383), IFNG (3458), IGF2 (3481), IGF2AS (51214), IL10 (3586), IL10RA (3587), IL10RB (3588), IL11 (3589), IL12A (3592), IL12B (3593), IL13 (3596), IL15RA (3601), IL17B (27190), IL19 (29949), IL1A (3552), IL1B (3553), IL1R1 (3554), IL1R2 (7850), IL1RN (3557), IL2 (3558), IL20 (50604), IL21R (50615), IL22 (50616), IL24 (11009), IL2RB (3560), IL3 (3562), IL4 (3565), IL4R (3566), IL5 (3567), IL6 (3569), IL8 (3576), IL9 (3578), IL9R (3581), IRAK4 (51135), ITGA2 (3673), ITGA8 (8516), JAK3 (3718), KEL (3792), KLK1 (3816), KLKB1 (3818), KNG (3827), LTA (4049), LTB (4050), MAP3K8 (1326), MC1R (4157), MMP3 (4314), MMP9 (4318), NOS3 (4846), PFC (5199), PLAT (5327), PLAU (5328), PLAUR (5329), PLG (5340), PON1 (5444), PON2 (5445), PPARA (5465), PPARG (5468), PROC (5624), PROCR (10544), PROS1 (5627), PROZ (8858), PTGS2 (5743), SCYA2 (6347), SELE (6401), SELL (6402), SELP (6403), SELPLG (6404), SERPINA5 (5104), SERPINC1 (462), SERPINE1 (5054), SFTPA1 (6435), SFTPA2 (6436), SFTPB (6439), SFTPC (6440), SFTPD (6441), SMP1 (23585), STAT4 (6775), STAT6 (6778), TF (7018), TFPI (7035), TGFB3 (7043), THBD (7056), TIRAP (114609), TNF (7124), TNFAIP1 (7126), TNFAIP2 (7127), TNFAIP3 (7126), TNFRSF1A (7132), TNFRSF1B (7133), TRAF6 (7189), TRPV5 (56302), TRPV6 (55503), VCAM1 (7412), VEGF (7422), and VTN (7448).LocusLink ID numbers (http://coriell.undmj.edu/) repository numbers for human genomic DNAs sequenced for this study are as follows. DNAs from African-Americans were NA17101–NA17116 and NA17133–NA17140. DNAs from European-Americans were NA06990, NA07019, NA07348, NA07349, NA10830, NA10831, NA10842–NA10845, NA10848, NA10850–NA10854, NA10857, NA10858, NA10860, NA10861, NA12547, NA12548, and NA12560.Coriell (


Subcutaneous leiomyosarcoma is a rare condition that accounts for 1% to 2% of all superficial soft tissue malignancies. Approximately 10% of cases arise in the trunk, although the extremities are the most commonly affected.We report herein the case of a 31-year-old man with a subcutaneous leiomyosarcoma, measuring 124 × 105 mm, arising in the left inguinal region. A wide local excision (with a resection margin ≥ 20 mm) was performed. Histological examination of the resected specimen revealed a leiomyosarcoma with high cellularity and two mitoses per 10 high-power fields. The patient remains well with no evidence of disease 5 years and 8 months after the operation.This is the first reported case of subcutaneous leiomyosarcoma arising in the inguinal region and also one of the largest tumors reported. The experience of this case and a review of the English-language literature  suggest that a resection margin of ≥ 10 mm is recommended when excising this rare tumor. Subcutaneous leiomyosarcoma arises from smooth muscle in the walls of arterioles and veins. It is a rare tumor accounting for 1% to 2% of all superficial soft tissue malignancies . It usuaA 31-year-old man presented with a painless left inguinal tumor, which had gradually grown during the past six months. Physical examination on admission revealed a fist-sized subcutaneous tumor in the inguinal region. The overlying skin appeared normal without ulceration Figure . With a The resected tumor, measuring 124 × 105 mm, was solid, encapsulated, and a homogeneous yellowish white in color, without central necrosis and hemorrhage on its cut surface. Routine histological examination with hematoxylin-and-eosin revealed that the tumor comprised spindle-shaped cells with high cellularity in parts Figure  and two The patient had an uneventful recovery and was discharged on the 9th postoperative day. As the resection margin was negative, no adjuvant treatment was given. He remains well with no evidence of disease 5 years and 8 months after excision.Although subcutaneous leiomyosarcoma commonly arises in the lower extremities, it occasionally affects the trunk ,2,4. A ret al., [As subcutaneous leiomyosarcoma is resistant to radiotherapy and chemotherapy ,3,9, suret al.,  demonstrThe author(s) declare that they have no competing interests.KY, YS, NF, and DS took part in the operation, performed the literature search and drafted the manuscript for submission. HU performed histological examination. KH supervised the preparation of the manuscript and edited the final version for publication. All authors read and approved the final manuscript.


