Substitution of histidine 30 by asparagine in manganese superoxide dismutase alters biophysical properties and supports proliferation in a K562 leukemia cell line.

We have generated a mutant of C. elegans manganese superoxide dismutase at histidine 30 by site-directed mutagenesis. The structure was solved at a resolution of 1.52 Å by X-ray crystallography (pdb: 6S0D). His30 was targeted, as it forms as a gateway residue at the top of the solvent access funnel to the active site, together with Tyr34. In the wild-type protein, these gateway residues are involved in the hydrogen-bonding network providing the protons necessary for the catalytic reaction at the metal center. However, biophysical characterization and cell viability experiments reveal that a mutation from histidine to asparagine in the H30N mutant modifies metal selectivity in the protein, favoring the uptake of iron over manganese in minimal media conditions, alters active-site coordination from the characteristic trigonal bipyramidal to octahedral geometry, and encourages cellular proliferation in K562 cells, when added exogenously to the cells.

Ethical aspects pertaining to the use of pharmacogenetic tests.

Administering medication safely and with confidence is important for both the patient and the prescriber. The individualised adjustment of a medicine dose, based solely on clinical outcomes or the change of a prescribed drug, possibly delays positive patient outcomes. This could lead to suboptimal patient management. Additionally, it could also have a negative pharmacoeconomic impact. The application of pharmacogenetics addresses this matter by refining and improving the safety and efficacy of medicines through a genotype-based prediction of responses. It also stratifies clinical trial populations in drug development in order to identify which patient genotypes benefit most from the drug under study. Although this emerging science presents a lot of prospects, it also raises a significant number of ethical questions. The problem with stratifying patient populations is addressed by promoting responsible and accountable scientific and intellectual liberty. This will avoid discrimination towards vulnerable populations. Therefore, there is a need to encourage informed consent and confidentiality, as well as to promote autonomy, justice, and equity by developing worldwide equivalent ethical, legal, and regulatory frameworks.

Correction: A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

[This corrects the article DOI: 10.1371/journal.pone.0162866.].

Genome-wide association analyses for lung function and chronic obstructive pulmonary disease identify new loci and potential druggable targets.

Chronic obstructive pulmonary disease (COPD) is characterized by reduced lung function and is the third leading cause of death globally. Through genome-wide association discovery in 48,943 individuals, selected from extremes of the lung function distribution in UK Biobank, and follow-up in 95,375 individuals, we increased the yield of independent signals for lung function from 54 to 97. A genetic risk score was associated with COPD susceptibility (odds ratio per 1 s.d. of the risk score (∼6 alleles) (95% confidence interval) = 1.24 (1.20-1.27), P = 5.05 × 10-49), and we observed a 3.7-fold difference in COPD risk between individuals in the highest and lowest genetic risk score deciles in UK Biobank. The 97 signals show enrichment in genes for development, elastic fibers and epigenetic regulation pathways. We highlight targets for drugs and compounds in development for COPD and asthma (genes in the inositol phosphate metabolism pathway and CHRM3) and describe targets for potential drug repositioning from other clinical indications.

A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant inter-population pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective.

Deregulation of the protein phosphatase 2A, PP2A in cancer: complexity and therapeutic options.

The complexity of the phosphatase, PP2A, is being unravelled and current research is increasingly providing information on the association of deregulated PP2A function with cancer initiation and progression. It has been reported that decreased activity of PP2A is a recurrent observation in many types of cancer, including colorectal and breast cancer (Baldacchino et al. EPMA J. 5:3, 2014; Cristobal et al. Mol Cancer Ther. 13:938-947, 2014). Since deregulation of PP2A and its regulatory subunits is a common event in cancer, PP2A is a potential target for therapy (Baldacchino et al. EPMA J. 5:3, 2014). In this review, the structural components of the PP2A complex are described, giving an in depth overview of the diversity of regulatory subunits. Regulation of the active PP2A trimeric complex, through phosphorylation and methylation, can be targeted using known compounds, to reactivate the complex. The endogenous inhibitors of the PP2A complex are highly deregulated in cancer, representing cases that are eligible to PP2A-activating drugs. Pharmacological opportunities to target low PP2A activity are available and preclinical data support the efficacy of these drugs, but clinical trials are lacking. We highlight the importance of PP2A deregulation in cancer and the current trends in targeting the phosphatase.

The effect of turmeric (Curcumin) supplementation on cytokine and inflammatory marker responses following 2 hours of endurance cycling.

