Steady-state and time-resolved fluorescence studies indicate an unusual conformation of 2-aminopurine within ATAT and TATA duplex DNA sequences

2-Aminopurine (2-AP), a fluorescent analog of adenine, has been widely used as a probe for local DNA conformation, since excitation and emission characteristics and fluoresence lifetimes of 2-AP vary in a sequence-dependent manner within DNA. Using steady-state and time-resolved fluorescence techniques, we report that 2-AP appears to be unusually stacked in the internal positions of ATAT and TATA in duplex DNA. The excitation wavelength maxima for 2-AP within these contexts were red shifted, indicating reduced solvent exposure for the fluorophore. Furthermore, in these contexts, 2-AP fluorescence was resistant to acrylamide-dependent collisional quenching, suggesting that the fluorophore is protected by its stacked position within the duplex. This conclusion was further reinforced by the presence of a secondary peak at 275 nm in the fluorescence excitation spectra that is indicative of efficient excitation energy transfer from nearby non-fluorescent DNA bases. Fluorescence anisotropy decay and internal angular ‘wobbling’ motion measurements of 2-AP within these alternating AT contexts were also consistent with the fluorophore being highly constrained and immobile within the base stack. When these fluorescence characteristics are compared with those of 2-AP within other duplex DNA sequence contexts, they are unique.     

Affinity chromatography of neoglycoproteins

Glycoproteins, as a class of biomolecules, exhibit much more heterogeneous structures than non-glycosylated proteins. They present a challenging area of research. Model glycoproteins with well-defined protein and carbohydrate structures are helpful in the search for high-resolution methods for the separation of glycoproteins. Neoglycoproteins, maltose-modified chymotrypsin and lactose-modified chymotrypsin, were synthesised by modifying chymotrypsin with maltose and lactose, respectively, using the reductive amination method. Boronate chromatography was applied to isolate the neoglycoproteins from non-glycosylated substances. The use of Tris–HCl as a shielding reagent during the boronate chromatography proved to be efficient in eliminating unwanted interactions between the boronate ligand and the peptide backbone of chymotrypsin. The retention time of neoglycoproteins on the boronate column was increased with increasing the degree of modification.     

Intraindividual and Interspecies Variation in the 5S rDNA of Coregonid Fish

This study was designed to characterize further the nontranscribed intergenic spacers (NTSs) of the 5S rRNA genes of fish and evaluate this marker as a tool for comparative studies. Two members of the closely related North American Great Lakes cisco species complex (Coregonus artedi and C. zenithicus) were chosen for comparison. Fluorescence in situ hybridization found the ciscoes to have a single multicopy 5S locus located in a C band-positive region of the largest submetacentric chromosome. The entire NTS was amplified from the two species by polymerase chain reaction with oligonucleotide primers anchored in the conserved 5S coding region. Complete sequences were determined for 25 clones from four individuals representing two discrete NTS length variants. Sequence analysis found the length variants to result from presence of a 130-bp direct repeat. No two sequences from a single fish were identical. Examination of sequence from the coding region revealed two types of 5S genes in addition to pseudogenes. This suggests the presence of both somatic and germline (oocyte) forms of the 5S gene in the genome of Coregonus. The amount of variation present among NTS sequences indicates that accumulation of variation (mutation) is greater in this multicopy gene than is gene conversion (homogenization). The high level of sequence variation makes the 5S NTS an inappropriate DNA sequence for comparisons of closely related taxa. Received: 22 August 1997 / Accepted: 31 October 1997     

Widespread Changes in White Matter Microstructure after a Day of Waking and Sleep Deprivation

BackgroundElucidating the neurobiological effects of sleep and waking remains an important goal of the neurosciences. Recently, animal studies indicated that sleep is important for cell membrane and myelin maintenance in the brain and that these structures are particularly susceptible to insufficient sleep. Here, we tested the hypothesis that a day of waking and sleep deprivation would be associated with changes in diffusion tensor imaging (DTI) indices of white matter microstructure sensitive to axonal membrane and myelin alterations.MethodsTwenty-one healthy adult males underwent DTI in the morning [7:30AM; time point (TP)1], after 14 hours of waking (TP2), and then after another 9 hours of waking (TP3). Whole brain voxel-wise analysis was performed with tract based spatial statistics.ResultsA day of waking was associated with widespread increases in white matter fractional anisotropy, which were mainly driven by radial diffusivity reductions, and sleep deprivation was associated with widespread fractional anisotropy decreases, which were mainly explained by reductions in axial diffusivity. In addition, larger decreases in axial diffusivity after sleep deprivation were associated with greater sleepiness. All DTI changes remained significant after adjusting for hydration measures.ConclusionsThis is the first DTI study of sleep deprivation in humans. Although previous studies have observed localized changes in DTI indices of cerebral microstructure over the course of a few hours, further studies are needed to confirm widespread DTI changes within hours of waking and to clarify whether such changes in white matter microstructure serve as neurobiological substrates of sleepiness.     

