Novel DNA methylation signatures of tobacco smoking with trans-ethnic effects.

BACKGROUND: Smoking remains one of the leading preventable causes of death. Smoking leaves a strong signature on the blood methylome as shown in multiple studies using the Infinium HumanMethylation450 BeadChip. Here, we explore novel blood methylation smoking signals on the Illumina MethylationEPIC BeadChip (EPIC) array, which also targets novel CpG-sites in enhancers. METHOD: A smoking-methylation meta-analysis was carried out using EPIC DNA methylation profiles in 1407 blood samples from four UK population-based cohorts, including the MRC National Survey for Health and Development (NSHD) or 1946 British birth cohort, the National Child Development Study (NCDS) or 1958 birth cohort, the 1970 British Cohort Study (BCS70), and the TwinsUK cohort (TwinsUK). The overall discovery sample included 269 current, 497 former, and 643 never smokers. Replication was pursued in 3425 trans-ethnic samples, including 2325 American Indian individuals participating in the Strong Heart Study (SHS) in 1989-1991 and 1100 African-American participants in the Genetic Epidemiology Network of Arteriopathy Study (GENOA). RESULTS: Altogether 952 CpG-sites in 500 genes were differentially methylated between smokers and never smokers after Bonferroni correction. There were 526 novel smoking-associated CpG-sites only profiled by the EPIC array, of which 486 (92%) replicated in a meta-analysis of the American Indian and African-American samples. Novel CpG sites mapped both to genes containing previously identified smoking-methylation signals and to 80 novel genes not previously linked to smoking, with the strongest novel signal in SLAMF7. Comparison of former versus never smokers identified that 37 of these sites were persistently differentially methylated after cessation, where 16 represented novel signals only profiled by the EPIC array. We observed a depletion of smoking-associated signals in CpG islands and an enrichment in enhancer regions, consistent with previous results. CONCLUSION: This study identified novel smoking-associated signals as possible biomarkers of exposure to smoking and may help improve our understanding of smoking-related disease risk.

The Escherichia coli GTPase CgtAE cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase.

CgtA(E)/Obg(E)/YhbZ is an Escherichia coli guanine nucleotide binding protein of the Obg/GTP1 subfamily whose members have been implicated in a number of cellular functions including GTP-GDP sensing, sporulation initiation, and translation. Here we describe a kinetic analysis of CgtA(E) with guanine nucleotides and show that its properties are similar to those of the Caulobacter crescentus homolog CgtA(C). CgtA(E) binds both GTP and GDP with moderate affinity, shows high guanine nucleotide exchange rate constants for both nucleotides, and has a relatively low GTP hydrolysis rate. We show that CgtA(E) is associated predominantly with the 50S ribosomal subunit. Interestingly, CgtA(E) copurifies with SpoT, a ribosome-associated ppGpp hydrolase/synthetase involved in the stress response. The interaction between CgtA(E) and SpoT was confirmed by reciprocal coprecipitation experiments and by two-hybrid assays. These studies raise the possibility that the ribosome-associated CgtA(E) is involved in the SpoT-mediated stress response.

Impact of skin cancer prevention on outdoor aquatics staff: the Pool Cool program in Hawaii and Massachusetts.

BACKGROUND: Aquatic staff, including lifeguards, are exposed to intense sunlight for many hours each day and are likely to be at a relatively high risk for developing skin cancer. However, no interventions have been specifically directed to staff at outdoor swimming pool sites. METHODS: We conducted a randomized controlled trial among aquatic staff at 28 outdoor pool sites in Hawaii and Massachusetts. Intervention pools received sun protection education and control pools received education on child injury prevention. Staff in both arms received orientation sessions and led instruction during swim lessons. Analysis of covariance was used to compare and test for changes in outcome variables (sun protection habits and sunburning rates of aquatic staff) and pool protection policies. Surveys were completed at the beginning and end of the summer. RESULTS: Surveys were completed by 220 aquatics staff at baseline; 194 surveys were completed at posttest. Compared with staff at control pools, sun protection policies (P < 0.04) and sunburning rates (P < 0.05) improved at sun protection pools from baseline to posttest. However, the difference in the mean score of all sun protection habits between the two study groups was nonsignificant. CONCLUSION: The Pool Cool sun protection intervention had significant effects on lifeguards' sunburn rates and pool sun safety policies but did not improve reported sun protection behaviors. More intensive strategies may be needed to influence aquatics workers who have already begun to adopt skin cancer prevention practices.

Environmental risk increase due to heavy metal contamination caused by a copper mining activity in Southern Brazil.

