No association of alcohol use and the risk of ulcerative colitis or Crohn's disease: data from a European Prospective cohort study (EPIC).

BACKGROUND/OBJECTIVES: The role of long-term alcohol consumption for the risk of developing ulcerative colitis (UC) and Crohn's disease (CD) is unclear. For the first time, to prospectively assess the role of pre-disease alcohol consumption on the risk of developing UC or CD. SUBJECTS/METHODS: Nested within the European Prospective Investigation into Cancer and Nutrition (EPIC-IBD), incident UC and CD cases and matched controls where included. At recruitment, participants completed validated food frequency and lifestyle questionnaires. Alcohol consumption was classified as either: non-use, former, light (⩽0.5 and 1 drink per week), below the recommended limits (BRL) (⩽1 and 2 drinks per day), moderate (⩽2.5 and 5 drinks per day), or heavy use (>2.5 and >5 drinks per day) for women and men, respectively; and was expressed as consumption at enrolment and during lifetime. Conditional logistic regression was applied adjusting for smoking and education, taking light users as the reference. RESULTS: Out of 262 451 participants in six countries, 198 UC incident cases/792 controls and 84 CD cases/336 controls were included. At enrolment, 8%/27%/32%/23%/11% UC cases and 7%/29%/40%/19%/5% CD cases were: non-users, light, BRL, moderate and heavy users, respectively. The corresponding figures for lifetime non-use, former, light, BRL, moderate and heavy use were: 3%/5%/23%/44%/19%/6% and 5%/2%/25%/44%/23%/1% for UC and CD cases, respectively. There were no associations between any categories of alcohol consumption and risk of UC or CD in the unadjusted and adjusted odds ratios. CONCLUSION: There was no evidence of associations between alcohol use and the odds of developing either UC or CD.

Association between high dietary intake of the n-3 polyunsaturated fatty acid docosahexaenoic acid and reduced risk of Crohn's disease.

BACKGROUND: There are plausible mechanisms for how dietary docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, could prevent Crohn's disease (CD). AIM: To conduct a prospective study to investigate the association between increased intake of DHA and risk of CD. METHODS: Overall, 229 702 participants were recruited from nine European centres between 1991 and 1998. At recruitment, dietary intakes of DHA and fatty acids were measured using validated food frequency questionnaires. The cohort was monitored through to June 2004 to identify participants who developed incident CD. In a nested case-control analysis, each case was matched with four controls; odds ratios (ORs) were calculated for quintiles of DHA intake, adjusted for total energy intake, smoking, other dietary fatty acids, dietary vitamin D and body mass index. RESULTS: Seventy-three participants developed incident CD. All higher quintiles of DHA intake were inversely associated with development of CD; the highest quintile had the greatest effect size (OR = 0.07; 95% CI = 0.02-0.81). The OR trend across quintiles of DHA was 0.54 (95% CI = 0.30-0.99, Ptrend  = 0.04). Including BMI in the multivariate analysis, due to its correlation with dietary fat showed similar associations. There were no associations with the other dietary fatty acids studied. CONCLUSION: There were inverse associations, with a biological gradient between increasing dietary docosahexaenoic acid intakes and incident Crohn's disease. Further studies in other populations should measure docosahexaenoic acid to determine if the association is consistent and the hypothesis tested in randomised controlled trials of purely docosahexaenoic acid supplementation.

A novel microscopy-based high-throughput screening method to identify proteins that regulate global histone modification levels.

Posttranslational modifications of histones play an important role in the regulation of gene expression and chromatin structure in eukaryotes. The balance between chromatin factors depositing (writers) and removing (erasers) histone marks regulates the steady-state levels of chromatin modifications. Here we describe a novel microscopy-based screening method to identify proteins that regulate histone modification levels in a high-throughput fashion. We named our method CROSS, for Chromatin Regulation Ontology SiRNA Screening. CROSS is based on an siRNA library targeting the expression of 529 proteins involved in chromatin regulation. As a proof of principle, we used CROSS to identify chromatin factors involved in histone H3 methylation on either lysine-4 or lysine-27. Furthermore, we show that CROSS can be used to identify chromatin factors that affect growth in cancer cell lines. Taken together, CROSS is a powerful method to identify the writers and erasers of novel and known chromatin marks and facilitates the identification of drugs targeting epigenetic modifications.