The Dynamics of Incomplete Lineage Sorting across the Ancient Adaptive Radiation of Neoavian Birds

The diversification of neoavian birds is one of the most rapid adaptive radiations of extant organisms. Recent whole-genome sequence analyses have much improved the resolution of the neoavian radiation and suggest concurrence with the Cretaceous-Paleogene (K-Pg) boundary, yet the causes of the remaining genome-level irresolvabilities appear unclear. Here we show that genome-level analyses of 2,118 retrotransposon presence/absence markers converge at a largely consistent Neoaves phylogeny and detect a highly differential temporal prevalence of incomplete lineage sorting (ILS), i.e., the persistence of ancestral genetic variation as polymorphisms during speciation events. We found that ILS-derived incongruences are spread over the genome and involve 35% and 34% of the analyzed loci on the autosomes and the Z chromosome, respectively. Surprisingly, Neoaves diversification comprises three adaptive radiations, an initial near-K-Pg super-radiation with highly discordant phylogenetic signals from near-simultaneous speciation events, followed by two post-K-Pg radiations of core landbirds and core waterbirds with much less pronounced ILS. We provide evidence that, given the extreme level of up to 100% ILS per branch in super-radiations, particularly rapid speciation events may neither resemble a fully bifurcating tree nor are they resolvable as such. As a consequence, their complex demographic history is more accurately represented as local networks within a species tree.     

Circulating Autoantibodies against the Apolipoprotein B-100 Peptides p45 and p210 in Relation to the Occurrence of Carotid Plaques in 64-Year-Old Women

Objectives

Immune responses against oxidized low density lipoprotein (LDL) play a key role in atherosclerosis. Previous studies have indicated inverse associations between autoantibodies to epitopes in oxidized LDL and cardiovascular disease. In this study we investigated the associations between autoantibodies against the apolipoprotein B-100 (apoB-100) peptides p45 and p210 and occurrence of carotid plaques.

Design

The study cohort consisted of a population-based sample of 64-year-old women with varying degrees of glucose tolerance (n=594). To identify and record the occurrence of carotid atherosclerotic plaques ultrasonography was used. Measurements of plasma IgM and IgG autoantibodies against the native and malondialdehyde (MDA)-modified apoB-100 peptides p45 and p210 were performed by ELISA.

Results

Women with carotid plaques were found to have lower levels of IgM MDA-p210 autoantibodies compared to plaque-free women. The number of carotid plaques in each subject and the total carotid plaque area correlated inversely with IgM MDA-p210 levels (r=-0.11, P=0.009 and r=-0.11, P=0.013, respectively). Furthermore, levels of IgM MDA-p210 above the lowest tertile were associated with an odds ratio of 0.55 (95% CI 0.38-0.79, P=0.001) for occurrence of carotid plaques, independently of other risk markers and statin treatment. Associations between apo-B100 peptide autoantibodies and cardiovascular risk factors were generally weak but subjects with impaired glucose tolerance had higher levels of IgM against MDA-p210.

Conclusion

The present study demonstrates that high levels of IgM against MDA-p210 are associated with less severe carotid disease in women. These findings provide additional support for a role of immune responses against oxidized LDL in cardiovascular disease.     

Trypanothione Reductase: A Target Protein for a Combined In Vitro and In Silico Screening Approach

With the goal to identify novel trypanothione reductase (TR) inhibitors, we performed a combination of in vitro and in silico screening approaches. Starting from a highly diverse compound set of 2,816 compounds, 21 novel TR inhibiting compounds could be identified in the initial in vitro screening campaign against T. cruzi TR. All 21 in vitro hits were used in a subsequent similarity search-based in silico screening on a database containing 200,000 physically available compounds. The similarity search resulted in a data set containing 1,204 potential TR inhibitors, which was subjected to a second in vitro screening campaign leading to 61 additional active compounds. This corresponds to an approximately 10-fold enrichment compared to the initial pure in vitro screening. In total, 82 novel TR inhibitors with activities down to the nM range could be identified proving the validity of our combined in vitro/in silico approach. Moreover, the four most active compounds, showing IC₅₀ values of <1 μM, were selected for determining the inhibitor constant. In first on parasites assays, three compounds inhibited the proliferation of bloodstream T. brucei cell line 449 with EC₅₀ values down to 2 μM.     