To investigate the genetic underpinnings of a particular biological process, geneticists screen large collections of mutant organisms to characterize their physical defects. By comparing the genetic makeup of nonmutant  organisms to mutants, it's possible to tease out the genes responsible for a defective appearance, or phenotype. In a classic study in the fruitfly, Christiane Nüsslein-Volhard and Eric Weischaus bred many lines of flies with mutations that were lethal: the fly embryos died, but not before displaying a wide range of developmental defects. Since it was known that the fruitfly needed only a single wild-type copy of these genes to survive, the mutations in these “embryonic lethals” had to be recessive, meaning that both copies, or alleles, of the gene had to be mutated for the lethal defect to appear. Nüsslein-Volhard and Weischaus's work revealed many such recessive genes crucial to early development and earned them a Nobel Prize.Among the model systems for studying development, the zebrafish has become prized because its transparent embryo develops outside the mother's body. The zebrafish has helped biologists identify many genes involved in embryogenesis and, because it's a vertebrate animal, has become a valuable resource for identifying genes involved in human disease. Now, a team led by Nancy Hopkins of the Massachusetts Institute of Technology, has created over 500 lines of zebrafish with lesions in key embryogenic genes and used them to identify a group of genes that predispose the fish to cancer, with some surprising results.(rp)—that is, each line carried one healthy version and one defective version of a different rp gene. These proteins are components of ribosomes—the massive molecular complexes within cells that mediate protein synthesis—and are essential for embryonic development.All of the 500 lines created by the researchers carried a recessive embryonic lethal mutation; for about 400 of the lines, mutations in 300 distinct genes were identified as the cause of the embryonic phenotype. During the process of cultivating some of these mutant lines, the Hopkins team noticed that an abnormally large percentage of fish experienced early mortality , while the surviving fish in these lines developed large, highly invasive malignant tumors; both phenotypes persisted over successive generations. The tumors resembled malignant peripheral nerve sheath tumors (MPNSTs) that have been found in other fish species as well as in mammals. Suspecting that these mutant lines had elevated rates of cancer, the researchers investigated the genetic makeup of the fish and discovered to their surprise that each line was heterozygous for a mutation in a different ribosomal protein gene rp mutations, the researchers report, either reduced or eliminated expression of the corresponding rp gene. In the case of “classic” tumor suppressor genes, the wild-type allele must be lost for the defective allele to set the stage for cancer. Here, the wild-type allele appeared to remain intact in the tumor cells, implicating the proteins as “haploinsufficient” tumor suppressors—a reduction from two gene copies to one functional copy seems to be enough to increase the risk of cancer. Apart from the mutations in rp genes, the authors also found a loss-of-function mutation in a gene  that acts as a tumor suppressor in mammals—establishing the soundness of this approach for identifying mammalian cancer genes.All of the rp genes whose mutations caused cancer from the five other rp genes whose mutations did not, the authors raise a number of possibilities for future study. And given the high degree of conservation of genes and pathways among vertebrates, it's likely that rp mutations also raise cancer risk in humans. Together, these results demonstrate that the tiny freshwater workhorse of developmental biology has a promising future as a model system for human cancer.While these experiments do not explore how these mutations lead to cancer, the results suggest that some shared, ribosome-associated function allows these genes to act as tumor suppressors and that disrupting this function somehow leads to tumor formation. Though it's not clear what distinguishes the 11


With billions of cells in the adult human body, all replicating and dividing in an environment laden with toxins, radiation, and free radicals, a certain amount of DNA damage is guaranteed to occur. Fortunately, all organisms have built-in checkpoints throughout the cell cycle that prevent such mistakes from propagating. At the G1 checkpoint during cell division, for example, molecules survey nuclear DNA for errors and breaks before the cell is deemed fit to undergo S phase, the DNA replication stage. If damage is found, enzymes either work to repair it or, in some cases, trigger programmed cell death, or apoptosis. But when checkpoints fail, and DNA damage is left unrepaired, disease such as cancer can result. A better understanding of these events, as provided, for example, by Vincenzo Costanzo and colleagues in this issue, will consequently lead to a better understanding of the mechanisms that give rise to cancer.A serious form of DNA damage, called a double strand break (DSB), cuts the helix clean through—a far worse scenario than if just one strand slips free. In response to a DSB, the cell recruits a signaling protein called ATM and a three-protein complex called MRN, whose components selectively bind to broken DNA ends. A malfunction of this signal and repair pathway is dire. People who suffer from the genetic disease ataxia-telangiectasia (A-T) lack a functioning ATM molecule and therefore cannot properly handle DSBs or successfully navigate the G1 checkpoint. This condition leads to a host of problems, including abnormal chromosomes, deficient immune function, and a predisposition to cancer. A-T-like disease (ATLD), another rare genetic condition, has very similar symptoms. The only difference is that the protein missing is Mre11, a subunit of MRN. While recent work on the cellular level has indicated that MRN activates ATM, the biochemical relationship between these proteins has yet to be fully understood.Studying these two molecules using traditional biochemical assays is difficult because knocking out the activity of these proteins is lethal to many cells. Costanzo and colleagues used a novel test system of cell-free frog extracts and found that Mre11 is necessary for both ATM activation and for the formation of large protein–DNA complexes apparently responsible for triggering the cascade of signaling molecules underlying the DNA damage response at the G1 checkpoint.The frog extract system allowed the team to manipulate the presence or absence of Mre11 and accurately measure the response triggered by the addition of fragmented bits of DNA . As predicted, without a functional Mre11 protein, ATM was not activated and there was no response. By simply adding the protein Mre11 back to the mixture, the damage response was restored. But when the researchers added a mutant form of Mre11, still capable of performing its essential tasks in another stage of the cell cycle—DNA replication—the G1 damage response remained suppressed. This mutant form of the Mre11 protein lacks the C-terminal, or DNA-binding, end.Costanzo and colleagues also found that this DNA-binding end is required for the assembly of DNA–ATM–MRN complexes in the presence of fragmented DNA and seems to direct the entire damage response. This work helps to explain the similarity between patients with A-T and those with ATLD, and hints at the formation of a large “signaling” complex that helps to orchestrate the crucial response to DBSs in DNA.


