Alcohol and delirium tremens: effects of average number of drinks per day and beverage type.

OBJECTIVE: Associations of amount of alcohol intake and beverage type with the risk of delirium tremens (DT) have not been studied. This longitudinal study investigated if the average number of drinks per day and beverage type predict DT. METHODS: A cohort of 3 582 alcohol-dependent men and women aged 19-82 without previous DT were interviewed about alcohol intake and beverage type at baseline in 1994-2005 and followed through record linkage in Danish nationwide registers to identify incident DT. Data were analyzed by means of Cox regression models. RESULTS: An average number of drinks per day of 20-30 or >30 was associated with hazard ratios (HRs) of 1.38 (95% CI 1.03-1.84) and 1.64 (95% CI 1.19-2.27) relative to the reference category (1-9 drinks). Independently of amount consumed and covariates (age, gender, civil status and work status), beverage type (spirits vs. mixed alcohol) was associated with a HR of 1.63 (95% CI 1.08-2.46). Male gender was robustly associated with increased risk (HR = 1.62 (95% CI 1.25-2.08). CONCLUSIONS: In alcohol-dependent men and women, daily alcohol intake above a threshold of 20 beverages or 240 g alcohol and a preference for spirits increase the risk of developing DT.

Parental alcohol use disorder with and without other mental disorders and offspring alcohol use disorder.

OBJECTIVE: To examine the association between parental alcohol use disorder (AUD) with and without other mental disorders and offspring AUD. METHODS: Using data from Danish nationwide registers, we identified 15 477 offspring with parental AUD and 154 392 reference individuals from the general population. Parental AUD was defined as registration for AUD treatment. Parental mental disorders were identified in medical registers and comprised psychotic, mood, anxiety, personality, drug use, and other non-alcohol-related mental disorders. AUD in offspring was identified from medical, pharmacy, treatment and cause of death registers. Hazard ratios (HRs) of AUD were estimated using Cox regression models. RESULTS: AUD in one or both parents was associated with higher risks of AUD in offspring compared with reference individuals. Paternal AUD plus other mental disorder (HR = 2.27, 95% confidence interval (CI): 2.10-2.46) and paternal AUD alone (HR = 2.21, 95% CI: 2.07-2.36) were associated with higher offspring AUD risk. Similarly, maternal AUD plus other mental disorder (HR = 3.02, 95% CI: 2.66-3.43) and maternal AUD alone (HR = 2.57, 95% CI: 2.20-3.01) were associated with higher offspring AUD risk. CONCLUSIONS: Offspring with parental AUD are at increased risk of AUD irrespective of exposure to other parental mental disorders.

Long-term weight changes in obese young adult men and subsequent all-cause mortality.

BACKGROUND: Although the expectation is that weight gain increases mortality and weight loss among those overweight reduces mortality, results on weight gain and mortality in young adults are conflicting, and weight loss is less explored. We investigated the association between long-term weight change and all-cause mortality in a broad range of body mass index (BMI) in young men. METHODS: Among 362200 Danish draftees, examined between 1943 and 1977, all obese (BMI 31.0 kg m(-2); n=1930), and a random 1% sample of the others (n=3601) were identified at a mean age of 20 years (range: 18-25 years). All the obese and half the controls were re-examined between 4 and 40 years later (mean age 35 years). Weight changes were defined as: weight loss <-0.1 kg m(-2) per year, weight stability within ±0.1 kg m(-2) per year and weight gain >0.1 kg m(-2) per year. Hazard ratios (HR) and 95% confidence intervals (CI) were estimated using Cox regression. RESULTS: Among the 908 obese and 1073 controls followed for 30 years after re-examination 220 and 232 died. HR of the weight stable obese was 2.32 (CI: 1.56-3.44) compared with the weight stable controls. In the obese cohort there was no association between weight loss, adjusted for initial BMI, and mortality (HR: 0.99; CI: 0.68-1.45) compared with weight stable obese. Too few controls lost weight to allow assessment of weight loss. Weight gain was associated with increased mortality in the obese (HR: 1.50; CI: 1.07-2.10) and controls (HR: 1.54; CI: 1.14-2.09) compared with weight stable obese and controls, respectively. Neither the time between the two examinations, life-style factors nor exclusion of diseased individuals influenced the results. CONCLUSIONS: Although there were increased mortality of the weight-stable obese compared with controls, there was no association between weight loss and mortality in the obese. Weight gain increased mortality regardless of the initial weight.