The Camaquã Copper Mines (CCM) were the main sulphide deposit in Southern Brazil and have been in operation from last century to 1996. To evaluate water contamination and environmental risk increase by heavy metals from mining operations, two points on the João Dias Creek were sampled (Station 1, background area and Station 2, contaminated area). Mining activity increased the natural weakly heavy metal fluxes by approximately 5424 kg. (approximately 60%) of the total metal flux, 1542 kg. (approximately 49%) of dissolved and 3881 kg (approximately 66%) of particulate metal flux. Total metal flux of anthropic origin was mostly due to Fe followed by Cu > Zn > Mn whereas Cd, As and Pb fluxes were negligible. The potential human health hazards and risk assessment related to daily intake of water from João Dias Creek are mostly due to Mn and should be of concern for the contaminated area. The ingestion of water from station 2 represents incremental risks of 130% and 59% respectively, considering the non-carcinogenic and the carcinogenic effects. The real increase of human health hazards may be greater than those related to the total concentrations since Mn and As dissolved concentrations were 5.5 and 2.0 higher than acceptable, respectively.

A randomized trial of the Hawaii SunSmart program's impact on outdoor recreation staff.

BACKGROUND: Skin cancer is the most common form of cancer in the United States and one of the most preventable. Prevention programs for children at outdoor recreation sites may influence not only the youth, but the staff, or caregivers, as well. By teaching children about sun protection, staff may also change their sun protection behaviors. OBJECTIVE: We report on the impact of a childhood skin cancer prevention program (SunSmart) on staff at outdoor recreation sites where a child-focused intervention was conducted. METHODS: The intervention included staff training, on-site activities delivered by staff, distribution of sunscreen, and the promotion of sun-safe environments. It was hypothesized that by teaching children about sun protection, staff would change their sun protection behaviors. A randomized trial at 14 recreation sites (n = 176 staff) in Hawaii tested the efficacy of education only, and education plus environmental changes, compared with a control condition. RESULTS: Results showed significant positive changes in knowledge, sun protection habits, norms, and sun protection policies. The education plus environment group was not superior to education alone. CONCLUSION: Changes in staff behavior and attitudes are important for their own health, as positive role models, and for the dissemination of skin cancer control programs.

Bacterial division: Finding the dividing line.

Division of a cell - whether eukaryotic or prokaryotic - requires accurate spatial coordination. Recent work on the bacterium Escherichia coli has shown that correct placement of the cell division site at the midcell position occurs by a combination of selection against potential polar sites and selection of the midcell site.

Sun protection behaviors and stages of change for the primary prevention of skin cancers among beachgoers in southeastern New England.

Sun exposure is the most important avoidable cause of skin cancers. We report characteristics of a representative sample (N = 2,324) of beachgoers in Southeastern New England during the summer of 1995. This sample was not employing adequate sun protection behaviors (83% did not often avoid the sun during midday and only 45% often used sunscreen). Important demographic and skin cancer risk factor differences in sun protective behaviors and stages of change for sun protection were found, especially differences based on age, gender, and degree of sun sensitivity. Consistent with previous research, increased age, female gender, and greater sun sensitivity were each independently associated with more sun protective behaviors. These findings underscore the need for interventions targeting high-risk populations, such as those receiving high-intensity sun exposures at the beach.

Identification of the fliI and fliJ components of the Caulobacter flagellar type III protein secretion system.

Caulobacter crescentus is motile by virtue of a polar flagellum assembled during the predivisional stage of the cell cycle. Three mutant strains in which flagellar assembly was blocked at an early stage were isolated. The mutations in these strains mapped to an operon of two genes, fliI and fliJ, both of which are necessary for motility. fliI encodes a 50-kDa polypeptide whose sequence is closely related to that of the Salmonella typhimurium FliI protein, an ATPase thought to energize the export of flagellar subunits across the cytoplasmic membrane through a type III protein secretion system. fliJ encodes a 16-kDa hydrophilic protein of unknown function. Epistasis experiments demonstrated that the fliIJ operon is located in class II of the C. crescentus flagellar regulatory hierarchy, suggesting that the gene products act at an early stage in flagellar assembly. The expression of fliIJ is induced midway through the cell cycle, coincident with other class II operons, but the FliI protein remains present throughout the cell cycle. Subcellular fractionation showed that FliI is present both in the cytoplasm and in association with the membrane. Mutational analysis of FliI showed that two highly conserved amino acid residues in a bipartite ATP binding motif are necessary for flagellar assembly.