BACKGROUND: Endurance exercise induces IL-6 production from myocytes that is thought to impair intracellular defence mechanisms. Curcumin inhibits NF-κB and activator protein 1, responsible for cytokine transcription, in cell lines. The aim of this study was to investigate the effect of curcumin supplementation on the cytokine and stress responses following 2 h of cycling. METHODS: Eleven male recreational athletes (35.5 ± 5.7 years; Wmax 275 ± 6 W; 87.2 ± 10.3 kg) consuming a low carbohydrate diet of 2.3 ± 0.2 g/kg/day underwent three double blind trials with curcumin supplementation, placebo supplementation, and no supplementation (control) to observe the response of serum interleukins (IL-6, IL1-RA, IL-10), cortisol, c-reactive protein (CRP), and subjective assessment of training stress. Exercise was set at 95% lactate threshold (54 ± 7% Wmax) to ensure that all athletes completed the trial protocol. RESULTS: The trial protocol elicted a rise in IL-6 and IL1-RA, but not IL-10. The supplementation regimen failed to produce statistically significant results when compared to placebo and control. IL-6 serum concentrations one hour following exercise were (Median (IQR): 2.0 (1.8-3.6) Curcumin; 4.8 (2.1-7.3) Placebo; 3.5 (1.9-7.7) Control). Differences between supplementation and placebo failed to reach statistical significance (p = 0.18) with the median test. Repeated measures ANOVA time-trial interaction was at p = 0.06 between curcumin supplementation and placebo. A positive correlation (p = 0.02) between absolute exercise intensity and 1 h post-exercise for IL-6 concentration was observed. Participants reported "better than usual" scores in the subjective assessment of psychological stress when supplementing with curcumin, indicating that they felt less stressed during training days (p = 0.04) compared to placebo even though there was no difference in RPE during any of the training days or trials. CONCLUSION: The limitations of the current regimen and trial involved a number of factors including sample size, mode of exercise, intensity of exercise, and dose of curcumin. Nevertheless these results provide insight for future studies with larger samples, and multiple curcumin dosages to investigate if different curcumin regimens can lead to statistically different interleukin levels when compared to a control and placebo.

Deregulation of the phosphatase, PP2A is a common event in breast cancer, predicting sensitivity to FTY720.

BACKGROUND: The most commonly used biomarkers to predict the response of breast cancer patients to therapy are the oestrogen receptor (ER), progesterone receptor (PgR), and human epidermal growth factor receptor 2 (HER2). Patients positive for these biomarkers are eligible for specific therapies such as endocrine treatment in the event of ER and PgR positivity, and the monoclonal antibody, trastuzumab, in the case of HER2-positive patients. Patients who are negative for these three biomarkers, the so-called triple negatives, however, derive little benefit from such therapies and are associated with a worse prognosis. Deregulation of the protein serine/threonine phosphatase type 2A (PP2A) and its regulatory subunits is a common event in breast cancer, providing a possible target for therapy. METHODS: The data portal, cBioPortal for Cancer Genomics was used to investigate the incidence of conditions that are associated with low phosphatase activity. Four (4) adherent human breast cancer cell lines, MDA-MB-468, MDA-MB-436, Hs578T and BT-20 were cultured to assess their viability when exposed to various dosages of rapamycin or FTY720. In addition, RNA was extracted and cDNA was synthesised to amplify the coding sequence of PPP2CA. Amplification was followed by high-resolution melting to identify variations. RESULTS AND CONCLUSION: The sequence of PPP2CA was found to be conserved across a diverse panel of solid tumour and haematological cell lines, suggesting that low expression of PPP2CA and differential binding of inhibitory PPP2CA regulators are the main mechanisms of PP2A deregulation. Interestingly, the cBioPortal for Cancer Genomics shows that PP2A is deregulated in 59.6% of basal breast tumours. Viability assays performed to determine the sensitivity of a panel of breast cancer cell lines to FTY720, a PP2A activator, indicated that cell lines associated with ER loss are sensitive to lower doses of FTY720. The subset of patients with suppressed PP2A activity is potentially eligible for treatment using therapies which target the PI3K/AKT/mTOR pathway, such as phosphatase activators.

Golden Helix Pharmacogenomics Days: educational activities on pharmacogenomics and personalized medicine.

The Golden Helix Pharmacogenomics Days are high-profile international educational scientific meetings discussing pharmacogenomics and personalized medicine. Here, we provide an overview of the scientific lectures and the topics discussed during the 4th Golden Helix Pharmacogenomics Day, held in Cagliari, Italy, on 7 October 2011, and the 5th Golden Helix Pharmacogenomics Day, that was held in Msida, Malta, on 3 December 2011. The scientific programs of both events included scientific and company lectures on pharmacogenomics, bioinformatics and personalized medicine by local and international speakers from Europe and the USA.