Weight and weight gain during early infancy predict childhood obesity: a case-cohort study.

BACKGROUND: Infant weight and weight gain are positively associated with later obesity, but whether there is a particular critical time during infancy remains uncertain. OBJECTIVE: The aim was to investigate when and how weight and weight gain during infancy become associated with childhood obesity. METHODS: In a cohort representing 28 340 children born from 1959-67 and measured in Copenhagen schools, 962 obese children (2007 World Health Organization criteria), were compared with a 5% randomly selected sub-cohort of 1417 children. Information on weight at birth, 2 weeks, 1, 2, 3, 4, 6 and 9 months was retrieved from health visitors' records. Odds ratios and 95% confidence intervals (CI) for childhood obesity by tertiles of weight at each age and by change in tertiles of weight between two consecutive measurements were estimated using multivariate logistic regression with adjustment for indicators of socioeconomic status, preterm birth, and breastfeeding. RESULTS: Compared with children in the middle weight-tertile, children with a weight in the upper tertile had a 1.36-fold (CI, 1.10-1.69) to 1.72-fold (CI, 1.36-2.18) higher risk of childhood obesity from birth through 9 months, whereas children in the lower weight-tertile had almost half the risk of obesity from 2 through 9 months. The risk of childhood obesity associated with change in weight-tertile in each interval was stable at ∼1.5-fold per weight-tertile increase throughout infancy. CONCLUSIONS: Infant weight and weight gain are associated with obesity in childhood already during the first months of life. Determinants of weight gain shortly after birth may be a suitable target for prevention of obesity.

Circulating ACE is a predictor of weight loss maintenance not only in overweight and obese women, but also in men.

BACKGROUND: Circulating angiotensin-converting enzyme (ACE) was identified as a predictor of weight loss maintenance in overweight/obese women of the Diogenes project. OBJECTIVE: To investigate whether ACE acted also as a predictor in men of the Diogenes study and to compare it with that in women. DESIGN: Subjects, who lost ≥ 8% of body weight induced by low-caloric diet in an 8-week weight loss period, were assigned to weight loss maintenance with dietary intervention for 6 months. SUBJECTS: 125 overweight/obese healthy men from eight European countries who completed whole intervention. MEASUREMENTS: Concentrations and activity of serum ACE at baseline and after the 8-week weight loss, in addition to anthropometric and physiological parameters. RESULTS: Serum ACE concentration decreased by 11.3 ± 10.6% during the weight loss period in men. A greater reduction is associated with less body weight regain during the maintenance period (r=0.227, P=0.012). ACE change was able to predict a weight regain ≤ 20% after 6 months, with an odds ratio of 1.59 (95% confidence interval (CI): 1.09-2.33, P=0.016) for every 10% reduction, which was independent of body mass index and weight loss. The prediction power was weaker in men than in women, but without a significant sex difference (P=0.137). In pooled subjects (N=218), the odds ratio was 1.96 (95% CI: 1.46-2.64, P<0.001). CONCLUSIONS: A greater reduction of ACE during weight loss is favorable for weight maintenance in both men and women. This can offer useful information for personalized advice to improve weight loss maintenance. It also confirms the role of ACE in the metabolic pathways of weight regulation.

Lifelong doubling of mortality in men entering adult life as obese.

BACKGROUND: The association between obesity in adults and excess morbidity and mortality is well established, but the impact of being obese in early adulthood on health throughout adult life needs elucidation. We investigated the all-cause mortality until 80 years of age in men starting adult life as obese. METHODS: Among 362,200 Danish young men, examined for military service between 1943 and 1977, all obese (defined as body mass index (BMI ≥ 31.0 kg m(-2)), and, as controls, a random 1% sample of the remaining population were identified. A total of 1862 obese, corresponding to all men above the 99.5 percentile in this population, and 3476 controls were included, at a median age of 19 years (range: 18-25 years of age). They were followed until 2007 and Cox regression models were used to estimate the mortality in the obese relative to the controls. In addition, two reference groups were used: normal weight men (BMI: 18.5-24.9 kg m(-2)) and the men with the lowest mortality in this cohort (BMI: 22.0-24.9 kg m(-2)). RESULTS: During the 65 years of follow-up, 1191 men died. At all ages from 18 to 80 years, the mortality in the obese was twice that of the controls (hazard ratio (HR): 2.10; 95% confidence interval (CI): 1.84-2.39). The median survival proportion (0.5) was reached about 8 years earlier in the obese than in either of the reference groups. Relative to the normal weight and men with the lowest mortality HRs of 2.14 (95% CI: 1.86-2.45) and 2.38 (95% CI: 2.00-2.85), respectively, were estimated for the obese. Neither year of birth nor education significantly influenced the excess mortality. CONCLUSION: Men entering adult life as obese experience a lifelong doubling of mortality, a finding that strongly supports the continued need to avoid beginning adult life as obese.