The final stages of spliceosome maturation require Spp2p that can interact with the DEAH box protein Prp2p and promote step 1 of splicing.

Pre-mRNA processing occurs by assembly of splicing factors on the substrate to form the spliceosome followed by two consecutive RNA cleavage-ligation reactions. The Prp2 protein hydrolyzes ATP and is required for the first reaction (Yean SL, Lin RJ, 1991, Mol Cell Biol 11:5571-5577; Kim SH, Smith J, Claude A, Lin RJ, 1992, EMBO J 11:2319-2326). The Saccharomyces cerevisiae SPP2 gene was previously identified as a high-copy suppressor of temperature-sensitive prp2 mutants (Last RL, Maddock JR, Woolford JL Jr, 1987, Genetics 117:619-631). We have characterized the function of Spp2p in vivo and in vitro. Spp2p is an essential protein required for the first RNA cleavage reaction in vivo. Depletion of Spp2p from yeast cells results in accumulation of unspliced pre-mRNAs. A temperature-sensitive spp2-1 mutant accumulates pre-mRNAs in vivo and is unable to undergo the first splicing reaction in vitro. However, spliceosomal complexes are assembled in extracts prepared from the mutant. We show that Spp2p function is required after spliceosome assembly but prior to the first reaction. Spp2p associates with the spliceosome before the first RNA cleavage reaction and is likely to be released from the spliceosome following ATP hydrolysis by Prp2p. The Prp2 and Spp2 proteins are capable of physically interacting with each other. These results suggest that Spp2p interacts with Prp2p in the spliceosome prior to the first cleavage-ligation reaction. Spp2p is the first protein that has been found to interact with a DEAD/H box splicing factor.

Simultaneous determination of clebopride and a major metabolite N-desbenzylclebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry.

A procedure for the simultaneous assay of clebopride and its major metabolite N-desbenzylclebopride in plasma has been developed. The method utilizes capillary gas chromatography-negative-ion chemical ionization mass spectrometry with selected-ion monitoring of characteristic ions. Employing 2-ethoxy analogues as internal standards, the benzamides were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyric anhydride produced volatile mono- and diheptafluorobutyryl derivatives of clebopride and N-desbenzylclebopride, respectively. The methane negative-ion mass spectra of these derivatives exhibited intense high-mass ions ideal for specific quantitation of low levels in biological fluids. Using this procedure the recovery of the drug and metabolite from human plasma was found to be 84.4 +/- 1.5% (n = 3) and 77.4 +/- 4.7% (n = 3), respectively, at 0.5 ng/ml. Measurement of both compounds down to 0.10 ng/ml with a coefficient of variation of less than 10.5% is described. Plasma levels are reported in four volunteers up to 24 h following oral administration of 1 mg of clebopride malate salt.

Determination of clebopride in plasma by capillary gas chromatography-negative-ion chemical ionization mass spectrometry.

A procedure for the analysis of clebopride in plasma using capillary gas chromatography-negative-ion chemical ionization mass spectrometry has been developed. Employing an ethoxy analogue as internal standard, the two compounds were extracted from basified plasma using dichloromethane. Subsequent reaction with heptafluorobutyryl imidazole produced volatile monoheptafluorobutyryl derivatives whose ammonia negative-ion mass spectra proved ideal for selected-ion monitoring. The recovery of clebopride from plasma at 0.536 nmol/l was found to be 85.5 +/- 0.9% (n = 3) whilst measurement down to 0.268 nmol/l was possible with a coefficient of variation of 7.9%. Plasma levels of the compound are reported in two volunteers following ingestion of 1 mg of clebopride as the malate salt.

Improved method for the determination of N-acetylcysteine in human plasma by high-performance liquid chromatography.

An improved method for the determination of N-acetylcysteine by paired-ion reversed-phase high-performance liquid chromatography has been developed. Following incubation with dithiothreitol to release bound N-acetylcysteine, free N-acetylcysteine and the internal standard N-acetylpenicillamine were derivatised with 2,4-dinitro-1-fluorobenzene. The samples were then deproteinised by ultrafiltration. The dinitrophenyl derivatives were extracted from acidified ultrafiltrate into diethyl ether and purified by using a back-extraction step. They were then separated from naturally occurring plasma components and reagent impurities by high-performance liquid chromatography, utilising an Ultrasphere ODS (5 microns) reversed-phase column and detection at 360 nm.

High-performance liquid chromatographic assay for N-acetylcysteine in plasma and urine.