Anxiety and the management of asthma in an adult outpatient population.

BACKGROUND: Review of the literature suggests that anxiety is more common among patients with asthma than among the general population, yet it does not appear to be given the attention it deserves as part of the overall management of asthma. The aim of this study was to investigate the relationship between anxiety and asthma management, in terms of Global Initiative for Asthma steps, lung function and medication. METHODS: A total of 201 consecutive patients with respiratory physician-diagnosed asthma were recruited from an adult outpatient asthma clinic. Participants underwent a sociodemographic review, and a medical interview which included a detailed drug history. Forced expiratory volume in 1 second (FEV(1)) and peak expiratory flow (PEF) values were recorded using a Micro Medical((R)) portable spirometer. The level of anxiety was assessed using the Beck Anxiety Inventory (BAI). RESULTS: A total of 51.5% of participants registered clinically significant levels of anxiety. Of these only 21% had already been diagnosed and were receiving treatment. Females reported significantly higher BAI scores than males (p < 0.01). More females (66.3%) registered clinically significant levels of anxiety as compared with males (33.7%) (p < 0.05). There was a positive correlation between the BAI score and the prescribed dose of inhaled glucocorticoids (r(s) = 0.150, p < 0.05) and between anxiety and GINA treatment step (r(s) = 0.139, p < 0.05). There was also a positive correlation between anxiety and the number of medicines taken by patients (r(s) = 0.259, p < 0.001). CONCLUSIONS: Physicians treating patients with asthma should be sensitised to the association between asthma and anxiety, and should also consider assessing patients for the possibility of anxiety disorders as part of asthma management plans.

An analysis of gender differences in self-reported health, use of medicines and access to information sources about medicines among adolescents.

Adolescence is a key period in the emergence of gender differences in health that persist into adulthood. Knowledge about health-related gender differences among adolescents could be instrumental in informing policy and practice to effect health improvement among this age group. The main objectives of this study were therefore to investigate gender differences in self-reported health complaints and use of medicines during the preceding 3 months and access to information sources about medicines among adolescents in Malta. A self-administered questionnaire was distributed among adolescents attending secondary schools in Malta. A stratified random sample design generated a sample size of 514 students. Common health complaints, use of medicines, and sources of information about medicines were investigated by means of author-designed checklists. The analytical sample was formed from 474 usable questionnaires collected from students aged 14-16 years, of which 53.8% were females. In general, a female excess emerged for health complaints and use of medicines except for sport injuries and use of vitamins. Gender differences were statistically significant for a female excess in the retrieval of information from the package insert (p < .001), parents or adult relatives (p < .01) and friends or schoolmates (p < .05). The findings of this study underscore the need for awareness of gender differences in the health of adolescents among health care providers, policy makers, and educators. An important implication of the findings is that the health needs of adolescents could be more equitably addressed by adopting a holistic patient-oriented approach particular to the health needs of each individual.

Knowledge and sources of information about medicines among adolescents in Malta

Objective: To investigate knowledge of medicines and sources of information about medicines among adolescents in Malta. Methods: A self-administered questionnaire was used to survey knowledge of medicines and information sources among adolescents attending secondary schools in Malta. A random stratified sample design generated a sample size of 514 students. Knowledge of medicines was investigated by questions that included topics about the efficacy of medicines, proper communication during a medical consultation, the safety of medicines, antibiotic use and pictograms. Results: The analytical sample, of which 53.8% were girls, was made up of 474 students, aged 14-16 years. The students obtained a mean score of 22.92 points (SD = 4.31) out of a maximum of 32 points for medicine knowledge. More than 30.0% of the respondents did not mark the correct answer for 40.6% of the questions that investigated knowledge of medicines. The family physician, community pharmacist and parents were the sources of information that were mostly cited. A proportion of 4.4% had obtained information from the teacher. A quarter of the respondents (24.7%) had obtained information from friends/schoolmates, young relatives or the media, generally television (17.3%). Conclusion: Although the level of knowledge about medicines among this study sample of Maltese students was good, there appeared to be particular misconceptions with regard to important aspects associated with the proper use of medicines. These findings highlight the need to educate adolescents about the proper use of medicines. The most important information providers about medicines appeared to be the physician, pharmacist and parents. It is being suggested that education campaigns should be organized in order to help parents guide adolescents on how to use medicines appropriately. In addition, adolescents would benefit if more information about the proper use of medicines would be disseminated by means of television programs and school-based health education curricula (AU)