The determination of budesonide and fluticasone in human sputum samples collected from COPD patients using LC-MS/MS.

A bioanalytical method for the quantitative determination of budesonide and fluticasone in human sputum was developed. Sputolysin(®) Reagent was added to the sputum samples. After incubation (37°C; 60-70 min under shaking) and automated solid phase extraction the extracts were analysed using LC-MS/MS. Budesonide and fluticasone showed good linearity (r>0.99) over the range 0.1-100 nM in the first and second validation batch, and over the range 0.25-10,000 nM in the third and fourth validation batch. The lower limit of quantification (LLOQ) achieved was 5 nM for budesonide and fluticasone in 100 µL human sputum. Intra-run and inter-run RSD for four quality control levels (5-100 nM) were within 6.9% (budesonide) and 8.0% (fluticasone). The accuracy ranged from -11.4% to -1.6% (budesonide), and from -11.8% to 0.4% (fluticasone). The validated method was applied to clinical sputum samples from COPD patients.

Mitogenic signaling of insulin-like growth factor I in MCF-7 human breast cancer cells requires phosphatidylinositol 3-kinase and is independent of mitogen-activated protein kinase.

Addition of insulin-like growth factor I (IGF-I) to quiescent breast tumor-derived MCF-7 cells causes stimulation of cyclin D1 synthesis, hyperphosphorylation of the retinoblastoma protein pRb, DNA synthesis, and cell division. All of these effects are independent of the mitogen-activated protein kinase (MAPK) pathway since none of them is blocked by PD098059, the specific inhibitor of the MAPK activating kinase MEK1. This observation is consistent with the finding that the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a strong inducer of MAPK activity in MCF-7 cells, effectively inhibits proliferation. The anti-proliferative effect of TPA in these cells may be accounted for, at least in part, by the MAPK-dependent stimulation of the synthesis of p21(WAF1/CIP1), an inhibitor of cyclin/cyclin-dependent kinase complexes. In contrast, all of the observed stimulatory effects of IGF-I on cell cycle progression, cyclin D1 synthesis, and pRb hyperphosphorylation were blocked by the specific phosphatidylinositol 3-kinase inhibitor LY294002, suggesting that phosphatidylinositol 3-kinase activity but not MAPK activity is required for transduction of the mitogenic IGF-I signal in MCF-7 cells.

beta-Carotene absorption and cleavage in rats is affected by the vitamin A concentration of the diet.

The purpose of this study was to examine whether intestinal beta-carotene cleavage activity, measured with the dioxygenase assay, is affected by vitamin A intake and whether this in vitro activity is a determinant of beta-carotene cleavage in vivo, measured in lymph-cannulated rats. Six groups of 10-20 rats were fed a diet with a low, normal or high retinyl palmitate concentration (120 RE, 1200 RE and 12,000 RE per kg, respectively) for 14 to 18 wk, either supplemented or not with 50 mg beta-carotene/kg in the last 6 wk. Intestinal dioxygenase activity was 90% higher (P < 0.05) in the animals fed the unsupplemented low vitamin A diet than in the animals fed the unsupplemented high vitamin A diet, whereas in beta-carotene-supplemented rats intestinal dioxygenase activity was significantly lower than in unsupplemented rats. The molar ratio between retinyl esters and beta-carotene in lymph collected over 8 h after a single intestinal dose of beta-carotene (250 micrograms) to beta-carotene-unsupplemented rats fed the three levels of vitamin A was correlated with intestinal dioxygenase activity (r = 0.66, P = 0.003). Dioxygenase activity in the liver was not affected by the vitamin A concentration of the diet but was 70% higher in the beta-carotene-supplemented rats. Based on the difference in liver vitamin A contents between beta-carotene-supplemented and unsupplemented rats we estimated beta-carotene conversion factors of 9:1 for the rats fed the high vitamin A diet and 4:1 for the rats fed the normal and low vitamin A diets. Intestinal beta-carotene cleavage activity is higher in vitamin A-deficient rats than in rats with a high intake of either vitamin A or beta-carotene. The intestinal dioxygenase activity as measured in vitro is an adequate indicator of in vivo beta-carotene cleavage activity.