Endothelial cell dysfunction may be implicated in the development of multiple organ failure (MOF) by a number of mechanisms. Among these, altered fibrinolysis promotes fibrin deposition, which may create microvascular alterations during inflammation. Elevated concentrations of C-reactive protein (CRP), especially when these persist over time, are correlated with an increased risk of MOF and death. CRP may inhibit fibrinolysis by inducing plasminogen activator inhibitor-1 (PAI-1) release from human aortic endothelial cells. Moreover, the administration of recombinant CRP in volunteers may increase circulating PAI-1 levels.In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis in intensive care patients with and without sepsis.We studied the association of inflammation and abnormal fibrinolysis in intensive care unit (ICU) patients with (n = 11) and without (n = 21) sepsis. The inflammatory response was assessed by serum concentration of C-reactive protein (CRP), a marker of the acute phase reaction, which increase rapidly in the inflammatory response, and the plasma fibrinolytic capacity was evaluated by the Euglobulin Clot Lysis Time (ECLT), determined by a new semi-automatic method.2 = 0.45; p < 0.001). In a multivariate analysis, CRP was the strongest predictor of ECLT . In addition, the overall ICU length of stay was significantly correlated with CRP  and ECLT .ECLT was significantly higher in septic than non-septic patients  and was significantly correlated with CRP concentration (RIn critically ill patients a significant correlation thus exists between plasma fibrinolytic capacity and serum CRP levels. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. This finding is compatible with a link between inflammation and abnormal fibrinolysis, and may explain the negative prognostic value of CRP in critically ill patients. Endothelial cells have a key role in the control of vascular permeability and vessel tone, coagulation and fibrinolysis, and inflammatory response . There iEndothelial dysfunction/or activation is associated with an imbalance in hemostatic functions. Endothelial cells are responsible for the release of tissue plasminogen activator (t-PA) and contribute to the release of plasminogen activator inhibitor-1 (PAI-1). Inhibition of the fibrinolytic system amplifies the pathogenic role of fibrin deposition during severe inflammation . MultiplIn infected patients, elevated concentrations of serum CRP are correlated with a risk of MOF and death , especiaCRP can act directly on endothelial cells, inducing, for example, the expression of intercellular adhesion molecule (ICAM)-1  and the In this study, we tested the hypothesis that CRP is associated with hypofibrinolysis as measured by ECLT in intensive care patients with and without sepsis.After approval by the A. Vésale hospital ethics committee, we studied 32 ICU patients with severe sepsis n = 11) or other diagnoses (n = 21). Infection definition required isolation of a microorganism from a normally sterile body site, concurrent with accompanying signs and symptoms of sepsis and decision of antibiotic therapy. Criteria for severe sepsis included signs of at least one organ dysfunction attributed to sepsis . All pat or other®. Fibrinogen was determined by thrombin time on a STA® automate (STAGO). Leukocyte and platelet counts were determined on a hemocytometer . All tests were performed on blood obtained from the same venipuncture.Blood samples were obtained during the first 24 hours of sepsis or on the first day of admission for non-septic patients. Serum samples were collected in vacuum tubes without anticoagulant. Plasma samples were harvested in citrated vacuum tubes and put in melting ice. Whole blood was collected on EDTA-treated tubes. CRP was evaluated by antibody-binding and turbidity measurement on SYNCHRON LXThe Euglobulin Clot Lysis Time (ECLT), which is the most common test used to estimate the plasma fibrinolytic capacity, represents the balance between t-PA and PAI-1 activities . ECLT wa® software package (Jandle Scientific). The data are presented as mean ± SD. Correlation between variables was analyzed using a Pearson correlation test. A multivariate analysis was used with stepwise backward selection of the explicative variables. Sepsis was considered as a dichotomous variable while all other data were considered as continuous . ECLT was the dependent variable. A probability level of p < 0.05 was considered as statistically significant.We used SigmaStatThe major cause of severe sepsis (9 patients) or septic shock (2 patients) was pneumonia (8 patients); angiocholitis was the cause in 1 patient, and in 2 patients the cause was not identified. In 8 patients (7 with pneumonia and 1 with angiocholitis), the infection was due to a Gram negative bacteria. Only 1 patient had documented bacteremia. Non-septic patients were admitted for postoperative surveillance (7 patients), intracerebral hemorrhage (3 patients), heart failure (3 patients), drug intoxication (3 patients), or aggravated chronic obstructive pulmonary disease (5 patients).2 = 0.45; p < 0.001) with no perceptible threshold . In multivariate analysis, ECLT was best predicted by the CRP level  and not significantly by sepsis or the fibrinogen concentration. Interestingly, the ICU length of stay was significantly correlated with CRP  and ECLT  in all patients, and in the survivors .As expected, inflammatory parameters such as white blood cells and CRP levels, the SAPS II score and ECLT were higher in the septic than the non-septic population Table . In an uMany studies have demonstrated that sepsis is associated with endothelial cell dysfunction and promotes coagulation activation . Althougin vivo. Our data suggest that CRP could itself be involved in the processes leading to endothelium dysfunction. The observed relationship does not prove a direct biological link between increasing CRP and hypofibrinolysis; however, indirect arguments exist in support of the concept.Inhibition of the fibrinolytic system may contribute to MOF, as fibrin can activate endothelial cells leading to a disorganization of the monolayer and the release of inflammatory cytokines . Increasin vitro studies have reported the direct effects of CRP on endothelial cells [In vivo, Cleland et al. [G-monomethyl-L-arginine (L-NMMA), reflecting endothelial dysfunction. Bisoendial et al. reported that the administration of CRP in volunteers impairs the fibrinolytic balance [Several al cells -8. In vid et al.  reported balance . In addi balance . In a no balance .in-vitro and in-vivo. This mechanism seems to be important in sepsis, as high plasma levels of PAI-1 are associated with poor outcome [CRP could also act indirectly on endothelial cells via the action of monocytes and the release of tumor necrosis factor-α (TNF-α). TNF-α is a strong inducer of PAI-1 production  outcome . Moreove outcome . CRP can outcome .CRP also has essential biological functions. No polymorphism of either the gene coding sequence or of the protein itself has been described in humans . Also, hThis work is a pilot study. We have chosen to include patients in the first day of ICU admission to limit the possible rapid effect of inflammatory response on fibrinolysis, especially in septic patients. Indeed, this particular patient population has inflammatory reaction before signs of severe sepsis and thus before their admission to ICU. In fact, we have studied ECLT test at the onset of the organ dysfunction and the inflammatory reaction. Other studies with serial measurement of ECLT in patients who developed nosocomial ICU infections are needed to study the time course of these events. Moreover, we could not definitively exclude that all patients in the non-septic groups were non infected. For example, some non-septic patients with decompensated COPD may have minor infections, despite the negative microbiology cultures and the absence of antibiotic therapy. Viral infections were also possible in some patients.Moreover, it would be of great interest to determine the interactions between CRP and ECLT with IL-6, TNF-α and endothelium dysfunction markers such as soluble thrombomodulin and soluble von Willebrand factor.Despite accumulating evidence that the inflammatory and coagulation systems are activated in sepsis, little is known about the mechanisms that ultimately lead to organ dysfunction and death. Our data were obtained in the first 24 hours of ICU admission or of sepsis, thus, the relation between CRP and hypofibrinolysis appeared very quickly. Prospective studies including the time course of CRP and hypofibrinolysis would provide additional information about this relationship.KZB: Laboratory analysis, writing of the manuscript and design of the study.MP: patients recruitment and design of the study.DB: coordination and design analysis of the results.MG: design of the study.PC: laboratory analysis.JLV: design of the study and analysis of the results.CR: design of the study and analysis of the results.YB: patients recruitment.MV: statistical analysis and coordination.


