An assessment of swinger techniques for the playground swing oscillatory motion

Much attention has been devoted to how playground swing amplitudes are built up by swinger techniques, i.e. body actions. However, very little attention has been given to the requirements that such swinger techniques place on the swinger himself. The purpose of this study was to find out whether different swinger techniques yield significantly different maximum torques, endurance and coordinative skills, and also to identify preferable techniques. We modelled the seated swinger as a rigid dumbbell and compared three different techniques. A series of computer simulations were run with each technique, testing the performance with different body rotational speeds, delayed onset of body rotation and different body mass distributions, as swing amplitudes were brought up towards 90°. One technique was found to be extremely sensitive to the timing of body actions, limiting swing amplitudes to 50° and 8° when body action was delayed by 0.03 and 0.3?s, respectively. Two other more robust techniques reached 90° even with the largest of these delays, although more time (and endurance) was needed. However, these two methods also differed with respect to maximum torque and endurance, and none was preferable in both these aspects, being dependent on the swinger goals and abilities.     

Putative drug binding conformations of monoamine transporters

Structural information about monoamine transporters and their interactions with psychotropic drugs is important for understanding their molecular mechanisms of action and for drug development. The crystal structure of a Major Facilitator Superfamily (MFS) transporter, the lactose permease symporter (lac permease), has provided insight into the three-dimensional structure and mechanisms of secondary transporters. Based on the hypothesis that the 12 transmembrane alpha-helix (TMH) secondary transporters belong to a common folding class, the lac permease structure was used for molecular modeling of the serotonin transporter (SERT), the dopamine transporter (DAT), and the noradrenaline transporter (NET). The molecular modeling methods used included amino acid sequence alignment, homology modeling, and molecular mechanical energy calculations. The lac permease crystal structure has an inward-facing conformation, and construction of outward-facing SERT, DAT, and NET conformations allowing ligand binding was the most challenging step of the modeling procedure. The psychomotor stimulants cocaine and S-amphetamine, and the selective serotonin reuptake inhibitor (SSRI) S-citalopram, were docked into putative binding sites on the transporters to examine their molecular binding mechanisms. In the inward-facing conformation of SERT the translocation pore was closed towards the extracellular side by hydrophobic interactions between the conserved amino acids Phe105, Pro106, Phe117, and Ala372. An unconserved amino acid, Asp499 in TMH10 in NET, may contribute to the low affinity of S-citalopram to NET.     

Secretion of proteases in serglycin transfected Madin-Darby canine kidney cells

Madin-Darby canine kidney (MDCK) cells, which do not normally express the proteoglycan (PG) serglycin, were stably transfected with cDNA for human serglycin fused to a polyhistidine tag (His-tag). Clones with different levels of serglycin mRNA expression were generated. One clone with lower and one with higher serglycin mRNA expression were selected for this study. 35S-labelled serglycin in cell fractions and conditioned media was isolated using HisTrap affinity chromatography. Serglycin could also be detected in conditioned media using western blotting. To investigate the possible importance of serglycin linked to protease secretion, enzyme activities using chromogenic substrates and zymography were measured in cell fractions and serum-free conditioned media of the different clones. Cells were cultured in both the absence and presence of phorbol 12-myristate 13-acetate (PMA). In general, enzyme secretion was strongly enhanced by treatment with PMA. Our analyses revealed that the clone with the highest serglycin mRNA expression, level of HisTrap isolated 35S-labelled serglycin, and amount of serglycin core protein as detected by western blotting, also showed the highest secretion of proteases. Transfection of serglycin into MDCK cells clearly leads to changes in secretion levels of secreted endogenous proteases, and could provide further insight into the biosynthesis and secretion of serglycin and potential partner molecules.     

Quantitation of heteroplasmy of mtDNA sequence variants identified in a population of AD patients and controls by array-based resequencing

The role of mitochondrial dysfunction in the pathogenesis of Alzheimer's disease (AD) has been well documented. Though evidence for the role of mitochondria in AD seems incontrovertible, the impact of mitochondrial DNA (mtDNA) mutations in AD etiology remains controversial. Though mutations in mitochondrially encoded genes have repeatedly been implicated in the pathogenesis of AD, many of these studies have been plagued by lack of replication as well as potential contamination of nuclear-encoded mitochondrial pseudogenes. To assess the role of mtDNA mutations in the pathogenesis of AD, while avoiding the pitfalls of nuclear-encoded mitochondrial pseudogenes encountered in previous investigations and showcasing the benefits of a novel resequencing technology, we sequenced the entire coding region (15,452 bp) of mtDNA from 19 extremely well-characterized AD patients and 18 age-matched, unaffected controls utilizing a new, reliable, high-throughput array-based resequencing technique, the Human MitoChip. High-throughput, array-based DNA resequencing of the entire mtDNA coding region from platelets of 37 subjects revealed the presence of 208 loci displaying a total of 917 sequence variants. There were no statistically significant differences in overall mutational burden between cases and controls, however, 265 independent sites of statistically significant change between cases and controls were identified. Changed sites were found in genes associated with complexes I (30.2%), III (3.0%), IV (33.2%), and V (9.1%) as well as tRNA (10.6%) and rRNA (14.0%). Despite their statistical significance, the subtle nature of the observed changes makes it difficult to determine whether they represent true functional variants involved in AD etiology or merely naturally occurring dissimilarity. Regardless, this study demonstrates the tremendous value of this novel mtDNA resequencing platform, which avoids the pitfalls of erroneously amplifying nuclear-encoded mtDNA pseudogenes, and our proposed analysis paradigm, which utilizes the availability of raw signal intensity values for each of the four potential alleles to facilitate quantitative estimates of mtDNA heteroplasmy. This information provides a potential new target for burgeoning diagnostics and therapeutics that could truly assist those suffering from this devastating disorder.     

