Gas phase dimerization of neuropeptide head activator analogs useful for the noncovalent constraint of peptides

We have used electrospray mass spectrometry to examine the dimerization of EFLIVKS, a reversed sequence analog of part of the neuropeptide head activator, and other similar analogs. Observation of the EFLIVKS gas phase dimer is concentration-dependent, with a half-saturation concentration for relative dimer formation of 7.8 microM, similar to that of SKVILFE of 12 microM. The lowest energy conformers from quenched molecular dynamics simulations suggest EFLIVKS may dimerize in the gas phase by formation of multiple ion pairs across the dimer interface. Alanine-scan mutants also dimerize in the gas phase, with replacement of the interior residues FLIVK diminishing dimerization. The concentration-dependence of the EFLIVKS circular dichroism spectrum at pH 7.5 suggests the existence of different conformation states at different concentrations, but does not provide evidence supporting the saturable dimer formation in solution. Different analogs of EFLIVKS, when fused to each end of a 18mer unfolded peptide, induce solution structures with T(m)s of 42-50 degrees C. These peptides and analogs may thus be useful for the noncovalent constraint of peptides and peptide library members used in cellular screens.     

A fluorescent GTP analog as a specific, high-precision label of microtubules

Fluorescent imaging of cytoskeletal structures permits studies of both organization within the cell and dynamic reorganization of the cytoskeleton itself. Traditional fluorescent labels of microtubules, part of the cytoskeleton, have been used to study microtubule localization, structure, and dynamics, both in vivo and in vitro. However, shortcomings of existing labels make imaging of microtubules with high precision light microscopy difficult. In this paper, we report a new fluorescent labeling technique for microtubules, which involves a GTP analog modified with a bright, organic fluorophore (TAMRA, Cy3, or Cy5). This fluorescent GTP binds to a specific site, the exchangeable site, on tubulin in solution with a dissociation constant of 1.0±0.4 μM. Furthermore, the label becomes permanently incorporated into the microtubule lattice once tubulin polymerizes. We show that this label is usable as a single molecule fluorescence probe with nanometer precision and expect it to be useful for modern subdiffraction optical microscopy of microtubules and the cytoskeleton.     

Shotgun DNA sequencing using cloned DNase I-generated fragments.

A method for DNA sequencing has been developed that utilises libraries of cloned randomly-fragmented DNA. The DNA to be sequenced is first subjected to limit attach by a non-specific endonuclease (DNase I in the presence of Mn++), fractionated by size and cloned in a single-stranded phage vector. Clones are then picked at random and used to provide a template for sequencing by the dideoxynucleotide chain termination method. This technique was used to sequence completely a 4257 bp EcoRI fragment of bovine mitochondrial DNA. The cloned fragments were evenly distributed with respect to the EcoRI fragment, and completion of the entire sequence required the construction of only a single library. In general, once a clone library has been prepared, the speed of this approach (greater than 1000 nucleotides of randomly selected sequence per day) is limited mainly by the rate at which the data can be processed. Because the clones are selected randomly, however, the average amount of new sequence information per clone is substantially diminished as the sequence near completion.     

Variability in the Effect of 5-HTTLPR on Depression in a Large European Population: The Role of Age,Symptom Profile,Type and Intensity of Life Stressors

Background

Although 5-HTTLPR has been shown to influence the risk of life stress-induced depression in the majority of studies, others have produced contradictory results, possibly due to weak effects and/or sample heterogeneity.

Methods

In the present study we investigated how age, type and intensity of life-stressors modulate the effect of 5-HTTLPR on depression and anxiety in a European population cohort of over 2300 subjects. Recent negative life events (RLE), childhood adversity (CHA), lifetime depression, Brief Symptoms Inventory (BSI) depression and anxiety scores were determined in each subject. Besides traditional statistical analysis we calculated Bayesian effect strength and relevance of 5-HTTLPR genotypes in specified models.

Results

The short (s) low expressing allele showed association with increased risk of depression related phenotypes, but all nominally significant effects would turn to non-significant after correction for multiple testing in the traditional analysis. Bayesian effect strength and relevance analysis, however, confirmed the role of 5-HTTLPR. Regarding current (BSI) and lifetime depression 5-HTTLPR-by-RLE interactions were confirmed. Main effect, with other words direct association, was supported with BSI anxiety. With more frequent RLE the prevalence or symptoms of depression increased in ss carriers. Although CHA failed to show an interaction with 5-HTTLPR, in young subjects CHA sensitized towards the depression promoting effect of even mild RLE. Furthermore, the direct association of anxiety with the s allele was driven by young (≤30) individuals.

Limitations

Our study is cross-sectional and applies self-report questionnaires.

Conclusions

Albeit 5-HTTLPR has only weak/moderate effects, the s allele is directly associated with anxiety and modulates development of depression in homogeneous subgroups.     

Trace metal bioavailability in municipal solid waste and sewage sludge composts

A sequential extraction procedure was employed to determine the soil fractions, and assess plant availability of Cr, Cu, Ni, Pb and Zn in a Glynwood silt loam amended with five rates (0, 30, 60, 120 and 240 metric tons/ha) of composted municipal solid waste (CMSW) or composted sewage sludge (CSS) cropped to oats (Avena sativa). The application of the composts tended to shift the solid phase forms of the metals away from those extractable with HNO₃ to those extractable with NaOH and EDTA. The more labile fractions (KNO₃ and H₂O extracts) of the metals typically decreased with application of CMSW and CSS. Crop dry matter increased at the 30 and 60 MT/ha CMSW rates by 142 and 152%, respectively, after which yields declined to below control values. Yields at all rates of CSS declined. The CMSW and CSS had an insignificant effect on concentrations of Cr and Pb in oat tissue, but tissue levels of Cu, Ni and Zn increased with increased rate of compost application.     

Canopy interactions and physical stress gradients in subtidal communities

Species interactions are integral drivers of community structure and can change from competitive to facilitative with increasing environmental stress. In subtidal marine ecosystems, however, interactions along physical stress gradients have seldom been tested. We observed seaweed canopy interactions across depth and latitudinal gradients to test whether light and temperature stress structured interaction patterns. We also quantified interspecific and intraspecific interactions among nine subtidal canopy seaweed species across three continents to examine the general nature of interactions in subtidal systems under low consumer pressure. We reveal that positive and neutral interactions are widespread throughout global seaweed communities and the nature of interactions can change from competitive to facilitative with increasing light stress in shallow marine systems. These findings provide support for the stress gradient hypothesis within subtidal seaweed communities and highlight the importance of canopy interactions for the maintenance of subtidal marine habitats experiencing environmental stress.     

Epithelial heterogeneity in the murine thymus: fucose-specific lectins bind medullary epithelial cells

By means of histochemical techniques, two lectins with nominal specificity for L-fucose, Tetragonolobus purpureas agglutinin (TPA), and Ulex europeus agglutinin (UEA) were found to specifically label the medullary area of murine thymuses. Although the binding of both lectins was restricted to the medullary area of the thymus, each staining pattern was unique. Cells binding UEA formed a reticular network throughout the medulla, whereas cells binding TPA occurred as single cells or small clumps of cells and resembled Hassall's corpuscles. The cells binding either lectin were identified as epithelial on the basis of ultrastructural features (tonofilaments, desmosomes, and keratohyalin bodies) and resembled Ia+ medullary epithelial cells described previously. An age-related decline in UEA binding was observed, whereas labeling with TPA remained unchanged. On the basis of the labeling patterns obtained with UEA and TPA and the reported specificities of these two lectins, it is suggested that the majority of the fucose detected is associated with type 1 carbohydrate chains.