Objetivo: Investigar el conocimiento y las fuentes de información sobre medicamentos entre adolescentes en Malta. Métodos: Se usó un cuestionario auto-administrado para investigar el conocimiento sobre medicamentos y las fuentes de información entre adolescentes de escuelas secundarias en Malta. Un diseño de muestra aleatorio estratificado generó una muestra de 514 estudiantes. El conocimiento sobre medicamentos se investigó con preguntas que incluían temas sobre la eficacia de los medicamentos, la correcta comunicación en la consulta médica, la seguridad de los medicamentos, el uso de antibióticos y los pictogramas. Resultados: La muestra analítica, de las cuales 53,8% eran mujeres, se construyó con 474 estudiantes de 14 a 16 años. Los estudiantes obtuvieron una puntuación media de 22,92 puntos (DE=4,31) de un máximo de 32 puntos para el conocimiento de medicamentos. Más del 30,0% de los respondentes no marcó la respuesta correcta para el 40,6% de las preguntas del cuestionario. El médico de familia, el farmacéutico comunitario y los padres fueron las fuentes de información más citadas. Un 4,4% había obtenido la información del profesor. Un cuarto de los respondentes (24,7%) había obtenido la información de amigos/compañeros, parientes jóvenes o los medios de comunicación, generalmente la televisión (17,3%). Concusión: Aunque el nivel de conocimientos en esta muestra de estudiantes malteses fue bueno, pareció que había errores de concepto específicos en torno a importantes aspectos relacionados con el uso correcto de medicamentos. Estos hallazgos ensalzan la necesidad de educar a los adolescentes en al uso correcto de medicamentos Los proveedores de información sobre medicamentos más importantes parecieron ser el médico, el farmacéutico y los padres. Se sugiere que deberían organizarse campañas de información para ayudar a que los padres guíen a los adolescentes en cómo utilizar los medicamentos apropiadamente. Además, los adolescentes se beneficiarían si se diseminase más información sobre el uso correcto de medicamentos por televisión y en el currículo de la educación escolar (AU)

Health complaints and use of medicines among adolescents in Malta

Objective: To investigate self-reported health complaints and the use of medicines among adolescents in Malta. Methods: A self-administered questionnaire was used to survey self-reported health complaints, the use and the sources of medicines that had been accessed, during the preceding 3 months among adolescents attending secondary schools in Malta. A stratified random sample design generated a sample size of 514 students. The health complaints and use of medicines that were investigated included ear problems/hay fever/cold/cough, headache, skin problems, sport injuries, indigestion/diarrhoea/constipation, eye problems and menstrual pain (for girls). The use of vitamins and antibiotics was also investigated. Results: A total of 477 students participated in the final data collection. Correct information was submitted by 474 students, (aged 14-16 years), who formed the analytical sample, of which 53.8% were girls. The students reported a mean number of 2.70 (SD = 1.39) out of a total of 7 health complaints and 90.3% reported using at least 1 medicine during the preceding 3 months. The community pharmacy was cited as the most commonly accessed source for most of the medicines that were investigated. A proportion of 24.3% of the students had taken at least 1 medicine without adult guidance during the preceding 3 months. Almost 10% of those who had taken antibiotics, had accessed them from the home medicine cabinet. Conclusion: A high proportion of adolescents in Malta reported the use of medicines to alleviate the symptoms of common health complaints. This result is concordant with previous research carried out in the United Kingdom, Germany, Slovakia and Kuwait. A considerable proportion of students in this study had obtained medicines without adult guidance and accessed antibiotics from the home medicine cabinet. This highlights the importance of carefully designed education programs for adolescents that will integrate information about the proper use of medicines (AU)