Common variants near MC4R in relation to body fat, body fat distribution, metabolic traits and energy expenditure.

OBJECTIVE: Common variants near melanocortin receptor 4 (MC4R) have been related to fatness and type 2 diabetes. We examined the associations of rs17782313 and rs17700633 in relation to body fat, body fat distribution, metabolic traits, weight development and energy expenditure. METHODS: Obese young men (n = 753, BMI > or = 31.0 kg m(-2)) and a randomly selected group (n = 874) identified from a population of 174 800 men were re-examined in three surveys at mean ages 35, 46 and 49 years (S-35, S-46 and S-49). Measurements were available at upto eight times from birth to adulthood. Logistic regression analysis was used to assess odds ratio (OR) for the presence of the carrier allele for a given difference in phenotypic values. RESULTS: Rs17782313 minor C-allele was associated with overall, abdominal and peripheral fatness (range of OR = 1.06-1.14 per z-score units) at all three surveys, although only consistently significant at S-35 and S-46. Rs17700633 minor A-allele was also associated with the fatness measures, but significantly so only at S-49 for overall and abdominal fatness (range of OR = 1.03-1.15 per z-score units), and peripheral fatness (OR = 1.15-1.20 per z-score units). There were only few significant associations with metabolic traits. The rs17782313 C-allele and the rs17700633 A-allele were both associated with lower high-density lipoprotein cholesterol (range of OR = 0.64-0.84 per mol l(-1)), significantly at S-46. The rs17700633 A-allele was significantly associated with insulin (OR = 1.25 per 50 pmol l(-1)), leptin (OR = 1.42 per 10 ng microl(-1)) and insulin sensitivity (OR = 0.81 per model unit). The rs17782313 C-allele and the rs17700633 A-allele were both associated with BMI in childhood and adolescence (range of OR = 1.04-1.17 per z-score units), significant for the rs17782313 C-allele at the age of 13-19 years and for rs17700633 A-allele at age 7, 10, 13 and 19 years. No significant associations were found for energy expenditure. CONCLUSION: Near MC4R variants appear to contribute to body fat, body fat distribution, some metabolic traits, weight development during childhood, but not to energy expenditure.

Apoptosis induced by the potential chemotherapeutic drug N1, N11-Diethylnorspermine in a neuroblastoma cell line.

Neuroblastoma is a highly malignant neoplasm found in young children. Although children with high-risk neuroblastoma respond to chemotherapy, relapses are common. On account of poor treatment outcome, new treatment strategies are constantly sought for neuroblastoma. Polyamine analogues are potentially novel substances for treatment of neuroblastoma. In this study, we have treated two neuroblastoma cell lines, SH-SY5Y and LA-N-1, with the spermine analogue N1, N11-Diethylnorspermine (DENSPM). SH-SY5Y was the most sensitive cell line, in which DENSPM treatment resulted in an inhibition of cell proliferation and an induction of cell death. The cell death induced by DENSPM treatment was apoptotic, as evidenced by cleavage of procaspase 3 and induction of caspase-3 activity. In contrast, DENSPM treatment only resulted in a slight inhibition of cell proliferation in LA-N-1 cells. There were several possible causes for the lower sensitivity to DENSPM treatment in the latter cell line when compared with SH-SY5Y cells. DENSPM-induced polyamine depletion was more extensive in SH-SY5Y cells than in LA-N-1 cells. This was partly because of a higher induction of the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase in the cell line SH-SY5Y. The DENSPM-induced polyamine depletion was also caused by the inhibition of ornithine decarboxylase. LA-N-1 cells contained a higher level of the prosurvival protein survivin, which was further increased after DENSPM treatment. In contrast, DENSPM treatment resulted in a decreased survivin level in SH-SY5Y cells.

Key parameters for the development of a NIR microscopic method for the quantification of processed by-products of animal origin in compound feedingstuffs.

