Cysteine-scanning Analysis of Helices TM8, TM9a,and TM9b and Intervening Loops in the YgfO Xanthine Permease: A CARBOXYL GROUP IS ESSENTIAL AT ASP-276

Bacterial and fungal members of the ubiquitous nucleobase-ascorbate transporter (NAT/NCS2) family use the NAT signature motif, a conserved 11-amino acid sequence between amphipathic helices TM9a and TM9b, to define function and selectivity of the purine binding site. To examine the role of flanking helices TM9a, TM9b, and TM8, we employed Cys-scanning analysis of the xanthine-specific homolog YgfO from Escherichia coli. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequences ²⁵⁹FLVVGTIYLLSVLEAVGDITATAMVSRRPIQGEEYQSRLKGGVLADGLVSVIASAV³¹⁴ and ³⁴²TIAVMLVILGLFP³⁵⁴ including these TMs (underlined) was replaced individually with Cys, except the irreplaceable Glu-272 and Asp-304, which had been studied previously. Of 67 single Cys mutants, 55 accumulate xanthine to 35–140% of the steady state observed with C-less, five (I265C, D276C, I277C, G299C, L350C) accumulate to low levels (10–20%) and seven (T278C, A279C, T280C, A281C, G305C, G351C, P354C) show negligible expression in the membrane. Extensive mutagenesis reveals that a carboxyl group is needed at Asp-276 for high activity and that D276E differs from wild type as it recognizes 8-methylxanthine (K_i 79 μm) but fails to recognize 2-thioxanthine, 3-methylxanthine or 6-thioxanthine; bulky replacements of Ala-279 or Thr-280 and replacements of Gly-305, Gly-351, or Pro-354 impair activity or expression. Single Cys mutants V261C, A273C, G275C, and S284C are sensitive to inactivation by N-ethylmaleimide and sensitivity of G275C (IC₅₀ 15 μm) is enhanced in the presence of substrate. The data suggest that residues crucial for the transport mechanism cluster in two conserved motifs, at the cytoplasmic end of TM8 (EXXGDXXAT) and in TM9a (GXXXDG).     

Multiscale Hy3S: Hybrid stochastic simulation for supercomputers

Background

DNA copy number alterations are one of the main characteristics of the cancer cell karyotype and can contribute to the complex phenotype of these cells. These alterations can lead to gains in cellular oncogenes as well as losses in tumor suppressor genes and can span small intervals as well as involve entire chromosomes. The ability to accurately detect these changes is central to understanding how they impact the biology of the cell.

Results

We describe a novel algorithm called CARAT (Copy Number Analysis with Regression And Tree) that uses probe intensity information to infer copy number in an allele-specific manner from high density DNA oligonuceotide arrays designed to genotype over 100, 000 SNPs. Total and allele-specific copy number estimations using CARAT are independently evaluated for a subset of SNPs using quantitative PCR and allelic TaqMan reactions with several human breast cancer cell lines. The sensitivity and specificity of the algorithm are characterized using DNA samples containing differing numbers of X chromosomes as well as a test set of normal individuals. Results from the algorithm show a high degree of agreement with results from independent verification methods.

Conclusion

Overall, CARAT automatically detects regions with copy number variations and assigns a significance score to each alteration as well as generating allele-specific output. When coupled with SNP genotype calls from the same array, CARAT provides additional detail into the structure of genome wide alterations that can contribute to allelic imbalance.     

Dipeptides containing the alpha-aminoisobutyric residue (Aib) as ligands: preparation,spectroscopic studies and crystal structures of copper(II) complexes with H-Aib-X-OH (X=Gly,L-Leu,L-Phe)

Synthetic procedures are described that allow access to new copper(II) complexes with dipeptides containing the alpha-aminoisobutyric residue (Aib) as ligands. The solid complexes [Cu(H(-1)L(A))](n).nH(2)O (1) (L(A)H=H-Aib-Gly-OH), [Cu(H(-1)L(B))(MeOH)](n).nMeOH (2) (L(B)H=H-Aib-L-Leu-OH) and [Cu(H(-1)L(C))](n) (3) (L(C)H=H-Aib-L-Phe-OH) have been isolated and characterized by single-crystal X-ray crystallography, solid-state IR spectra and UV-Vis spectroscopy in solution (H(-1)L(2-) is the dianionic form of the corresponding dipeptide). Complexes 1 and 3 are three-dimensional coordination polymers with similar structures. The doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate), O'(carboxylate), O(peptide) mu(3) ligand and binds to one Cu(II) atom at its amino and peptide nitrogens and at one carboxylate oxygen, to a second metal at the other carboxylate oxygen, while a third Cu(II) atom is attached to the peptide oxygen. The geometry around copper(II) is distorted square pyramidal with the peptide oxygen at the apex of the pyramid. The structure of 2 consists of zigzag polymeric chains, where the doubly deprotonated dipeptide behaves as a N(amino), N(peptide), O(carboxylate), O'(carboxylate) mu(2) ligand. The geometry at copper(II) is square pyramidal with the methanol oxygen at the apex. The IR data are discussed in terms of the nature of bonding and known structures. The UV-Vis spectra show that the solid-state structures of 1, 2 and 3 do not persist in H(2)O.     