A sensitive high-performance liquid chromatographic (HPLC) assay for N-acetylcysteine in human plasma and urine has been developed. The method employs a prechromatographic stage to produce the dinitrophenyl derivative of N-acetylcysteine, which is chromatographed on a 5-microns C18 bonded reverse-phase column using an external standard method. The derivatized N-acetylcysteine molecule has a retention time of congruent to 13 min at a flow rate of 1.0 mL/min for a mobile phase of methanol-aqueous 0.05 M citrate-0.001 M EDTA buffer pH 7.0 (30:70). In a multistep extraction procedure, which is slightly modified for the N-acetylcysteine assay in urine, the limits of sensitivity for the plasma and urine assays were found to be congruent to 60 ng/mL and congruent to 200 micrograms/mL, respectively. Preliminary data from pilot studies in human subjects are presented.

Effects of progestogens on serum lipids in the post-menopause.

Measurement of serum cholesterol, triglycerides, and phospholipids was undertaken in patients with a natural menopause or following oöphorectomy who received cyclical oestrogen regimens for six consecutive months. The regimen employed was either a 'high' or 'low' dose of equine conjugated oestrogens, piperazine oestrone sulphate or oestradiol valerate. After six months a change was made to cyclical sequential oestrogen/progestogen regimens for varying periods. Patients with a natural menopause given either 'high' or 'low' dose regimens showed no significant changes in serum cholesterol, triglyceride or phospholipid levels compared to baseline values. However, the serum cholesterol levels of the oöphorectomized subjects were significantly elevated in comparison with the perimenopausal group. The addition of progestogen to the oestrogen did not cause any significant alterations.

Endometrial factors under treatment with oestrogen and oestrogen/progestogen combinations.

In a continuing prospective study, uterine curettage was undertaken on sixty-four patients attending a Menopause Clinic prior to consideration of gonadal hormone therapy. Two of these patients (3.1%) were found to have endometrial hyperplasia, and subsequently they were not given gonadal hormone therapy. Sixty-two patients with normal endometrium at pre-treatment curettage received cyclical oestrogen regimens or sequential oestrogen/progestogen treatments. Four (30.8%) of the thirteen patients in receipt of cyclical 'high-dose' oestrogens developed cystic glandular hyperplasia, whereas none of the patients taking either cyclical 'low-dose' oestrogens (thirty patients) or cyclical-sequential oestrogen/progestogen regimens (nineteen patients) developed endometrial hyperplasia. Among the patients with a normal endometrium, both before and during cyclical gonadal hormone therapy, regular withdrawal bleeding was experienced by thirty-two patients (51.6%). Breakthrough bleeding occurred in nine (14.5%), while twenty-one patients (33.9%) had no vaginal bleeding. Of the four patients with normal endometrium at pre-treatment curettage who subsequently developed endometrial hyperplasia during cyclical 'high-dose' oestrogen therapy, regular withdrawal bleeding was experienced by two patients, and in one of these breakthrough bleeding also occurred. Furthermore, in the four patients who developed endometrial hyperplasia, this condition occurred within six months in two patients and within 9 and 10 months respectively in the remaining two. In the nineteen patients receiving cyclical sequential oestrogen/progestogen regimens, all had regular withdrawal bleeding, while one patient had breakthrough bleeding during sequential therapy. It is concluded that in those climacteric patients who present with severe menopausal symptoms which necessitate the administration of high-dose oestrogen regimes it is necessary either to undertake both pretreatment uterine curettage or to add a progestogen to the oestrogen in a sequential regimen.

Clinical therapeutic evaluation of methylcysteine hydrochloride in patients with chronic obstructive bronchitis: a balanced double-blind trial with placebo control.

A double-blind, between-patient, placebo controlled trial was carried out to investigate the effects of methylcysteine hydrochloride in patients with chronic obstructive bronchitis. After a 2-week washout period on placebo, 30 patients were allocated at random to treatment for 6 weeks with either methylcysteine (1200 mg daily in Week 1, 800 mg daily in Week 2, then 600 mg daily) or with identical placebo tablets on the same regimen. During the post-treatment period, all patients returned to a single-blind placebo regimen (6 tablets daily) for a further 14 days. Assessments were made at the start, at regular intervals during the trial, and at the end of the post-treatment period, of subjective and objective measures of clinical response, and measurements of pulmonary function and certain physico-chemical properties of sputum. The results showed that methylcysteine increased sputum volume, reduced the viscidity of sputum, and significantly improved the subjective assessments of ease of expectoration and severity and frequency of cough, leading to a definite improvement in the patients' clinical state. No side-effects of clinical significance were reported and no abnormalities were found in any of the haematological, hepatic and renal function tests carried out.