In vitro measurement of beta-carotene cleavage activity: methodological considerations and the effect of other carotenoids on beta-carotene cleavage.

In view of controversies about assessment of the beta-carotene cleavage activity, methodological aspects and problems of the dioxygenase assay are described. Using rat and hamster intestinal preparations the method was optimized on retinal formation, the only cleavage product we could demonstrate. It appeared that the cell fraction with the highest cleavage activity was the 9,000 g supernatant (S-9). Maximal retinal formation was obtained with SDS, taurocholate and egg lecithin in the buffer and 3 micrograms beta-carotene dissolved in acetone. Ethanol, THF/DMSO (1:1) or propylene glycol as solvent for beta-carotene reduced retinal formation to 55, 24, and 19%, respectively. Retinal formation increased proportionally with the amount of protein S-9 used and was linear up to 40-60 minutes of incubation. Incubation with alpha-carotene or beta-cryptoxanthin resulted in a retinal formation of 29 and 55% of the amount formed from beta-carotene. Addition of 9 micrograms of lutein to an incubation with 3 micrograms beta-carotene reduced retinal formation, while lycopene had no effect. In conclusion, the beta-carotene cleavage assay with S-9 as enzyme source described in this report, seems a useful tool to study (dietary) determinants of beta-carotene cleavage activity, but for other purposes adaptation of the method is required.

The promoter of the salmon insulin-like growth factor I gene is activated by hepatocyte nuclear factor 1.

Hepatocyte nuclear factor 1 (HNF-1) was found to have a potent stimulatory action on the activity of the promoter of the salmon insulin-like growth factor I (IGF-I) gene in transient transfection experiments. This liver-enriched transcription factor was shown to bind to an element in the proximal region of the promoter with distinct nucleotide sequence homology to the HNF-1 consensus binding sequence. Mutating this sequence to a variant no longer capable of HNF-1 binding resulted in the loss of the stimulatory effect. Since the sequence of the HNF-1 binding site is conserved in all mammalian, avian, and amphibian species from which the IGF-I promoter sequences have been derived to date, we propose that HNF-1 may be an important regulator of IGF-I gene expression in all of these species.

Expression of the insulin-like growth factor I gene is stimulated by the liver-enriched transcription factors C/EBP alpha and LAP.

The expression of the human insulin-like growth factor I (hIGF-I) gene is regulated in a developmental stage- and tissue-specific manner. Postnatally, the liver becomes the main endocrine source of this important growth factor. The hIGF-I gene contains two alternatively used leader exons, exon 1 and exon 2. In human adult liver, exon 1 sequences are represented in about 80% of the transcripts. In this study we have investigated the role of promoter 1 (P1), located upstream of leader exon 1, in the tissue-specific expression of the IGF-I gene in human adult liver. Factors involved in this process have not been described to date. In this report we show, employing transient transfection experiments in Hep3B cells, that two liver-enriched transcription factors, CCAAT/enhancer binding protein alpha (C/EBP alpha) and liver-enriched activating protein (LAP), enhance the activity of IGF-I P1. DNase I footprinting experiments demonstrate that a C/EBP-LAP binding site is located 119 base pairs upstream of the major transcription start site in exon 1. Comparison with other C/EBP-LAP binding sites reveals that the binding site in P1 is a high affinity binding site. Mutations of the C/EBP-LAP binding site completely abolished the enhancing effect of C/EBP alpha and LAP, indicating that their activating signal is indeed conferred by this binding site. These results suggest that both C/EBP alpha and LAP play important roles in the liver-specific expression of the hIGF-I gene and provide the first clues in the elucidation of its complicated developmental stage- and tissue-specific expression pattern.

Functional analysis of the human IGF-I gene promoters.

The two putative promoter regions of the human insulin-like growth factor-I (IGF-I) gene, located upstream of the two alternatively used leader exons 1 and 2, were tested for promoter activity in transient transfection assays. Both regions were shown to possess promoter activity. The results further indicate cell-type-specific regulation of the activities of the two promoters in endogenously IGF-I producing human cell lines of different origin.

