Leather tanning workers: chromosomal aberrations in peripheral lymphocytes and micronuclei in exfoliated cells in urine

A cytogenetic study was performed on workers of a leather tanning industry. Two different approaches for the biological monitoring of the individuals were used: chromosomal aberration analysis in peripheral lymphocytes and the frequency of micronucleated cells exfoliated in urine samples. 26 men working in the sections considered to present a greater risk were included in the study. Controls were 20 men that were not exposed to chemicals. The percentage of abnormal cells was higher in workers than in controls. Smokers showed higher values of chromosome breaks than non-smokers in both groups. These differences were not statistically significant. The percentage of cells with chromatid and chromosome gaps in workers and controls was different (p less than 0.01). A slight but not significant increase in the mean percentage of micronuclei was observed in the exposed group. We conclude that exposure to chemicals during leather tanning did not produce genotoxic effects measured by chromosomal aberrations in peripheral lymphocytes and micronuclei in urine in this group of workers.     

Altered brain-derived neurotrophic factor blood levels and gene variability are associated with anorexia and bulimia

Murine models and association studies in eating disorder (ED) patients have shown a role for the brain-derived neurotrophic factor (BDNF) in eating behavior. Some studies have shown association of BDNF -270C/T single-nucleotide polymorphism (SNP) with bulimia nervosa (BN), while BDNF Val66Met variant has been shown to be associated with both BN and anorexia nervosa (AN). To further test the role of this neurotrophin in humans, we screened 36 SNPs in the BDNF gene and tested for their association with ED and plasma BDNF levels as a quantitative trait. We performed a family-based association study in 106 ED nuclear families and analyzed BDNF blood levels in 110 ED patients and in 50 sib pairs discordant for ED. The rs7124442T/rs11030102C/rs11030119G haplotype was found associated with high BDNF levels (mean BDNF TCG haplotype carriers = 43.6 ng/ml vs. mean others 23.0 ng/ml, P = 0.016) and BN (Z = 2.64; P recessive = 0.008), and the rs7934165A/270T haplotype was associated with AN (Z =-2.64; P additive = 0.008). The comparison of BDNF levels in 50 ED discordant sib pairs showed elevated plasma BDNF levels for the ED group (mean controls = 41.0 vs. mean ED = 52.7; P = 0.004). Our data strongly suggest that altered BDNF levels modulated by BDNF gene variability are associated with the susceptibility to ED, providing physiological evidence that BDNF plays a role in the development of AN and BN, and strongly arguing for its involvement in eating behavior and body weight regulation.     

The proliferative response of NB69 human neuroblastoma cells to a 50 Hz magnetic field is mediated by ERK1/2 signaling

A number of studies have reported that extremely low frequency magnetic fields (ELF-MF) can modulate proliferative processes in vitro; however, the transduction mechanisms implicated in such phenomena remain to be identified. The present study was aimed to determine whether a 50 Hz, 100 μT MF can induce cell proliferation in the human neuroblastoma line NB69, and whether the signaling pathway MAPK-ERK1/2 (Mitogen-Activated Protein Kinase - Extracellular-Signal-Regulated Kinase 1 and 2) is involved in that proliferative response. The cultures were exposed intermittently or continuously to the MF for a 63-hour duration. The continuous treatment did not induce significant changes in cell proliferation. In contrast, intermittent exposure caused statistically significant increase in the percent of cells in phase S of the cell cycle, followed by a significant increase in cell number. The intermittent treatment also induced an early, transient and repetitive activation of ERK1/2 that could be involved in the proliferative effects. In fact, both the proliferative response and the repeated activation of ERK1/2 were blocked by PD98059, the specific inhibitor of MEK (ERK kinases 1 and 2). Taken together, the described results indicate that a 50 Hz, 100 μT MF can stimulate proliferation in NB69 cells by triggering MAPK-ERK1/ 2 signaling at each of the "On" periods of an intermittent exposure.     

Hypercholesterolemia impaired sperm functionality in rabbits

Hypercholesterolemia represents a high risk factor for frequent diseases and it has also been associated with poor semen quality that may lead to male infertility. The aim of this study was to analyze semen and sperm function in diet-induced hypercholesterolemic rabbits. Twelve adult White New Zealand male rabbits were fed ad libitum a control diet or a diet supplemented with 0.05% cholesterol. Rabbits under cholesterol-enriched diet significantly increased total cholesterol level in the serum. Semen examination revealed a significant reduction in semen volume and sperm motility in hypercholesterolemic rabbits (HCR). Sperm cell morphology was seriously affected, displaying primarily a "folded head"-head fold along the major axe-, and the presence of cytoplasmic droplet on sperm flagellum. Cholesterol was particularly increased in acrosomal region when detected by filipin probe. The rise in cholesterol concentration in sperm cells was determined quantitatively by Gas chromatographic-mass spectrometric analyses. We also found a reduction of protein tyrosine phosphorylation in sperm incubated under capacitating conditions from HCR. Interestingly, the addition of Protein Kinase A pathway activators -dibutyryl-cyclic AMP and iso-butylmethylxanthine- to the medium restored sperm capacitation. Finally, it was also reported a significant decrease in the percentage of reacted sperm in the presence of progesterone. In conclusion, our data showed that diet-induced hypercholesterolemia adversely affects semen quality and sperm motility, capacitation and acrosomal reaction in rabbits; probably due to an increase in cellular cholesterol content that alters membrane related events.     