Objetivo: Investigar los problemas de salud y el uso de medicamentos auto-comunicado entre adolescentes en Malta. Métodos: Se usó un cuestionario auto-administrado para indagar los problemas de salud auto-comunicados, el uso y las fuentes de medicamentos a los que habían accedido durante los 3 meses precedentes al estudio, adolescentes que asisten a escuelas secundarias en Malta. Un muestreo estratificado aleatorio generó un tamaño de muestra de 514 estudiantes. Los problemas de salud y el uso de medicamentos que se investigaron incluían problemas de oído/fiebre del heno/resfriado/tos, dolor de cabeza, problema sde piel, heridas de deportes, indigestión/diarrea/estreñimiento, problemas de ojos, y dolor menstrual (en niñas). También se investigo el uso de vitaminas y antibióticos. Resultados: Un total de 477 estudiantes participaron en el final de la recogida de datos. Se envió información correcta en 474 estudiantes (edad 14-16 años) que formaron la muestra estudiada, de los que el 53,8% eran niñas. Los estudiantes comunicaron una media de 2,70 (DE=1,39) de los 7 problemas de salud y el 90,3% comunicó que usaba al menos un medicamento en los últimos 3 meses. La farmacia comunitaria fue la fuente más comúnmente usada para la mayoría de los medicamentos investigados. Un 24,3% de los estudiantes había tomando al menos 1 medicamento sin control de un adulto en los últimos 3 meses. Casi el 10% de los que habían tomado antibióticos, los había conseguido en el botiquín casero. Conclusión: Una alta proporción de adolescentes en Malta comunicó el uso de medicamentos para aliviar síntomas de problemas de salud comunes. Este estudio es concordante con investigaciones anteriores realizadas en Reino Unido, Alemania, Eslovaquia y Kuwait. Una considerable proporción de estudiantes en este estudio había obtenido medicamentos sin control de adultos y accedió a los antibióticos en el botiquín casero. Esto remarca la importancia de el diseño cuidadoso de programas d educación para los adolescentes que integren información sobre el uso correcto de medicamentos (AU)

Knowledge and sources of information about medicines among adolescents in Malta.

OBJECTIVE: To investigate knowledge of medicines and sources of information about medicines among adolescents in Malta. METHODS: A self-administered questionnaire was used to survey knowledge of medicines and information sources among adolescents attending secondary schools in Malta. A random stratified sample design generated a sample size of 514 students. Knowledge of medicines was investigated by questions that included topics about the efficacy of medicines, proper communication during a medical consultation, the safety of medicines, antibiotic use and pictograms. RESULTS: The analytical sample, of which 53.8% were girls, was made up of 474 students, aged 1416 years. The students obtained a mean score of 22.92 points (SD = 4.31) out of a maximum of 32 points for medicine knowledge. More than 30.0% of the respondents did not mark the correct answer for 40.6% of the questions that investigated knowledge of medicines. The family physician, community pharmacist and parents were the sources of information that were mostly cited. A proportion of 4.4% had obtained information from the teacher. A quarter of the respondents (24.7%) had obtained information from friends/schoolmates, young relatives or the media, generally television (17.3%). CONCLUSION: Although the level of knowledge about medicines among this study sample of Maltese students was good, there appeared to be particular misconceptions with regard to important aspects associated with the proper use of medicines. These findings highlight the need to educate adolescents about the proper use of medicines. The most important information providers about medicines appeared to be the physician, pharmacist and parents. It is being suggested that education campaigns should be organized in order to help parents guide adolescents on how to use medicines appropriately. In addition, adolescents would benefit if more information about the proper use of medicines would be disseminated by means of television programs and school-based health education curricula.

Health complaints and use of medicines among adolescents in Malta.

OBJECTIVE: To investigate self-reported health complaints and the use of medicines among adolescents in Malta. METHODS: A self-administered questionnaire was used to survey self-reported health complaints, the use and the sources of medicines that had been accessed, during the preceding 3 months among adolescents attending secondary schools in Malta. A stratified random sample design generated a sample size of 514 students. The health complaints and use of medicines that were investigated included ear problems/hay fever/cold/cough, headache, skin problems, sport injuries, indigestion/diarrhoea/constipation, eye problems and menstrual pain (for girls). The use of vitamins and antibiotics was also investigated. RESULTS: A total of 477 students participated in the final data collection. Correct information was submitted by 474 students, (aged 14-16 years), who formed the analytical sample, of which 53.8% were girls. The students reported a mean number of 2.70 (SD = 1.39) out of a total of 7 health complaints and 90.3% reported using at least 1 medicine during the preceding 3 months. The community pharmacy was cited as the most commonly accessed source for most of the medicines that were investigated. A proportion of 24.3% of the students had taken at least 1 medicine without adult guidance during the preceding 3 months. Almost 10% of those who had taken antibiotics, had accessed them from the home medicine cabinet. CONCLUSION: A high proportion of adolescents in Malta reported the use of medicines to alleviate the symptoms of common health complaints. This result is concordant with previous research carried out in the United Kingdom, Germany, Slovakia and Kuwait. A considerable proportion of students in this study had obtained medicines without adult guidance and accessed antibiotics from the home medicine cabinet. This highlights the importance of carefully designed education programs for adolescents that will integrate information about the proper use of medicines.