The human IGF-I gene contains two cell type-specifically regulated promoters.

The human insulin-like growth factor-I (IGF-I) gene contains two alternative leader exons: exons 1 and 2. We have identified, by transient transfection experiments, the putative promoters P1 and P2 upstream of these leader exons. The promoter regions were cloned in front of the luciferase reporter gene and their promoter activities were measured in transfected SK-N-MC (human neuroepithelioma) and OVCAR-3 (human ovarian carcinoma) cells. Both of these cell lines express the IGF-I gene endogenously, resulting in normally sized IGF-I mRNAs of 7.6, 1.3 and 1.1 kb. In SK-N-MC cells, in which P1 is the most active IGF-I promoter, P2 displayed a three times lower promoter activity than P1. However, in OVCAR-3 cells, P2 is four times more active than P1, resulting in an overall 12-fold difference in the relative promoter activities of the two IGF-I gene promoters in these two cell types. This indicates that the IGF-I promoters show a cell type-specific expression pattern.

Initial characterization of the four promoters of the human insulin-like growth factor II gene.

The human insulin-like growth factor II (IGF-II) gene contains four promoters (P1-P4), which are expressed in a tissue-specific and development-dependent way. Analysis of IGF-II mRNAs in different tissues has revealed that promoters P3 and P4 are expressed in all fetal and in nonhepatic adult tissues. In adult liver, however, the promoters P2, P3 and P4 are completely shut off and another promoter, P1, is activated. To obtain more insight in the mechanisms involved in the regulation of IGF-II gene expression we have performed an initial characterization of the IGF-II promoters employing transient expression of IGF-II promoter constructs in Hep3B and HeLa cells. These studies have revealed that promoters P1, P3 and P4 are active in both cell lines tested, while no activity of promoter P2 could be detected. Employing gel retardation and DNaseI footprint analysis we have identified in the three IGF-II promoters a number of elements which are bound by nuclear proteins.

Determination of several retinoids, carotenoids and E vitamers by high-performance liquid chromatography. Application to plasma and tissues of rats fed a diet rich in either beta-carotene or canthaxanthin.

A method, using two different systems, is described for the high-performance liquid chromatographic analysis of retinol, retinal, retinoic acid, retinyl acetate, retinyl palmitate, alpha-, beta- and gamma-carotene, beta-apo-6'-, beta-apo-8', beta-apo-10'- and beta-apo-12'-carotenal, ethyl beta-apo-8'-carotenoate, alpha-tocopherol and alpha-tocopheryl acetate. The first system consists of a laboratory-packed Hypersil-ODS 3-microns column and a mobile phase of acetonitrile-methylene chloride-methanol-water (70:10:15:5, v/v). The second system consists of a laboratory-packed Hypersil-ODS 3-microns column and a mobile phase of acetonitrile-methylene chloride-methanol-water (70:10:15:5, v/v). The second system consists of a laboratory-packed Nucleosil C18 3-microns column and a mobile phase of acetonitrile-0.1 M ammonium acetate (80:20, v/v). The detection limits in standard solutions were 10 ng/ml for retinoids and carotenoids and 60 ng/ml for the E vitamers. Analysis of the tissues and plasma of rats, after 2 weeks on a diet supplemented with either beta-carotene or canthaxanthin (both 2 mg/g), led to the conclusion that the rats were able both to transport and store beta-carotene and canthaxanthin and to convert beta-carotene to retinol. Incubation of cytosol preparations from the mucosa of the small intestine of rat with 1 microgram of beta-carotene resulted in the formation of 10-20 ng of retinal within 1 h.

Isolated spinous process deviation. A pitfall in the interpretation of AP radiographs of the lumbar spine.