The aim of this analysis was, however, to identify the influencing factors on high catecholamine levels Various potential risk factors that could impact the humoral stress response before induction of anaesthesia were recorded in 84 males undergoing coronary aortic bypass surgery, and were entered into a stepwise linear regression analysis. The plasma level of norepinephrine measured immediately after radial artery canulation was chosen as a surrogate marker for the humoral stress response, and it was used as the dependent variable in the regression model. Accordingly, the mean arterial blood pressure, heart rate and the calculated pressure-rate product were taken as parameters of the hemodynamic situation.-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by more than 40% compared to no clonidine administration, from 1.26 to 0.75 nmol·l-1. There was no evidence for dose-responsiveness of clonidine. All other potential predictors were removed from the model as insignificant (p > 0.05). The use of beta-blocker, ace-inhibitors, ejection fraction, and body mass index were significant determinants for the hemodynamic situation  of the patient during the pre-induction period.Stepwise regression analysis revealed that the oral administration of low-dose clonidine (mean dose 1.75 μg·kgThe oral administration of clonidine is the only significant predictor for the observed variation of norepinephrine levels during the preoperative period. Lack of significant dose responsiveness suggests that even a low dose of the drug can attenuate the preoperative stress response and thus is recommended in cardiovascular high risk patients. Even ve drugs ,6-19.However, little attention has been paid to the stress response of cardiac high risk patients when entering the operating area, during initiation of routine monitoring, and finally during awake venous and arterial canulation. Especially the latter procedure can cause significant discomfort for patients even when performed under local anaesthesia . In seveAfter Ethics Committee approval was obtained, patients gave their written and informed consent. Eighty-four consecutive male patients undergoing CABG-surgery were enrolled into this observational study. The only exclusion criterion was emergency operation.-1 and the same-day coefficient of variation for norepinephrine measurements determined by repeated measures of a standardized probe was 3%.Due to the observational character of the study, drugs administered preoperatively (including clonidine) were given at the discretion of the anaesthetist performing the preoperative examination. Each patient received an oral premedication with clorazepate 20 mg in the evening before surgery and in the morning of surgery. In about half of the patients, benzodiazepine premedication was combined with clonidine 75–300 μg. Patients were maintained on their regular cardiac and antihypertensive medication up to the day of surgery but all inhibitors of platelet aggregation were discontinued 3–7 day preoperatively. After arrival at the operating theatre an i.v. line was initiated and 500 ml hydroxyethyl-starch  was infused. A 12-lead channel ECG with an automatic ST-segment analysis, oxygen saturation and invasive blood pressure monitoring were connected to the patients. A radial artery catheter was then inserted after local anaesthesia with 1 ml mepivacaine 1%. Heart rate (HR) and mean arterial blood pressure (MAP) were recorded every 15 seconds online using a Laptop computer connected to a Solar 9500 monitor . HR and MAP were multiplied to receive the pressure-rate product (PRP). These variables were used as a measure of the hemodynamic stress response. To determine humoral stress response, an arterial blood sample for the measurement of norepinephrine plasma level was taken immediately after placing the radial artery catheter (this measurement was the main outcome of the study) and 5, 15, and 60 minutes after endotracheal intubation was performed  and sufentanil (10 μg.kg-1.h-1). After loss of consciousness propofol was reduced to 3 mg·kg-1.h-1 and sufentanil to 1.5 μg·kg-1.h-1. Endotracheal intubation was performed after administration of pancuronium bromide 0.1 mg·kg-1.General anaesthesia was standardized. After administration of midazolam 0.05 mg·kgTo allow comprehensive analysis of potential factors associated with a reduced stress response, the following data were recorded prospectively:• Age• Bodyweight• Height• Body mass index (BMI)• Clorazepate dose per kilogram bodyweight• Clonidine (yes – no)• Clonidine per kilogram bodyweight• Time from morning premedication until observational period• Inhibitors of the angiotensine converting enzyme system (ace-inhibitors)• Beta-blocking drugs• Calcium antagonists• Angiotensin-2 receptor inhibitors• Left ventricular ejection fraction (EF)• Number of affected vessels2 of 0.3 and higher, attributed to 14 independent variables using an F-test with a significance level of 0.05. All potential relevant factors were subjected to a stepwise linear regression analysis using a backward technique. In each step the least significant factor was eliminated when F was lower than 3.96. The quality of the regression model was judged using the Durbin-Watson statistic , and by checking if the standardized residuals are normally distributed. All calculations were performed using SPSS 11.0 for Windows. All continuous data are presented as mean and standard deviation when normally distributed and as median (25th–75th percentile) when normal distribution had to be rejected using the Kolmogorov-Smirnov-test.A power analysis had revealed that 80 patients provide a power of more than 95% to detect an R-1) on the morning of surgery was the only significant predictor (p = 0.004) of the high variation in preoperative norepinephrine plasma levels. This intervention decreased norepinephrine levels by 40% compared to no clonidine administration (from 1.26 to 0.75 nmol·l-1). In this analysis, the dichotomous variable (clonidine administration: yes-no) was a better predictor for the norepinephrine levels than variables including the clonidine dose (absolute dose or dose per body weight), indicating that our data provide no evidence for a strong dose-responsiveness of clonidine in this context.Stepwise regression analysis revealed that the single administration of low-dose clonidine (mean dose 1.75 μg·kgAll other of the investigated factors (see methods) were removed from the regression model as not significant. The two factors that were eliminated with a p < 0.1 during the last but one and during the final step were body mass index (removed in step 11 with a p = 0.064) and age (removed in step 12 with a p = 0.076). Both factors were associated with increased norepinephrine levels.The overall quality of the regression model was excellent. The Durbin-Watson coefficient was 2.04 (very near to the optimum of 2.0) and the standardized residuals were normally distributed.For mean arterial blood pressure, heart rate and the calculated pressure rate product, however, preoperative clonidine administration was not an influencing factor.For the mean arterial pressure (MAP), a higher ejection fraction (EF) was a statistically significant predictor (p = 0.024). Each 10% increase of EF was associated with a 2.7 mmHg higher MAP. Administration of an ace-inhibitor was the second predictor in the final model of MAP (p = 0.03). These patients had a 7.5 mmHg lower MAP than patients without ace-inhibitors.