Chemical composition and biological activity of Nepeta parnassica oils and isolated nepetalactones

Essential oils of Nepeta parnassica, collected at different developmental stages, were analyzed by means of GC/MS. From the fifty-five identified constituents in samples A and B, representing 94.8% and 98.7% of the oils respectively, 4a(alpha),7alpha,7alpha(beta)-nepetalactone (22.0%), 1,8-cineole (21.1%), alpha-pinene (9.5%) and 4a(alpha),7,beta,7alpha(beta)-nepetalactone (7.9%) were the major components of sample A (vegetative stage), whereas in sample B (flowering stage) the main contributors were 1,8-cineole (34.6%), 4a(alpha),7alpha,7a(alpha)-nepetalactone (17.3%), alpha-pinene (11.4%) and 4a(alpha),7alpha,7alpha(beta)-nepetalactone (8.9%). The oils were tested on human health important insects such as the Pogonomyrmex sp. ants and the Culex pipiens molestus mosquitoes with promising results on insect repellency/toxicity.     

Cytotoxic lupane-type triterpenoids from Acacia mellifera

One new and eight previously described lupane-type metabolites were isolated for the first time from Acacia mellifera (Leguminosae). Based on spectral analyses, the structure of the new compound was elucidated as 28-hydroxy-3-oxo-lup-20-(29)-en-30-al (1), while the known compounds were identified as 3-oxo-lup-20-(29)-en-30-al (2), 3-hydroxy-lup-20-(29)-en-30-al (3), 28-hydroxy-lup-20-(29)-en-3-one (4), lupenone (5), lupeol (6), betulin (7), betulinic acid (8), and betulonic acid (9). Metabolites 2, 3, and 4 are reported for the first time in the Leguminosae family. The cytotoxicity of the isolated metabolites was evaluated on the NSCLC-N6 cell line, derived from a human non-small-cell bronchopulmonary carcinoma. Compounds 1 and 3 exhibited significant levels of activity.     

Solution conformation of the antibody-bound tyrosine phosphorylation site of the nicotinic acetylcholine receptor beta-subunit in its phosphorylated and nonphosphorylated states

Phosphorylation of the acetylcholine receptor (AChR) seems to be responsible for triggering several effects including its desensitization and aggregation at the postsynaptic membrane and probably initiates a signal transduction pathway at the postsynaptic membrane. To study the structural and functional role of the tyrosine phosphorylation site of the AChR beta-subunit and contribute to the in-depth understanding of the structural basis of the ion channel function, we synthesized four peptides containing the phosphorylated and nonphosphorylated sequences (380-391) of the human and Torpedo AChR beta-subunits and studied their interaction with a monoclonal antibody (mAb 148) that is known to bind to this region and that is capable of blocking ion channel function. All four peptides were efficient inhibitors of mAb 148 binding to AChR, although the nonphosphorylated human peptide was considerably less effective than the three others. We then investigated the conformation acquired by all four peptides in their antibody-bound state, which possibly illustrates the local conformation of the corresponding sites on the intact AChR molecule. The phosphorylated human and Torpedo peptides adopted a distorted 3(10) helix conformation. The nonphosphorylated Torpedo peptide, which is also an efficient inhibitor, was also folded. In contrast, the nonphosphorylated human peptide (a less efficient inhibitor) presented an extended structure. It is concluded that the phosphorylation of the AChR at its beta-subunit Tyr site leads to a significant change in its conformation, which may affect several functions of the AChR.     

Effect of a submaximal half-squats warm-up program on vertical jumping ability

The purpose of the current research was to study the effect of a warm-up program including submaximal half-squats on vertical jumping ability. Twenty physically active men participated in the study. Each subject performed 5 sets of half-squats with 2 repetitions at each of the following intensities: 20, 40, 60, 80, and 90% of the 1 repetition maximum (1RM) load. Prior to the first set and immediately after the end of the last set, the subjects performed 2 countermovement jumps on a Kistler force platform; the primary goal was to jump as high as possible. The results showed that mean vertical jumping ability improved by 2.39% after the warm-up period. Subjects were then divided into 2 groups according to their 1RM values for the half-squat. Subjects with greater maximal strength ability improved their vertical jumping ability (4.01%) more than did subjects with lower maximal strength (0.42%). A warm-up protocol including half-squats with submaximal loads and explosive execution can be used for short-term improvements of vertical jumping performance, and this effect is greater in athletes with a relatively high strength ability.     

Comparative 3-dimensional kinematic analysis of the snatch technique in elite male and female greek weightlifters

The purpose of the current research was the comparison of the snatch technique between elite male and female weightlifters. Two S-VHS cameras operating at 60 fields per second were used to record the snatch lifts of 6 male and 6 female Greek weightlifters under competitive conditions. The spatial coordinates of selected points on the body and the barbell were calculated using the direct linear transformation procedure, and the raw data were digitally filtered with a cutoff frequency of 4 Hz. Analyses of variance for dependent and independent samples were used to compare the selected variables in men with the corresponding variables in women. The results revealed that women flexed their knees significantly less and slower than men did during the transition phase (p < 0.05). Women also dropped under the barbell during the turnover and catch phases significantly less and slower than men did (p < 0.05). Moreover, the external mechanical work for the vertical displacement of the barbell in men was significantly greater in the first pull than in the second pull (p < 0.05). In contrast, women showed similar work outputs in the 2 phases. These differences between the 2 sexes might be because of the lower skill level of women in comparison with men, which is partly because of the recent participation of women in weightlifting.