Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.     

Antiproliferative effect of D-glucuronyl C5-epimerase in human breast cancer cells

Background  

D-glucuronyl C5-epimerase (GLCE) is one of the key enzymes in the biosynthesis of heparansulfate proteoglycans. Down-regulation of GLCE expression in human breast tumours suggests a possible involvement of the gene in carcinogenesis. In this study, an effect of GLCE ectopic expression on cell proliferation and viability of breast carcinoma cells MCF7 in vitro and its potential molecular mechanisms were investigated.     

Thiocyanate transport in resting and IL-4-stimulated human bronchial epithelial cells: role of pendrin and anion channels

SCN(-) (thiocyanate) is an important physiological anion involved in innate defense of mucosal surfaces. SCN(-) is oxidized by H(2)O(2), a reaction catalyzed by lactoperoxidase, to produce OSCN(-) (hypothiocyanite), a molecule with antimicrobial activity. Given the importance of the availability of SCN(-) in the airway surface fluid, we studied transepithelial SCN(-) transport in the human bronchial epithelium. We found evidence for at least three mechanisms for basolateral to apical SCN(-) flux. cAMP and Ca(2+) regulatory pathways controlled SCN(-) transport through cystic fibrosis transmembrane conductance regulator and Ca(2+)-activated Cl(-) channels, respectively, the latter mechanism being significantly increased by treatment with IL-4. Stimulation with IL-4 also induced the strong up-regulation of an electroneutral SCN(-)/Cl(-) exchange. Global gene expression analysis with microarrays and functional studies indicated pendrin (SLC26A4) as the protein responsible for this SCN(-) transport. Measurements of H(2)O(2) production at the apical surface of bronchial cells indicated that the extent of SCN(-) transport is important to modulate the conversion of this oxidant molecule by the lactoperoxidase system. Our studies indicate that the human bronchial epithelium expresses various SCN(-) transport mechanisms under resting and stimulated conditions. Defects in SCN(-) transport in the airways may be responsible for susceptibility to infections and/or decreased ability to scavenge oxidants.     

Sexually mature and immature yearling male European ground squirrels: a comparison of behavioral and physiological parameters

Early puberty may increase lifetime reproductive success, but may also entail diverse costs. In male European ground squirrels (Spermophilus citellus), age at sexual maturity varies among individuals. We compared sexually active and inactive yearling males under standardized conditions in outdoor enclosures. Non-reproductive yearling males hibernated significantly longer than mature males and emerged when the mating period had already started. Testosterone concentrations peaked in the precocious males during premating and mating, and were depressed in the non-reproductive individuals throughout the study period. Emergence body mass was similar in both groups, although sexually immature males were significantly heavier at the end of the active season. Additionally, non-reproductive yearlings grew faster than precocious individuals. Male–male aggression peaked during mating and the following 4 weeks. Intense aggressive interactions like chases and fights were mostly initiated by reproductive males and were directed towards mature competitors. Aggressive behavior in immature males was mainly expressed as spatial displacements. Non-agonistic interactions with females were only observed in sexually active males. Concentrations of fecal cortisol metabolites did not differ between the two male groups and were elevated in the second half of the active season. Leucocyte numbers were reduced during mating in mature males, whereas in immature animals, cell numbers did not change significantly. Our results indicate that early puberty in European ground squirrels involves lower growth rates, high intrasexual aggression, lower prehibernatory body mass, and probably worse immunological condition during mating.     

Endothelin stimulates vascular hydroxyl radical formation: effect of obesity

Reactive oxygen species (ROS) and endothelin-1 (ET-1) contribute to vascular pathophysiology in obesity. In this context, whether ET-1 modulates hydroxyl radical (*OH) formation and the function of ROS/*OH in obesity is not known. In the present study, formation and function of ROS, including *OH, were investigated in the aorta of lean and leptin-deficient obese ob/ob mice. Hydroxyl radical formation was detected ex vivo using terephthalic acid in intact aortic rings and the involvement of ROS in ET-1-mediated vasoreactivity was analyzed using the antioxidant EPC-K1, a combination of alpha-tocopherol and ascorbic acid. Generation of either *OH, *O(2)(-), and H(2)O(2) was strongly inhibited by EPC-K1 (all P < 0.05). In obese mice, basal vascular *OH formation and ROS activity were reduced by 3-fold and 5-fold, respectively (P < 0.05 vs. lean). ET-1 markedly enhanced *OH formation in lean (6-fold, P < 0.05 vs. untreated) but not in obese mice. Obesity increased ET-1-induced contractions (P < 0.05 vs. lean), and ROS scavenging further enhanced the response (P < 0.05 vs. untreated). Exogenous ROS, including *OH caused stronger vasodilation in obese animals (P < 0.05 vs. lean), whereas endothelium-dependent relaxation was similar between lean and obese animals. In conclusion, we present a sensitive method allowing ex vivo measurement of vascular *OH generation and provide evidence that ET-1 regulates vascular *OH formation. The data indicate that in obesity, vascular formation of ROS, including *OH is lower, whereas the sensitivity to ROS is increased, suggesting a novel and important role of ROS, including *OH in the regulation of vascular tone in disease status associated with increased body weight.     

Effect of whole body vibration training on lower limb performance in selected high-level ballet students

The aim of this study was to examine the effects of 8 weeks of whole body vibration (WBV) training on vertical jump ability (CMJ) and knee-extensor performance at selected external loads (50, 70, and 100 kg; leg-press exercise) in elite ballerinas. Twenty-two (age, 21.25 +/- 1.5 years) full-time ballerinas were assigned randomly to the experimental (E, n = 11) and control (C, n = 11) groups. The experimental group was submitted to WBV training 3 times per week before ballet practice. During the training period, the E and C groups undertook the same amount of ballet practice. Posttraining CMJ performance significantly increased in E group (6.3 +/- 3.8%, p < 0.001). Furthermore, E group showed significant (p < 0.05-0.001) posttraining average leg-press power and velocity improvements at all the external loads considered. Consequently, the force-velocity and power-velocity relationship shifted to the right after WBV training in the E group. The results of the present study show that WBV training is an effective short-term training methodology for inducing improvements in knee-extensor explosiveness in elite ballerinas.     

Leishmanicidal activity of a supercritical fluid fraction obtained from Tabernaemontana catharinensis

The branches and leaves of Tabernaemontana catharinensis were extracted with supercritical fluid using a mixture of CO(2) plus ethanol (SFE), and the indole alkaloid enriched fraction (AF3) was selected for anti-Leishmania activity studies. We found that AF3 exhibits a potent effect against intracellular amastigotes of Leishmania amazonensis, a causative agent of New World cutaneous leishmaniasis. AF3 inhibits Leishmania survival in a dose-dependent manner, and reached 88% inhibition of amastigote growth at 100 microg/mL. The anti-parasite effect was independent of nitric oxide (NO), since AF3 was able to inhibit NO production induced by IFN-gamma plus LPS. In addition, AF3 inhibited TGF-beta production, which could have facilitated AF3-mediated parasite killing. The AF3 fraction obtained from SFE was nontoxic for host macrophages, as assessed by plasma membrane integrity and mitochondrial activity. We conclude that SFE is an efficient method for obtaining bioactive indole alkaloids from plant extracts. Importantly, this method preserved the alkaloid properties associated with inhibition of Leishmania growth in macrophages without toxicity to host cells.