-2 (p = 0.001).For heart rate (HR) there were three significant predictors that remained in the model. Administration of beta-blockers and ace-inhibitors were both associated with a decreased HR (p = 0.004). Each of them decreased HR between 6–7 beats per minute (bpm). Additionally, a higher BMI was associated with a 1.3 bpm higher HR per kg·mSince the PRP is the product of HR and MAP, it is not surprising that similar variables contributed to its prediction. These were the administration of beta-blockers (p = 0.017) and ace-inhibitors (p = 0.004), each of them reducing the PRP, whereas the EF was associated with an increase in PRP (p = 0.014).No patient had signs of cardiac ischemia on arrival at the operating theatre until induction of anaesthesia (defined as ST-T change > 0.1 mV in any ECG lead). There were no major adverse events during the entire induction period and surgery.2-adrenergic receptor agonist clonidine acts by decreasing central sympathetic nervous system activity in all hyperadrenergic situations. In addition to its sedative, anxiolytic, analgesic and antihypertensive properties [The αoperties ,22 it haoperties ,24 and toperties ,26.In many investigations attention has been drawn to the stressful stimulus of endotracheal intubation ,4,6-19, Thus, it was the major aim of this observational study to identify factors that might contribute either to increased humoral stress or that might help to attenuate this response.Our results show, that a single application of low dose oral clonidine was the only factor that was associated with significantly decreased norepinephrine levels on arrival at the pre-induction area. The question that arises from this observation is, if this is simply an association  or if clonidine premedication is the cause for lower norepinephrine levels. In our opinion the latter is the case. Firstly, there were no differences considering any other variables between those patients who had received clonidine and those who had not (see table -1) was low compared to all other trials. Data concerning the appropriate dose of clonidine to attenuate the stress response to intubation vary considerably between 0.625 and 10 μg·kg-1. For example, one trial demonstrated that clonidine 0.625 and 1.25 μg·kg-1 i.v. were sufficient to attenuate pressure response to laryngoscopy and intubation [-1 clonidine i.v. was equally effective as placebo, and only 4 and 6 μg·kg-1 significantly attenuated hemodynamic and adrenergic reactions in an equal manner. It could also be shown that 4 or 6 μg·kg-1 were necessary to reduce norepinephrine levels before induction of anaesthesia, however 2 μg·kg-1 where not sufficient in this setting [However, it is interesting to notice that the mean dose administered to our patients . Furthermore, a post-hoc comparison between the patients receiving either 75 or 150μg clonidine did not show relevant differences (p = 0.91 using the Mann-Whitney U-test).Higher age and higher body mass index showed a non-significant tendency to increase the catecholamine concentration. No other of the investigated factors  had statistically significant impact on norepinephrine levels.An explorative post-hoc analysis of the impact of clonidine premedication (none versus any dose) and clonidine dose on norepinephrine levels during the entire induction period proves the results of the main analysis. There was a pronounced reduction of norepinephrine plasma levels after induction of general anaesthesia with lower values in the clonidine-group. However, a statistically significant interaction term p = 0.012) suggests that the fall of norepinephrine levels are more marked in the untreated group and thus mainly caused by induction of general anaesthesia rather than effects of clonidine (figure 2 suggestThis observational trial demonstrates that patients undergoing coronary artery bypass graft surgery have a great variation of norepinephrine levels when entering the operating theatre. We could identify oral clonidine premedication as the only predictor for increased humoral stress response. There was no strong evidence for a dose dependency, indicating that even small doses, like 75–150 μg attenuate the humoral stress response before coronary artery bypass graft surgery. Clonidine did not have a negative impact on hemodynamic parameters.None declared.AMM processed the data and wrote the manuscript.GG conceived the study, collected the clinical data and participated in its design.US collected the clinical data.MK designed the study and collected the clinical data.HAA performed the laboratory investigations.HW participated in the conception of the study.LHJE designed the study, performed the statistical analysis and extensively revised the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


While previous authors have emphasized the importance of integrating and reinforcing evidence-based medicine (EBM) skills in residency, there are few published examples of such curricula. We designed an EBM curriculum to train family practice interns in essential EBM skills for information mastery using clinical questions generated by the family practice inpatient service. We sought to evaluate the impact of this curriculum on interns, residents, and faculty.Interns (n = 13) were asked to self-assess their level of confidence in basic EBM skills before and after their 2-week EBM rotation. Residents (n = 21) and faculty (n = 12) were asked to assess how often the answers provided by the EBM intern to the inpatient service changed medical care. In addition, residents were asked to report how often they used their EBM skills and how often EBM concepts and tools were used in teaching by senior residents and faculty. Faculty were asked if the EBM curriculum had increased their use of EBM in practice and in teaching.Interns significantly increased their confidence over the course of the rotation. Residents and faculty felt that the answers provided by the EBM intern provided useful information and led to changes in patient care. Faculty reported incorporating EBM into their teaching (92%) and practice (75%). Residents reported applying the EBM skills they learned to patient care (86%) and that these skills were reinforced in the teaching they received outside of the rotation (81%). All residents and 11 of 12 faculty felt that the EBM curriculum had improved patient care.To our knowledge, this is the first published EBM curriculum using an individual block rotation format. As such, it may provide an alternative model for teaching and incorporating EBM into a residency program. Evidence-based medicine (EBM) strives to provide a systematic approach to integrating the best research evidence with clinician expertise and patient preferences to provide better patient care . While tWhile there have been several studies of residency EBM curricula ,9-11, noThe UCSF family practice residency program is based at San Francisco General Hospital (SFGH), a large county hospital serving the urban poor. The program runs a busy family practice inpatient service (at SFGH) as well as the Family Health Center, an outpatient clinic that includes both continuity practices and acute care services. Within this setting, we formulated 3 primary goals for our EBM curriculum: (1) to teach interns basic EBM concepts and skills; (2) to disseminate and reinforce EBM skills to second and third year residents and faculty; and (3) to apply EBM to the care of patients. This study was approved by the UCSF Committee on Human Research.The EBM curriculum for UCSF family practice residents began, in its current form, summer of 2001. The core of the curriculum is a 2-week, individual EBM rotation for interns. In contrast to the usual format of multiple lectures or learning modules scattered throughout residency, the two-week individual block format provides for a concentrated time in which to learn EBM. It also allows tailoring of the rotation to fit the residents' backgrounds and interests. During