A new type of spinous process deviation is described. This variant may cause confusion in the interpretation of anteroposterior (AP) radiographs of the lumbar spine. In the literature, two types of lumbar spinous process deviation (SPD) have been described: 1) SPD due to rotation of the entire vertebra (as in rotatory scoliosis and degenerative arthritis), and 2) SPD as a consequence of developmental asymmetries of the neural arch. The present study demonstrates that spinous process deviation in the AP radiograph is not a reliable diagnostic guide. The authors' quantitative morphologic analysis of computed tomographic (CT) sections of over 200 lower lumbar vertebrae in vivo revealed a third type of SPD, namely isolated deviation of the spinous process, ie, deviation without any associated rotation or asymmetry of the vertebral body or arch. Since the oval shadow cast by the spinous process in AP radiographs is caused by its tip, rather than by its base (as was demonstrated by in vitro tests), it is concluded that the position of the spinous process shadow in AP radiographs cannot be used as a reliable landmark to differentiate between the three types of SPD. This is only possible by means of a CT examination.

Differential expression of the human insulin-like growth factor II gene. Characterization of the IGF-II mRNAs and an mRNA encoding a putative IGF-II-associated protein.

Insulin-like growth factor II (IGF-II) is a polypeptide of 67 amino acids which is thought to play an important role in fetal growth and development. The human IGF-II gene is situated on chromosome 11, very close to the insulin gene. It extends over 30 kb of chromosomal DNA and consists of five noncoding exons (exons 1-4 and 4B) followed by three protein encoding exons (exons 5-7), one of which (exon 7) contains a long 3'-untranslated region. Here we show that differential initiation of transcription can occur at three distinct promoter sites, resulting in the appearance of mRNA species of different lengths. These promoters show a tissue-specific and a development-specific regulation of expression. Furthermore, we have determined the entire nucleotide sequence of the 3'-terminal exon, exon 7, which is about 4 kb long and contains 3.8 kb of 3'-untranslated sequences. This completes the elucidation of the human IGF-II gene structure. Surprisingly, Northern blot analysis of fetal and adult RNA with a probe derived from the 3'-nontranslated region of exon 7 detects a novel 1.8 kb mRNA which appears to be coordinately expressed with the IGF-II mRNAs. In vitro translation of this 1.8 kb mRNA results in the formation of a translation product of 8.3 kDa, which compares well with the size of a predicted translation product from a 252-nucleotides-long open reading frame.

Metabolic extraction of dopamine in the canine lungs.

Pulmonary metabolism has been demonstrated to be one of the mechanisms of intrapulmonary removal of dopamine. After a bolus injection of [14C]dopamine into the caval vein of an anaesthetized dog a removal of [14C]-radioactivity into the extravascular space was accompanied by an intravascular alteration of the composition of the [14C]compounds during a single transpulmonary passage. A removal of 22% of [14C]-radioactivity out of the bloodstream, together with a metabolic conversion of the remaining [14C]dopamine within the bloodstream, resulted in a total pulmonary extraction of 22% + (0.78 x 18%) = 36%. This report gives a design for further investigation of the metabolic function of the lung sustained by a scheme for problem solving. Also perspectives for clinical application have been compiled.

The human insulin-like growth factor II gene contains two development-specific promoters.

The insulin-like growth factors (IGF) play an important role in fetal and postnatal development. Recently, the nucleotide sequences of the cDNAs encoding IGF-I and IGF-II and part of the human IGF genes were reported. In this communication we describe two distinct IGF-II cDNAs isolated from a human adult liver and a human hepatoma cDNA library, respectively. Using these two cDNAs, we have established that the human IGF-II gene contains at least 7 exons. Two different IGF-II promoters have been identified, 19 kilobases (kb) apart, which are active in a development-specific manner. The promoter, active in the adult stage, is located only 1.4 kb downstream from the insulin gene.

Organization of the human genes for insulin-like growth factors I and II.

Recently, we have reported the isolation of cDNAs encoding the precursors of insulin-like growth factors I and II (IGF-I and II) [(1983) Nature 306, 609-611; (1985) FEBS Lett. 179, 243-246. These cDNAs were employed as specific probes to detect and isolate the corresponding genes from human cosmid DNA libraries. Three cosmids were detected, together containing the entire cDNA sequence of IGF-I, and one cosmid containing the sequence of IGF-II cDNA. Southern blot hybridization, physical mapping and nucleotide sequence analysis of these cosmids revealed that the IGF-I and -II genes have a discontinous structure. The IGF-I gene contains at least four exons spanning a region of probably more that 45 kilobasepairs (kb), while the IGF-II gene consists of at least five exons, spanning a region of 16 kb.