Functional polymorphism and differential regulation of CYSLTR1 transcription in human airway smooth muscle and monocytes.

Cysteinyl leukotrienes play an important role in the pathophysiology of many inflammatory disorders, including asthma. The aim of this study was to characterize the mechanisms underlying transcriptional regulation of the human cysteinyl leukotriene receptor 1 (hCYSLTR1) gene. 5'RACE was performed on human airway smooth muscle (HASM) and peripheral blood mononuclear cells. A1128-bp region of the hCYSLTR1 main putative promoter was screened for polymorphisms by sequencing of 48 individuals. Luciferase reporter gene assays were performed using fragments of the core promoter (232 bp to 1128 bp) in HASM and THP1 cells. Three hCYSLTR1 transcripts were found, one representing 90% of all messenger RNA identified. The genomic location of the transcription start sites suggested there are two putative hCYSLTR1 promoters. The majority of the transcriptional activity of the main putative promoter was detected between -232 and -679 bp. Four singlenucleotide polymorphisms in strong linkage disequilibrium were found in the region studied: -561 (rs7066737), -642 (rs2806489), -781 (rs2637204), and -940 (rs321029), with three haplotypes observed. In THP1 cells, the G allele (-642) caused a twofold decrease in luciferase expression compared to the Aallele. These data suggest that the majority of hCYSLTR1 transcripts in HASM and monocytes arise from a single promoter located immediately upstream of the 5\' untranslated region, although rarer transcripts can also occur. This study also raises the possibility that cell-type-dependent differences in transcriptional activity caused by the presence of specific haplotypes within the main CYSLTR1 promoter may be a predictor of disease risk or treatment response.

Eosinophil-mediated cholinergic nerve remodeling.

Eosinophils are observed to localize to cholinergic nerves in a variety of inflammatory conditions such as asthma, rhinitis, eosinophilic gastroenteritis, and inflammatory bowel disease, where they are also responsible for the induction of cell signaling. We hypothesized that a consequence of eosinophil localization to cholinergic nerves would involve a neural remodeling process. Eosinophil co-culture with cholinergic IMR32 cells led to increased expression of the M2 muscarinic receptor, with this induction being mediated via an adhesion-dependent release of eosinophil proteins, including major basic protein and nerve growth factor. Studies on the promoter sequence of the M2 receptor indicated that this induction was initiated at a transcription start site 145 kb upstream of the gene-coding region. This promoter site contains binding sites for a variety of transcription factors including SP1, AP1, and AP2. Eosinophils also induced the expression of several cholinergic genes involved in the synthesis, storage, and metabolism of acetylcholine, including the enzymes choline acetyltransferase, vesicular acetylcholine transferase, and acetylcholinesterase. The observed eosinophil-induced changes in enzyme content were associated with a reduction in intracellular neural acetylcholine but an increase in choline content, suggesting increased acetylcholine turnover and a reduction in acetylcholinesterase activity, in turn suggesting reduced catabolism of acetylcholine. Together these data suggest that eosinophil localization to cholinergic nerves induces neural remodeling, promoting a cholinergic phenotype.

Novel polymorphisms influencing transcription of the human CHRM2 gene in airway smooth muscle.

Muscarinic receptors are a functionally important family of G-protein-coupled receptors. Using a combination of rapid amplification of 5' cDNA ends and reporter gene assays, we characterized the 5' untranslated region of the CHRM2 gene as expressed in human airway smooth muscle (HASM) cells. A splice site is present 46 bp upstream from the ATG start codon. Five exons with alternative splicing patterns are present upstream of this splice site, separated by introns ranging from 87 bp to > 145 kb. There is evidence for the gene being under the control of a TATA-less promoter with Sp1, GATA, and activator protein-2 binding sites. Multiple transcription start sites (TSSs) were identified. We identified a novel 0.5-kb hypervariable region located 648 bp upstream of the most 5' TSS, a multiallelic (CA) tandem repeat 96 bp downstream of the most 5' TSS, and a common C-->A SNP located 136 bp upstream of the most 5' TSS. Functional studies in primary HASM cells and the BEAS-2B cell line demonstrated highest promoter activity to be upstream of the most 3' TSS, with potential repressor elements operating in a cell type-dependent manner, located upstream of the most 5' TSS. We present functional data to show that the CA repeat may influence the transcription of the gene in HASM and BEAS-2B cells.