each of the two weeks, residents have 3 half-day clinics, with the remainder of the time available for EBM. The EBM portion of the rotation fulfills the ACGME requirements for resident research and scholarly activity.patient/problem, intervention, comparison, and outcome). The entire process generally takes 5 to 10 minutes and includes modeling of how to formulate an appropriate 'answerable' question by senior residents and the inpatient attending faculty. The question(s) generated are then used by the librarian Co-director as material for a tutorial on developing search strategies and using high-quality web-based EBM resources. The emphasis of the tutorial is to introduce the intern to the concept of information mastery [The rotation begins with a meeting with the EBM faculty Director (DT) during which the structure and goals of the rotation are communicated, the intern's knowledge and experience related to EBM are assessed by review of his or her experience and by a pre-rotation test of EBM skills and knowledge (described below). Interns receive reference materials ,12 and a mastery , and lea mastery .During the first week, the intern completes a web-based EBM tutorial  which coDuring the initial 3 months of the curriculum, interns completed a written test of their EBM skills and knowledge before and immediately following the rotation. The tests were adapted, with permission, from a similar instrument developed by Sean Schafer, MD and Katie Ramos, PhD at the University of California Fresno Family Practice Residency Program . Scores As others have noted, progress in the use of EBM depends on the availability of information support services to resident and faculty at the point of patient care . In conjWe estimate that training 13 interns per year requires approximately 70 hours of librarian time and 200 hours of the faculty Director's time. In the event that the medical librarian Co-Director (JH) is not available, the faculty Director (DT) provides coverage for this portion of the rotation. Absence of the faculty Director for one or two days can usually be covered by schedule modifications and communication by telephone and e-mail. An extended absence of the faculty Director requires another faculty member assuming supervisory responsibility.We evaluated our EBM curriculum with respect to our 3 primary goals using pre- and post-rotation questionnaires (completed by each EBM intern) and by a survey of family practice residents and faculty.The EBM intern questionnaires consisted of pre- and post-rotation self-assessments of confidence in EBM knowledge and skills . Self-confidence in EBM knowledge and skills was assessed by asking the intern to rate his or her level of comfort from 1 = very uncomfortable to 5 = very comfortable for (1) MEDLINE searching to answer a clinical question; (2) use of Web-based EBM resources to answer a clinical question; and (3) use of EBM principles to critically evaluate articles. At the end of the rotation interns were asked to identify the most useful and least useful aspects of the rotation and what could make the rotation better. In addition, residents completed and returned to the Residency Coordinator a standard evaluation of the rotation.We evaluated the dissemination and reinforcement of EBM skills and knowledge to residents and faculty  and the application of EBM to patient care  by surveying residents  and faculty. These surveys were distributed and collected by a program assistant. Surveys were labeled with a code number for each resident and faculty and results were reported in aggregate to provide anonymity. The resident survey asked "How frequently have you continued to apply EBM concepts and tools from the rotation to answer clinical questions?" and "How frequently have EBM concepts and tools been reinforced via teaching by faculty or senior residents?" For both questions, response options were 1 = never, 2 = seldom (less than once per month on average); 3 = occasionally (1 to 3 times per month on average); 4 = often (1 or 2 times a week on average); and 5 = frequently (3 or more times per week on average). For faculty and residents who had had a clinical question answered by the EBM intern on the inpatient service were asked. "How often did the EBM answer provide useful information?" and "How often did the answer change your management of a patient?" For both questions, response options were 1 = less than 25% of the time; 2 = 25% to 75% of the time; and 3 = more than 75% of the time. Finally, all faculty and residents were asked if they believed that "the presence of the EBM rotation improves the quality of patient care within the family practice residency program."During the period from July 1, 2001 to September 30, 2003, 30 EBM interns presented 30 journal clubs and generated 74 CATs in response to questions from the inpatient service. Journal club presentations and CATS are electronically archived and make accessible via the residency website . InternsSurveys to evaluate goals 2 and 3 were completed by 21 of 25 residents (response rate of 84%) and 12 of 13 faculty (response rate or 92%). As seen in Table As shown in Table While the above findings support the acceptance and perceived utility of our EBM program, they do not provide a formal measure of its effectiveness. Changes in resident knowledge, skills and confident were measured over a short period of time. The frequency of reinforcement of EBM and the impact of EBM on clinical care was by resident and faculty report and may have been biased. No attempt was made to observe changes in physician behaviors or patient outcomes. There were no measures of EBM use prior to the introduction of the EBM rotation and no comparison group was available. It is therefore not possible to objectively determine to what degree current levels of awareness and utilization of EBM are the result of the rotation. It is also not possible to separate out the effects of the 2-week EBM rotation from the adjunct changes of establishing a medical information website or promoting the use of PDAs, except to the extent that the questions asked specifically about the 2-week EBM rotation.Our block EBM rotation differs substantially from the approaches reported in previous studies ,11,16 inInterns reported greater confidence in their search skills after the two week rotation. Two randomized controlled trials and a controlled before-after study have demonstrated benefits of training in electronic search skills -19. InteOne study that evaluated the impact of a 1-month pilot program to use EBM methods on an inpatient service, reported results roughly similar to ours . Our curPrevious studies have found only small increases in residents' knowledge and skills from journal clubs alone, leading to the suggestion that journal clubs should be used as a component of EBM training rather then being 'stand alone' activities -8. WhileOther investigators have reported educational interventions aimed at improving faculty knowledge and skills in medical informatics and EBM. One study reported increases in faculty self-rating of EBM skills following an intervention consisting of 2 half-day workshops and substantial amount of individual mentoring . We founOur EBM curriculum, based on an individual 2-week EBM rotation in the first year, appears to be successful in increasing resident's EBM skills and confidence. In addition, resident and faculty both perceive EBM as being incorporated and reinforced beyond the rotation, and that the presence of EBM in the residency improves the quality of patient care. We hope that our experience provides a useful model for teaching and integrating EBM into a busy, resource-limited, family practice residency.The authors declare that they have no competing interests.All authors participated in the development of the curriculum. DT conceived of and conducted the evaluation. JH and PSS reviewed the survey instrument. JH and PSS reviewed and suggested changes to the paper. JH drafted the subsection describing the teaching the use of electronic. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