The aim of this work is to show new advances in the analytical methods developed in the frame of the ban of processed animal by-products in compound feed that is currently applied within the European Union. With this aim, studies to develop a quantitative near infrared microscopy (NIRM) approach have been undertaken in order to fulfil future requirements of European legislation like the introduction of tolerance levels that would require for official control purposes the availability of specific quantitative methods. The capabilities of the NIRM method have been improved; no sample preparation is required and the acquisition parameters are optimised. Both the gross and the fine fractions of the samples are considered; the reflexion mode was used to analyse the gross raw fraction and the transmission mode was chosen to analyse the fine raw fraction. Parameters for reflexion analyses were already fixed in our previous studies while those of transmission mode have been determined in the present study. Because particles are too small, it is difficult to mark them; spectra were collected using the mapping technique. Quantitative analyses have been carried out for different percentages of adulteration (0.5, 1, 2 and 5%). Results were depending on the particle size distribution of the feed and of the fish meal which led to experimental values of adulteration varying between 0.13-0.92%, 0.93-3.7%, 2.42-5.83% and 1.95-9.39% for theoretical percentages of adulteration equal to 0.5, 1, 2 and 5%, respectively. The established protocol with the key parameters proposed has to be considered for the development of an accurate method of quantification.

eNOS phosphorylation and translocation are altered in male but not female mice by increased activation of the Gαq protein.

Little is known about sex-dependent physiological and pathophysiological differences in cardiac endothelial nitric oxide synthase (eNOS) expression and activation. Therefore, we investigated cardiac morphology and eNOS protein expression, including its translocation-dependent activation and phosphorylation, in cardiac tissue of male and female wild-type mice and transgenic heart-failure mice having a cardiac-specific, 5-fold overexpression of the Galphaq protein. In addition, we measured calcineurin protein expression. Heart-to-body weight ratio was increased in Galphaq mice. Female wild-type mice showed higher eNOS protein expression and activation (translocation and phosphorylation) than did wild-type males. In cardiac tissue of Galphaq mice, these sex-dependent differences remained or were enhanced. Protein expression of the catalytic subunit calcineurin A, which has been shown to dephosphorylate eNOS, was higher in wild-type males than in wild-type females. These differences were increased in the Galphaq mice model. We conclude that sex differences exist in cardiac eNOS protein expression and phosphorylation. Increased activation of the Galphaq protein appears to alter eNOS protein expression and phosphorylation only in males.

Macronutrient-specific effect of FTO rs9939609 in response to a 10-week randomized hypo-energetic diet among obese Europeans.

BACKGROUND: The A risk allele of rs9939609 of the fat mass- and obesity-associated gene (FTO) increases body fat mass. OBJECTIVE: To examine whether FTO rs9939609 affects obese individuals' response to a high-fat, low-carbohydrate (CHO) (HF) or low-fat, high-CHO (LF), hypo-energetic diet and whether the effect of the FTO variant depends on dietary fat and CHO content. DESIGN: In a 10-week, European, multi-centre dietary intervention study 771 obese women and men were randomized to either LF (20-25% of energy (%E) from fat, 60-65%E from CHO) or HF (40-45%E from fat, 40-45%E from CHO), hypo-energetic diet (measured resting metabolic rate multiplied by 1.3-600 kcal day(-1)). Body weight, fat mass (FM), fat-free mass (FFM), waist circumference (WC), resting energy expenditure (REE), fasting fat oxidation as % of REE (FatOx), insulin release (HOMA-beta) and a surrogate measure of insulin resistance (HOMA-IR) were measured at baseline and after the intervention. In all, 764 individuals were genotyped for FTO rs9939609. RESULTS: For A-allele carriers the drop-out rate was higher on HF than LF diet (in AT, P=0.002; in AT/AA combined, P=0.003). Among those individuals completing the intervention, we found no effect of FTO rs9939609 genotype on Deltaweight, DeltaFM, DeltaFFM, DeltaWC or DeltaFatOx. However, participants with TT had a smaller reduction in REE on LF than on HF diet (75 kcal/24 h; interaction: P=0.0055). These individuals also showed the greatest reduction in HOMA-beta and HOMA-IR (interaction: P=0.0083 and P=0.047). CONCLUSION: The FTO rs9939609 may interact with the macronutrient composition in weight loss diets in various ways; carriers of the A allele on LF diet appear to have a lower risk for drop out, and TT individuals have a smaller decrease in REE and greater decrease in HOMA-beta and HOMA-IR on LF than on HF diet.