Control of glycogen deposition

Traditionally, glycogen synthase (GS) has been considered to catalyze the key step of glycogen synthesis and to exercise most of the control over this metabolic pathway. However, recent advances have shown that other factors must be considered. Moreover, the control of glycogen deposition does not follow identical mechanisms in muscle and liver. Glucose must be phosphorylated to promote activation of GS. Glucose-6-phosphate (Glc-6-P) binds to GS, causing the allosteric activation of the enzyme probably through a conformational rearrangement that simultaneously converts it into a better substrate for protein phosphatases, which can then lead to the covalent activation of GS. The potency of Glc-6-P for activation of liver GS is determined by its source, since Glc-6-P arising from the catalytic action of glucokinase (GK) is much more effective in mediating the activation of the enzyme than the same metabolite produced by hexokinase I (HK I). As a result, hepatic glycogen deposition from glucose is subject to a system of control in which the 'controller', GS, is in turn controlled by GK. In contrast, in skeletal muscle, the control of glycogen synthesis is shared between glucose transport and GS. The characteristics of the two pairs of isoenzymes, liver GS/GK and muscle GS/HK I, and the relationships that they establish are tailored to suit specific metabolic roles of the tissues in which they are expressed. The key enzymes in glycogen metabolism change their intracellular localization in response to glucose. The changes in the intracellular distribution of liver GS and GK triggered by glucose correlate with stimulation of glycogen synthesis. The translocation of GS, which constitutes an additional mechanism of control, causes the orderly deposition of hepatic glycogen and probably represents a functional advantage in the metabolism of the polysaccharide.     

Glycogen synthase: towards a minimum catalytic unit?

Classically, alpha-1,4-glucan synthases have been divided into two families, animal/fungal glycogen synthases (GS) and bacterial/plant starch synthases (G(S)S), according to differences in sequence, sugar donor specificity and regulatory mechanisms. Detailed sequence analysis, predicted secondary structure comparison and threading analysis show that these two families are structurally related and that some domains of GSs were acquired to meet regulatory requirements. Archaeal G(S)S present structural and functional features that are conserved in one, the other or both families. Therefore, they are the link between GS and G(S)S and harbor the minimal sequence and structural features that constitute the minimum catalytic unit of the alpha-1,4-glucan synthase superfamily.     

Splice variants of a ClC-2 chloride channel with differing functional characteristics

We identified two ClC-2 clones in a guinea pigintestinal epithelial cDNA library, one of which carries a 30-bpdeletion in the NH₂ terminus. PCR using primersencompassing the deletion gave two products that furthermore wereamplified with specific primers confirming their authenticity. Thecorresponding genomic DNA sequence gave a structure of three exons andtwo introns. An internal donor site occurring within one of the exonsaccounts for the deletion, consistent with alternative splicing.Expression of the variants gpClC-2 and gpClC-277-86 in HEK-293cells generated inwardly rectifying chloride currents with similaractivation characteristics. Deactivation, however, occurred with fasterkinetics in gpClC-277-86. Site-directed mutagenesis suggeststhat a protein kinase C-mediated phosphorylation consensus site lost ingpClC-277-86 is not responsible for the observed change. Thedeletion-carrying variant is found in most tissues examined, and itappears more abundant in proximal colon, kidney, and testis. Thepresence of a splice variant of ClC-2 modified in itsNH₂-terminal domain could have functional consequences intissues where their relative expression levels are different.

     

A new approach for identifying non-pathogenic mutations. An analysis of the cystic fibrosis transmembrane regulator gene in normal individuals

Given q as the global frequency of the alleles causing a disease, any allele with a frequency higher than q minus the cumulative frequency of the previously known disease-causing mutations (threshold) cannot be the cause of that disease. This principle was applied to the analysis of cystic fibrosis transmembrane conductance regulator (CFTR) mutations in order to decide whether they are the cause of cystic fibrosis. A total of 191 DNA samples from random individuals from Italy, France, and Spain were investigated by DGGE (denaturing gradient gel electrophoresis) analysis of all the coding and proximal non-coding regions of the gene. The mutations detected by DGGE were identified by sequencing. The sample size was sufficient to select essentially all mutations with a frequency of at least 0.01. A total of 46 mutations was detected, 20 of which were missense mutations. Four new mutations were identified: 1341+28 C/T, 2082 C/T, L1096R, and I11131V. Thirteen mutations (125 G/C, 875+40 A/G, TTGAn, IVS8-6 5T, IVS8-6 9T, 1525-61 A/G, M470V, 2694 T/G, 3061-65 C/A, 4002 A/G, 4521 G/A, IVS8 TG10, IVS8 TG12) were classified as non-CF-causing alleles on the basis of their frequency. The remaining mutations have a cumulative frequency far exceeding q; therefore, most of them cannot be CF-causing mutations. This is the first random survey capable of detecting all the polymorphisms of the coding sequence of a gene.     

Novel 2-N,N-dimethylaminomethyl-2,3,3a,12b-tetrahydrodibenzo[b,f]furo[2,3-d]oxepin derivatives displaying combined norepinephrine reuptake inhibition and 5-HT2A/2C receptor antagonism

A novel series of cis-fused 2-N,N-dimethylaminomethyl-2,3,3a,12b-tetrahydrodibenzo[b,f]furo[2,3-d]oxepin derivatives modified at position C-11 was prepared and evaluated for its potential antidepressant/anxiolytic properties. In vitro affinities for the norepinephrine transporter and for 5-HT(2A) and 5-HT(2C) receptors, as well as the ED(50) values obtained in some in vivo assays predictive for antidepressant and anxiolytic potential are reported.