A new study adds to the mounting evidence implicating T cells as an important component of the inflammation in chronic obstructive pulmonary disease Chronic obstructive pulmonary disease (COPD) is a global epidemic of major proportions that is predicted to become the third most common cause of death and fifth most frequent cause of chronic disability by 2020. In developed countries it is mainly caused by cigarette smoking, but the reasons why only a proportion 10%–20%) of smokers develop progressive airflow limitation is currently unknown. The disease is characterized by a chronic inflammatory process predominantly in the small airways and lung parenchyma, with increased numbers of macrophages, neutrophils, and T lymphocytes [0%–20% of3 of lung and the extent of emphysema [+ (T helper) and CD8+ (suppressor/cytotoxic) T cells were increased in the airways and lung parenchyma of patients with COPD, with a predominance of CD8+ cells [+ cells, which are predominantly of the T helper 2 (Th2) pattern, with increased expression of interleukin (IL)-4, IL-5, and IL-13 (see Glossary), and which are associated with an increased number of eosinophils. In smokers who develop COPD there appears to be activation of adaptive immunity, with the infiltration of CD8+ and CD4+ cells in the alveolar walls and small airways and—in patients with the most severe disease—the presence of lymphoid follicles that contain a core of B lymphocytes surrounded by T cells [+ and, to an even greater extent, CD8+ cells.T lymphocytes were first reported to be increased in patients with COPD by Finkelstein and colleagues, who showed a correlation between the number of T lymphocytes/mm8+ cells ,5. This PLoS Medicine takes the story forward [+ and CD8+ cells appear to be fully activated, as they would be after being presented with antigens, and they show predominantly a T helper 1 (Th1)/cytotoxic T 1 (Tc1) pattern, with increased expression of interferon-γ (IFN-γ) and Th1 chemokines. This is consistent with the recent demonstration of increased expression of IL-12 in bronchial biopsies of patients with COPD and activation of the transcription factor STAT-4 in T cells, subsequent STAT-4 nuclear translocation, and IFN-γ gene induction, and thus a Th1 commitment in the T cells [The study by Grumelli et al. (2004) published in this issue of  forward . The CD4 T cells .+ cells [As well as producing the cytokines IL-2 and IFN-γ, Th1 and Tc1 cells also express the chemokine receptor CXCR3 and the ligands that activate this receptor, IFN-γ inducible protein 10 , monokine induced by IFN-γ (CXCL9), and IFN-inducible T cell α chemoattractant (CXCL11). There is an increase in the expression of IP-10 in the airways of patients with COPD and an increase in the number of CXCR3+ cells . CXCR3 i+ cells and the severity of emphysema [It is likely that Th1 cells are the major source of IFN-γ in the lungs of patients with COPD and therefore drive and maintain the T cell response and promote an “immune inflammation” with neutrophils and macrophages. However, it is the role of Tc1 cells that is of particular interest, as these cells are cytotoxic to epithelial cells through the release of granzymes and perforins, which induce apoptosis. Increased concentrations of perforins have recently been reported in the sputum of patients with COPD . In suppmphysema .The T cell inflammatory response appears in mild COPD but increases markedly with disease severity. It is possible that the initial immune response becomes self-perpetuating because of endogenous autoantigens resulting from inflammatory and oxidative lung injury. There are also antigens in tobacco, but the inflammatory response appears to become independent of smoking status, and there is intense inflammation even in patients who stopped smoking many years previously , as seenCOPD is characterized by destruction of the lung parenchyma and loss of elastin due to elastolytic enzymes, such as neutrophil elastase and certain matrix metalloproteinases (MMPs). The predominant MMP in COPD appears to be MMP9, which is released in much larger amounts from alveolar macrophages of patients with COPD than from those of smokers without the disease . The stuThere are currently no treatments that reduce the relentless progression of COPD, and none that have significant anti-inflammatory effects. However the recognition that an adaptive immune T cell response, most likely driven by antigens, may play an important pathophysiological role in the pathogenesis of COPD has important therapeutic implications. It is possible that T cell inhibitory strategies, such as the use of immunosuppressants, might be effective, although side effects may be a problem, and there is particular concern about increasing the risk of bacterial infection. Another approach might be to block the trafficking of Th1 and Tc1 cells to the lungs by blocking CXCR3, and there is now a search for small-molecule inhibitors of these receptors. Inhibition of IFN-γ signaling might be another approach.The mounting evidence implicating T cells, and thus an adaptive immune response, as an important component of the inflammation in COPD is overwhelming. A better understanding of the immune mechanisms involved in COPD is important, since it might lead us to new and more effective therapeutic approaches to this important disease.+ (helper) T cell:CD4 T lymphocyte that enhances the inflammatory response+ (cytotoxic/suppressor) T cell:CD8 T lymphocyte that suppresses the inflammatory responseCXCR3: Chemokine receptor that is selectively activated by IP-10, monokine induced by IFN-γ, and IFN-inducible T cell chemoattractantCytotoxic (Tc1) cell: T cell that is characterized by secretion of INF-γGranzyme: Enzyme released by cytotoxic T cellsInterferon-γ inducible protein 10 : Chemokine of 10 kDa that selectively activates CXCR3Interferon-inducible T cell γ chemoattractant : Chemokine that selectively activates CXCR3Interferon-γ (IFN-γ): Protein secreted by Th1 and Tc1 cellsInterleukin-4 (IL-4): Protein secreted by Th2 cells that is important in increasing IgE secretionInterleukin-5 (IL-5): Protein secreted by Th2 cells that is important for eosinophiliaInterleukin-12 (IL-12): Protein secreted by antigen-presenting cells that promotes differentiation of Th1 cellsInterleukin-13 (IL-13): Protein secreted by Th2 cells that is important for IgE secretionMatrix metalloproteinase (MMP): Proteolytic enzyme that degrades connective tissueMMP9, MMP12: MMPs that destroy elastin fibersMonokine induced by interferon-γ : Chemokine that selectively activates CXCR3Neutrophil elastase: Enzyme released from neutrophils that destroys elastin fibersPerforin: Protein released by cytotoxic T cells that induces apoptosisSTAT-4: Transcription factor specifically activated by IL-1T helper 1 (Th1) cell: T lymphocyte that is characterized by secretion of INF-γT helper (Th2) cell: T lymphocyte that is characterized by increased secretion of the cytokines IL-4, IL-5, and IL-13; characteristically increased in allergic inflammation