Altered olfactory receptor neuron responsiveness in rare Ostrinia nubilalis males attracted to the O. furnacalis pheromone blend

Three percent of E-strain Ostrinia nubilalis males fly upwind in response to the Ostrinia furnacalis pheromone blend [a 40:60 ratio of (E)-12-tetradecenyl acetate to (Z)-12-tetradecenyl acetate (E12-14:OAc to Z12-14:OAc)], in addition to their own pheromone blend [a 99:1 ratio of (E)-11-tetradecenyl acetate to (Z)-11-tetradecenyl acetate) (E11-14:OAc to Z11-14:OAc)]. We assessed the olfactory receptor neuron (ORN) responses of these behaviorally "rare" males versus those of normal males. For the three ORNs housed within each sensillum, we tested responsiveness to Z12-14:OAc, E12-14:OAc, Z11-14:OAc, E11-14:OAc, and the behavioral antagonist (Z)-9-tetradecenyl acetate (Z9-14:OAc). Z11-14:OAc, E11-14:OAc, and Z9-14:OAc stimulated ORNs exhibiting distinct small, large, and medium spike sizes, respectively. For rare and normal males, both Z12-14:OAc and E12-14:OAc usually elicited responses from the largest-spiking ORN. In many ORNs of normal males, Z12-14:OAc or E12-14:OAc stimulated the smaller-spiking ORN that is responsive to Z11-14:OAc. In rare males, detectable ORN responses from the smaller-spiking ORN in response to Z12- and E12-14:OAc were virtually non-existent. These differences in ORN tuning in rare males will tend to create an ORN firing ratio between the large- and small-spiking ORNs in response to the O. furnacalis blend that is similar to that elicited by the O. nubilalis blend.     

Evidence of olfactory antagonistic imposition as a facilitator of evolutionary shifts in pheromone blend usage in Ostrinia spp. (Lepidoptera: Crambidae)

Olfactory receptor neuron (ORN) response was measured to assess why some males ("rare males") of the Asian corn borer (ACB), Ostrinia furnacalis, have a broad behavioral response to fly upwind to both the ACB and the European corn borer (ECB), Ostrinia nubilalis, pheromone blends. We performed single-sensillum electrophysiological recordings on ACB males that had been behaviorally assessed for upwind flight response to the ACB blend [60:40 (Z)-12-tetradecenyl acetate (Z12-14:OAc) to (E)-12-tetradecenyl acetate (E12-14:OAc)], as well as to ECB (Z-strain) and ECB (E-strain) blends [3:97 and 99:1 (Z)-11-tetradecenyl acetate (Z11-14:OAc) to (E)-11-tetradecenyl acetate (E11-14:OAc)]. Sensilla from all types of males had large- and small-spike-sized ORNs responding strongly to Z12- or E12-14:OAc, but weakly to Z11- and E11-14:OAc. In the majority of males ("normal males") that flew upwind only to the ACB blend, Z11-14:OAc elicited responses in an intermediate spike-sized ORN associated with behavioral antagonism that is mainly tuned to (Z)-9-tetradecenyl acetate (Z9-14:OAc). In the rare-type ACB males that flew to both the ACB and ECB pheromone blends, Z11-14:OAc did not stimulate this ORN. Increased responsiveness to ancestral pheromone components by ORNs associated with behavioral antagonism could be instrumental in reproductive character displacement, or in reinforcement and reproductive isolation during speciation by helping to increase assortative mating between males and females in derived populations that use novel sex pheromone blends.     

Genome-wide association mapping in a wild avian population identifies a link between genetic and phenotypic variation in a life-history trait

Understanding the genetic basis of traits involved in adaptation is a major challenge in evolutionary biology but remains poorly understood. Here, we use genome-wide association mapping using a custom 50 k single nucleotide polymorphism (SNP) array in a natural population of collared flycatchers to examine the genetic basis of clutch size, an important life-history trait in many animal species. We found evidence for an association on chromosome 18 where one SNP significant at the genome-wide level explained 3.9% of the phenotypic variance. We also detected two suggestive quantitative trait loci (QTLs) on chromosomes 9 and 26. Fitness differences among genotypes were generally weak and not significant, although there was some indication of a sex-by-genotype interaction for lifetime reproductive success at the suggestive QTL on chromosome 26. This implies that sexual antagonism may play a role in maintaining genetic variation at this QTL. Our findings provide candidate regions for a classic avian life-history trait that will be useful for future studies examining the molecular and cellular function of, as well as evolutionary mechanisms operating at, these loci.