Experiences with Duddingtonia flagrans administration to parasitized small ruminants.

The aim of this study was to investigate the effect of the nematophagous fungus Duddingtonia flagrans applied orally to small ruminants in a field study in Germany. 20 female, pure breed goat kids and 20 female, pure breed lambs, all naturally infected with GIN, were kept on pasture and fed additionally with concentrates amended by 5 x 10(5) spores of D. flagrans per kilogram bodyweight daily for 3 months during pasture season. The equally sized control groups got the concentrate without spores. Every fortnight data of body weight, eggs per gram faeces and larval development in faecal cultures and on pasture were collected. Following 3 months of spore feeding the control goats showed arithmetic mean faecal egg counts of 1235 (+/-533) eggs per gram (epg) faeces, in comparison to 517 (+/-671) epg in the fungus-fed group (p<0.001). No statistically significant difference was found between the two sheep groups. The maximum in larval reduction in faeces was found at the end of the fungus-feeding period (81.3% in the sheep groups and to 67.9% in the goat groups), but without statistical significance. At the end of the study the mean body weight gain in the fungus-treated groups tended to be higher than in the control groups, but not showing statistically significant differences. Only regarding the first-year-grazing-goats, the bodyweights of the post-feeding period revealed significant (p<0.05) differences between fungus-fed and control group. However, no statistically significant differences were observed in qualitative faecal cultures, pasture larvae counts, serum pepsinogen-level and PCV. In the study presented here, no clear effect of fungus could be observed. Furthermore, climatic conditions during the study period may have demonstrated how vulnerable the fungus application is to such parameters.

Lack of association of fatness-related FTO gene variants with energy expenditure or physical activity.

CONTEXT: A common variant in the first intron of FTO (rs9939609, T/A) is associated with fatness in Caucasians. OBJECTIVE: FTO may regulate energy homeostasis through the hypothalamus, and we hypothesized that AA-genotypes of rs9939609 FTO have lower energy expenditure and/or a lower level of physical activity. METHODS: The study population included all obese young men (body mass index > or = 31 kg/m(2)) at the mandatory draft board examinations in the Copenhagen area from 1943 to 1977 and a randomly selected control group from this population. Subgroups of 234 obese and 323 controls were examined in 1998-2000 (median age 48 yr). Fat mass (FM), lean body mass (LBM), leisure-time physical activity (LTPA), maximum oxygen uptake (VO(2)max), resting energy expenditure (REE), and glucose-induced thermogenesis (GIT) were measured. The FTO rs9939609 variant was genotyped. A recessive transmission mode fit the data best. Logistic regression was used to assess the odds ratios of the AA-genotype in relation to LTPA, VO(2)max, REE, and GIT. RESULTS: The AA-genotype of FTO rs9939609 had higher REE in the age-adjusted model, but the association was eliminated when adjusting for FM and LBM. The AA-genotype was not associated with LTPA, VO(2)max, or GIT. This was not influenced by adjustment for age, FM, or LBM. The AA-genotype had increased FM, even with adjustment for age, LBM, REE, GIT, VO(2)max, and LTPA. Results were similar for FTO rs8050136 and rs7193144. CONCLUSIONS: Homozygous carriers of the A-allele of rs9939609 FTO do not have lower REE, GIT, VO(2)max, or LTPA but higher FM, irrespective of LBM, REE, GIT, VO(2)max, and LTPA.

Impact on weight dynamics and general growth of the common FTO rs9939609: a longitudinal Danish cohort study.

OBJECTIVE AND DESIGN: We investigated the impact of the fatness-related FTO rs9939609 A-allele on cross-sectional and longitudinal measures of body mass index (BMI), height and lean body mass (LBM) in a unique cohort representing a broad range of BMI. SUBJECTS AND MEASUREMENTS: A random sample of all men attending the Danish draft boards during 1943-1977 plus all men with a BMI>or=31.0 kg/m(2) (assuring representation of the right end of the distribution) was taken. Anthropometric measures were available at up to eight points in time from birth to adulthood in 1629 genotyped men. The odds ratio (OR) for being a carrier of FTO rs9939609 according to (1) one unit alteration in z-scores for BMI, height and LBM at given ages and (2) longitudinal changes in BMI and height z-scores were assessed by logistic regression. RESULTS: Except at birth, the AA genotype was associated with increased BMI z-scores at all point during the monitored lifespan, starting at the age of 7 years. This effect remained stable until early adulthood, where further weight gain occurred. The AA genotype was also--mainly through the effect on fatness--associated with accelerated linear growth in childhood (age 7 years; OR, 1.36; 95% confidence interval (CI), 1.06-1.74) and increased LBM in adulthood (OR, 1.24; 95% CI, 1.14-1.35). CONCLUSION: Fatness induced by FTO rs9939609 in early childhood is sustained until early adulthood, where further weight gain may occur. FTO rs9939609 may, however, also be associated with linear growth and LBM mainly through the effect on fat mass.