Chronic food restriction augments the rewarding effect of centrally administered psychostimulant drugs and this effect may involve a previously documented upregulation of D-1 dopamine receptor-mediated MAP kinase signaling in nucleus accumbens (NAc) and caudate-putamen (CPu). Psychostimulants are known to induce striatal glutamate release, and group I metabotropic glutamate receptors (mGluR) have been implicated in the cellular and behavioral responses to amphetamine. The purpose of the present study was to evaluate whether chronic food restriction increases striatal MAP kinase signaling in response to the group I mGluR agonist, DHPG.Western immunoblotting was used to demonstrate that intracerebroventricular (i.c.v.) injection of DHPG (500 nmol) produces greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted as compared to ad libitum fed rats. Fos-immunostaining induced by DHPG was also stronger in CPu and NAc core of food-restricted relative to ad libitum fed rats. However, i.c.v. injection of saline-vehicle produced greater activation of ERK1/2 and CREB in CPu and NAc of food-restricted relative to ad libitum fed rats, and this difference was not seen when subjects received no i.c.v. injection prior to sacrifice. In addition, although DHPG activated Akt, there was no difference in Akt activation between feeding groups. To probe whether the augmented ERK1/2 and CREB activation in vehicle-injected food-restricted rats are mediated by one or more GluR types, effects of an NMDA antagonist , AMPA antagonist , and group I mGluR antagonist  were compared to saline-vehicle. Antagonist injections did not diminish activation of ERK1/2 or CREB.These results indicate that a group I mGluR agonist induces phosphorylation of Akt, ERK1/2 and CREB in both CPu and NAc. However, group I mGluR-mediated signaling may not be upregulated in food-restricted rats. Rather, a physiological response to "i.c.v. injection stress" is augmented by food restriction and appears to summate with effects of the group I mGluR agonist in activating ERK1/2 and CREB. While the augmented cellular response of food-restricted rats to i.c.v. injection treatment represents additional evidence of enhanced CNS responsiveness in these subjects, the functional significance and underlying mechanism(s) of this effect remain to be elucidated. The twelve unoperated/uninjected rats were simply removed from home cages and exposed to CO2 followed by decapitation. Brains were rapidly removed and immediately frozen in powdered dry ice. Five hundred-micrometer sections were cut using an IEC Minotome cryostat, and CPu and NAc were micropunched, under an Olympus dissecting microscope, from a series of 8 consecutive frozen sections. The tissue was then homogenized in 10 volumes of 50 mM Tris-HCl, pH 7.5 containing 50 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 40 mM β-glycerophosphate, 50 mM NaF and 5 mM Na4P2O7, 1% Tx-100, 0.5 μM okadaic acid, 0.5% sodium deoxycholate and 0.1% SDS, followed by centrifugation and protein determination using BCA reagent kit as described by the manufacturer (Pierce) Supernatants were mixed with 5 × SDS-PAGE sample buffer, boiled for 5 min, cooled on ice and kept at -80°C until future use.Prior studies have indicated that MAP kinase activation is transient and the optimal time-point to study phosphorylation of ERK 1/2 after physiological or pharmacological treatment is 15–20 min . TherefoProtein (10–30 μg per lane) was separated by electrophoresis on precast 10% polyacrylamide gels . Precision Plus protein standard molecular weight markers  were also loaded to assure complete electrophoretic transfer and to estimate the size of bands of interest. The gels were transferred to nitrocellulose membrane (Osmonics) for 2 h, with a constant voltage of 100 volts. Membranes were blocked for 1 hr at room temperature with blocking buffer, 5% non fat dry milk in 50 mM Tris-HCl, pH 7.5 containing 150 mM NaCl and 0.1% Tween 20 (TBS-T), then probed overnight at 4°C using primary monoclonal antibodies for phospho-(Thr202/Tyr204)-p44/42 ERK1/2 , or polyclonal antibodies for phospho-Akt (Ser 473) , and phospho (Ser 133) CREB . Total levels of ERK1/2, Akt and CREB were detected on the same blots using anti-rabbit p42/44 ERK1/2 antibody 1:2000, , anti-rabbit total Akt , or anti-rabbit CREB antibody . After detection of phosphorylated ERK1/2, phospho-Akt and CREB blots were stripped with 25 mM Glycine, pH 2.0 containing 1% SDS for 30 min at room temperature, washed six times in TBS-T buffer, blocked in blocking buffer for 1 h and then incubated overnight at 4°C in total ERK1/2, total Akt or CREB antibody. After probing with primary antibodies and washing with TBS-T buffer (3 × 5 min), membranes were incubated with 1:2000 dilution horseradish peroxidase conjugated anti-mouse or 1: 2000 dilution anti-rabbit IgG . Proteins were visualized using a chemiluminescense ECL kit (Pierce). Densitometric analysis of the bands was performed using NIH image software. Phospho-p42/44 MAPK, phospho Akt and phospho CREB values were normalized to total p42/44 MAPK, Akt and CREB values respectively.Ninety min after DHPG injection rats were anesthetized with sodium pentobarbital  and transcardially perfused with isotonic phosphate buffered saline (PBS) followed by 4% paraformaldehyde in PBS. Brains were then removed and maintained in 20% sucrose at 4°C for 48 h. Forty μm sections were cut on an IEC Minotome cryostat and collected in a cryoprotective solution. Fos immunostaining was carried out using a rabbit polyclonal c-Fos antiserum  and the avidin-biotin peroxidase complex .Sections were washed in 1% sodium borohydride followed by PBS and incubated for 2 hrs in 4% normal goat serum plus 1% BSA in PBS containing 0.2% Triton X-100 (Sigma-Aldrich) to block nonspecific binding. This was followed by incubation, overnight, with rabbit polyclonal c-fos antiserum (1:5000 dilution). Following several PBS washes, sections were incubated with a secondary antiserum  for 60 min and subsequently reacted with avidin-biotin complex (ABC) (Vector). The peroxidase reaction was visualized with a chromogen solution containing 100 mM nickel sulfate, 125 mM sodium acetate, 10 mM imidazole, 0.03% diaminobenzidine (DAB), and 0.01% hydrogen peroxide at pH 6.5. Sections were then mounted on chrome-alum coated slides, dehydrated, and coverslipped.Objective counting of c-Fos positive cells in CPu at coronal levels +1.7 and -0.3 mm, and NAc core and shell at coronal level +1.5 mm in relation to bregma  was accoFor each Western blot, film exposure time was set as needed to visualize distinct bands in the control samples of each experiment. Immunoblots were analyzed using NIH imaging software. For each blot, relative phospho-protein levels were calculated from the ratio of optical density of the phosphorylated protein/total protein to correct for small differences in protein loading. In addition, tubulin levels were analyzed in several representative gels and no differences were observed between treatment groups. Results were expressed by comparison to the normalized control, which in Experiment 1 was defined as the ad libitum fed group injected with vehicle. In the first part of Experiment 2, unoperated/uninjected ad libitum fed rats served as control. In the second part of Experiment 2, food-restricted rats injected with i.c.v. saline vehicle served as control. Differences between treatment conditions in Experiment 1 were analyzed by two-way analysis of variance . Differences between treatment conditions in the first and second parts of Experiment 2 were analyzed by student's t-test and one-way ANOVA, respectively.YP conducted the majority of immunoblotting experiments plus the immunohistochemistry experiment, contributed to experimental design and assisted in manuscript preparation. YB provided technical supervision of immunoblotting experiments and assisted in manuscript preparation. KC contributed to design of the study, assisted in all experiments, and wrote the final draft of the manuscript.