Genetic population structure of Euphausia superba DANA in the Atlantic Sector of the Southern Ocean as demonstrated by different electrophoretic techniques

Summary Electrophoretic analyses of allele distributions at nine polymorphic gene loci were conducted in samples of Euphausia superba from the Bransfield Strait, off the South Orkney Islands, and from the south-eastern part of the Weddell Sea. The aim was to determine whether reported phenotypic differences between krill stocks from these areas could be linked to genotypic differences. Despite minor deviations of genotype distributions from Hardy-Weinberg expectations, the data gave no evidence of genetic heterogeneity over the sampled area, and the hypothesis of a single genetically homogeneous krill population could not be rejected. Genetic data for four selected loci were verified by using two different electrophoretic methods. Results from these two techniques yielded no discrepancies in interpretation of the data.     

In vivo analysis of the regulatory genes in the nystatin biosynthetic gene cluster of Streptomyces noursei ATCC 11455 reveals their differential control over antibiotic biosynthesis

Six putative regulatory genes are located at the flank of the nystatin biosynthetic gene cluster in Streptomyces noursei ATCC 11455. Gene inactivation and complementation experiments revealed that nysRI, nysRII, nysRIII, and nysRIV are necessary for efficient nystatin production, whereas no significant roles could be demonstrated for the other two regulatory genes. To determine the in vivo targets for the NysR regulators, chromosomal integration vectors with the xylE reporter gene under the control of seven putative promoter regions upstream of the nystatin structural and regulatory genes were constructed. Expression analyses of the resulting vectors in the S. noursei wild-type strain and regulatory mutants revealed that the four regulators differentially affect certain promoters. According to these analyses, genes responsible for initiation of nystatin biosynthesis and antibiotic transport were the major targets for regulation. Data from cross-complementation experiments showed that nysR genes could in some cases substitute for each other, suggesting a functional hierarchy of the regulators and implying a cascade-like mechanism of regulation of nystatin biosynthesis.     

Ilimaquinone inhibits gap-junctional communication prior to Golgi fragmentation and block in protein transport

Brefeldin A and ilimaquinone are compounds known to affect Golgi structure and function. In particular, the transport of proteins is blocked either at the level of exit from endoplasmic reticulum (brefeldin) or at cis-Golgi (ilimaquinone). Brefeldin caused a slow decrease in gap-junctional communication and a slow loss of all phosphorylated forms of connexin43 in hamster and rat fibroblasts, while ilimaquinone caused an abrupt decrease in gap-junctional communication and rapid loss of only the slowest migrating phosphorylated connexin43 band (P2). Ilimaquinone caused these effects prior to any significant Golgi fragmentation, especially in hamster fibroblasts. Concurrently, ilimaquinone minimally affected protein secretion, while brefeldin caused an instantaneous decrease. These results show that ilimaquinone inhibits gap-junctional communication in connexin43-expressing cells by a mechanism not dependent on Golgi fragmentation or block in protein transport.     

The Pseudomonas fluorescens AlgG protein,but not its mannuronan C-5-epimerase activity,is needed for alginate polymer formation

Bacterial alginates are produced as 1-4-linked beta-D-mannuronan, followed by epimerization of some of the mannuronic acid residues to alpha-L-guluronic acid. Here we report the isolation of four different epimerization-defective point mutants of the periplasmic Pseudomonas fluorescens mannuronan C-5-epimerase AlgG. All mutations affected amino acids conserved among AlgG-epimerases and were clustered in a part of the enzyme also sharing some sequence similarity to a group of secreted epimerases previously reported in Azotobacter vinelandii. An algG-deletion mutant was constructed and found to produce predominantly a dimer containing a 4-deoxy-L-erythro-hex-4-enepyranosyluronate residue at the nonreducing end and a mannuronic acid residue at the reducing end. The production of this dimer is the result of the activity of an alginate lyase, AlgL, whose in vivo activity is much more limited in the presence of AlgG. A strain expressing both an epimerase-defective (point mutation) and a wild-type epimerase was constructed and shown to produce two types of alginate molecules: one class being pure mannuronan and the other having the wild-type content of guluronic acid residues. This formation of two distinct classes of polymers in a genetically pure cell line can be explained by assuming that AlgG is part of a periplasmic protein complex.