High social class and suicide in persons at risk for schizophrenia.

OBJECTIVE: The relationship between suicide and social class has been equivocal. While some authors have reported that higher social class is related to higher rates of suicide, most other studies report that lower social class is associated with higher rates of suicide. Our study attempted to resolve these inconsistencies by using a High Risk for schizophrenia method. METHOD: Children of women with severe schizophrenia were assessed in 1962. In 2005, when subjects were a mean age of 58 years, we identified those who had committed suicide. RESULTS: A higher rate of suicide was associated with risk for schizophrenia in the High-Risk sample. Higher social class origin was associated with suicide in persons at risk for mental illness. CONCLUSION: Higher social class origin was associated with suicide in subjects at genetic risk for schizophrenia (but not those without risk).

Possible role of adiponectin and insulin sensitivity in mediating the favorable effects of lower body fat mass on blood lipids.

AIMS: The objective of this study was to investigate the role of insulin sensitivity and serum adiponectin concentration as determinants, in middle-aged men, of the relationship between lower body fat and blood lipids after truncal fat has been accounted for. METHODS: Men (443) aged 39-65 yr, body mass index 18-43 kg/m(2), participated in the study. The following variables were measured: regional body fat distribution as assessed by dual-energy x-ray absorptiometry, maximal oxygen uptake, physical activity, fasting levels of serum adiponectin, triglycerides, and high-density lipoprotein- and total cholesterol. Plasma glucose and serum insulin were measured in the fasting state and after an oral glucose load. RESULTS: Lower body fat mass was inversely associated with serum triglycerides and total cholesterol and positively with serum high-density lipoprotein-cholesterol after adjustment for age, lean tissue mass, truncal fat mass, weight history, maximal oxygen uptake, and the level of physical activity (P < 0.0005). Serum adiponectin level and Matsudas insulin sensitivity index were positively intercorrelated, and both were positively correlated to lower body fat mass. When including adiponectin and insulin sensitivity in the analyses, the relationships between lower body fat mass and serum lipids were partly explained. CONCLUSION: For a given level of truncal fat mass, a large lower body fat mass is associated with an advantageous blood lipid profile, which may be partially mediated by the relationships to both insulin sensitivity and serum adiponectin level.

Differential polyamine analogue effects in four human breast cancer cell lines.

Polyamine analogues have demonstrated anti-tumour activity in a number of solid tumour models. In the present study we compared the cytotoxicities of three polyamine analogues against four breast cancer cell lines. All cell lines are derived from tumours of women with breast cancer and, although we are sampling just a small number of tumours, they represent a spectrum of the genetic plethora of breast cancers. Cytotoxicity, over a dose range from 0.1 to 100 microM, was evaluated with three different cytotoxicity assays performed in 96-well plates. Comparing the effects of the analogues on polyamine pools with data from the cytotoxicity assays indicates that there was not a direct correlation between polyamine pool depletion and cytotoxicity. Flow cytometry was used to investigate analogue-induced cell death as measured by the appearance of a sub-G(1) peak. Induction of cell death by the analogues differed in the cell lines, however, cell death when induced was apoptotic, as demonstrated by detection of apoptotic bodies with immunofluorescence microscopy of propidium iodide-stained nuclei. Comparing the flow cytometry-derived data and the data from the cytotoxicity assays reveals that the analogues exert their effects by inhibiting cell growth and/or inducing cell death.

Optimisation of asymmetric flow field-flow fractionation for the characterisation of nanoparticles in coated polydisperse TiO2 with applications in food and feed.

Titanium dioxide (TiO2) has various applications in consumer products and is also used as an additive in food and feeding stuffs. For the characterisation of this product, including the determination of nanoparticles, there is a strong need for the availability of corresponding methods of analysis. This paper presents an optimisation process for the characterisation of polydisperse-coated TiO2 nanoparticles. As a first step, probe ultrasonication was optimised using a central composite design in which the amplitude and time were the selected variables to disperse, i.e., to break up agglomerates and/or aggregates of the material. The results showed that high amplitudes (60%) favoured a better dispersion and time was fixed in mid-values (5 min). In a next step, key factors of asymmetric flow field-flow fraction (AF4), namely cross-flow (CF), detector flow (DF), exponential decay of the cross-flow (CFexp) and focus time (Ft), were studied through experimental design. Firstly, a full-factorial design was employed to establish the statistically significant factors (p < 0.05). Then, the information obtained from the full-factorial design was utilised by applying a central composite design to obtain the following optimum conditions of the system: CF, 1.6 ml min-1; DF, 0.4 ml min-1; Ft, 5 min; and CFexp, 0.6. Once the optimum conditions were obtained, the stability of the dispersed sample was measured for 24 h by analysing 10 replicates with AF4 in order to assess the performance of the optimised dispersion protocol. Finally, the recovery of the optimised method, particle shape and particle size distribution were estimated.

5-HT2B receptor antagonists attenuate myofibroblast differentiation and subsequent fibrotic responses in vitro and in vivo.

Pulmonary fibrosis is characterized by excessive accumulation of connective tissue, along with activated extracellular matrix (ECM)-producing cells, myofibroblasts. The pathological mechanisms are not well known, however serotonin (5-HT) and 5-HT class 2 (5-HT2) receptors have been associated with fibrosis. The aim of the present study was to investigate the role of 5-HT2B receptors in fibrosis, using small molecular 5-HT2B receptor antagonists EXT5 and EXT9, with slightly different receptor affinity. Myofibroblast differentiation [production of alpha-smooth muscle actin (α-SMA)] and ECM synthesis were quantified in vitro, and the effects of the receptor antagonists were evaluated. Pulmonary fibrosis was also modeled in mice by subcutaneous bleomycin administrations (under light isoflurane anesthesia), and the effects of receptor antagonists on tissue density, collagen-producing cells, myofibroblasts and decorin expression were investigated. In addition, cytokine expression was analyzed in serum. Lung fibroblasts displayed an increased α-SMA (P < 0.05) and total proteoglycan production (P < 0.01) when cultured with TGF-ß1 together with 5-HT, which were significantly reduced with both receptor antagonists. Following treatment with EXT5 or EXT9, tissue density, expression of decorin, number of collagen-producing cells, and myofibroblasts were significantly decreased in vivo compared to bleomycin-treated mice. Receptor antagonization also significantly reduced systemic levels of TNF-α and IL-1ß, indicating a role in systemic inflammation. In conclusion, 5-HT2B receptor antagonists have potential to prevent myofibroblast differentiation, in vitro and in vivo, with subsequent effect on matrix deposition. The attenuating effects of 5-HT2B receptor antagonists on fibrotic tissue remodeling suggest these receptors as novel targets for the treatment of pulmonary fibrosis.

The human zinc finger protein EGR-4 acts as autoregulatory transcriptional repressor.

The human EGR-4 (AT133) gene represents one member of a family of four related zinc finger proteins, that are simultaneously and coordinately induced in resting cells upon growth stimulation. In order to characterise the function of the EGR-4 zinc finger protein, we have expressed the protein in the eukaryotic baculovirus system. The recombinant EGR-4 protein has a molecular mass of 78 kDa, as demonstrated by SDS-PAGE and Western blotting. DNA binding studies revealed that the EGR-4 protein binds to the EGR consensus motif GCGTGGGCG, but not to the G-rich regulatory ZIP-element of the human IL-2 gene, that is a binding site for EGR-1. EGR-4 functions as transcriptional repressor. Overexpression of EGR-4 mediates repression of a minimal c-fos promoter through a threefold EGR consensus site. Furthermore the EGR-4 protein displays autoregulatory activities. This protein downregulates expression of its own gene promoter in a dose dependent manner. A G-rich region in the EGR-4 promoter, located at position -106 to -82, could be identified as binding site for the recombinant EGR-4 protein. A comparison of the two related zinc finger proteins EGR-4 and EGR-1 revealed for each protein distinct and specific DNA binding- and transcriptional regulatory activities.