FRET-based biosensor for oleic acid in nanomolar range with quantum dots as an energy donor

A self-assembling sensor for oleic acid has been developed. The sensor consists of a self-assembling fluorescent dye labeled BSA and quantum dots CdSe/ZnS capped with 3-mercaptopropionic acid. The detection limit of the new sensor is 10-1000 nM. The influence of the quantum dot size on the FRET efficiency in the course of the interaction of the sensor system with the analyte has been studied. The pH dependence, aggregation stability. and electrophoretic properties of the sensor have been examined. The data suggest a new approach for the development of nanoscale FRET-based sensors operating effectively due to unique fluorescent properties of quantum dots as well as due to selective protein-ligand interactions.     

Antimalarial activity of azadipeptide nitriles

Azadipeptide nitriles—novel cysteine protease inhibitors—display structure-dependent antimalarial activity against both chloroquine-sensitive and chloroquine-resistant lines of cultured Plasmodium falciparum malaria parasites. Inhibition of parasite’s hemoglobin-degrading cysteine proteases was also investigated, revealing the azadipeptide nitriles as potent inhibitors of falcipain-2 and -3. A correlation between the cysteine protease-inhibiting activity and the antimalarial potential of the compounds was observed. These first generation azadipeptide nitriles represent a promising new class of compounds for antimalarial drug development.     

Network Models of Frequency Modulated Sweep Detection

Frequency modulated (FM) sweeps are common in species-specific vocalizations, including human speech. Auditory neurons selective for the direction and rate of frequency change in FM sweeps are present across species, but the synaptic mechanisms underlying such selectivity are only beginning to be understood. Even less is known about mechanisms of experience-dependent changes in FM sweep selectivity. We present three network models of synaptic mechanisms of FM sweep direction and rate selectivity that explains experimental data: (1) The ‘facilitation’ model contains frequency selective cells operating as coincidence detectors, summing up multiple excitatory inputs with different time delays. (2) The ‘duration tuned’ model depends on interactions between delayed excitation and early inhibition. The strength of delayed excitation determines the preferred duration. Inhibitory rebound can reinforce the delayed excitation. (3) The ‘inhibitory sideband’ model uses frequency selective inputs to a network of excitatory and inhibitory cells. The strength and asymmetry of these connections results in neurons responsive to sweeps in a single direction of sufficient sweep rate. Variations of these properties, can explain the diversity of rate-dependent direction selectivity seen across species. We show that the inhibitory sideband model can be trained using spike timing dependent plasticity (STDP) to develop direction selectivity from a non-selective network. These models provide a means to compare the proposed synaptic and spectrotemporal mechanisms of FM sweep processing and can be utilized to explore cellular mechanisms underlying experience- or training-dependent changes in spectrotemporal processing across animal models. Given the analogy between FM sweeps and visual motion, these models can serve a broader function in studying stimulus movement across sensory epithelia.     

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

Genetic enrichment of the arctic clonal plant Saxifraga cernua at its southern periphery via the alpine sexual Saxifraga sibirica

Isolation of populations at the margins of a species range may lead to decreasing genetic diversity via genetic drift and inbreeding. Hybridization between peripheral populations of two species can, however, counteract genetic impoverishment. The mainly clonal, polyploid plant Saxifraga cernua has a wide arctic distribution but also extends southwards into alpine sites. In the Ural Mountains, its peripheral distribution overlaps with that of its sexually reproducing, diploid relative Saxifraga sibirica, and fertile polyploids of more or less intermediate appearance are found in this overlap zone. We used amplified fragment length polymorphism (AFLP) analysis to address the potential impact of interspecific gene flow on genetic diversity in the peripheral populations. A total of 149 plants from 17 populations along a 1650 km south-north gradient were analysed for 253 markers. The results suggest that three Middle Ural populations containing fertile and morphologically more or less intermediate plants have been affected by hybridization. All of these plants formed a strongly supported (100%) group with S. cernua in a neighbour-joining tree, but their AFLP phenotypes assigned either to S. cernua or to artificial (simulated) F(1) hybrids between S. cernua and S. sibirica in multilocus assignment tests. The three populations were highly diverse with virtually every plant representing a distinct AFLP phenotype, providing additional evidence for formation of later-generation hybrids and/or backcrossing to S. cernua. In contrast, other peripheral populations of S. cernua were typically monoclonal, suggesting that hybridization with S. sibirica can increase genetic diversity in S. cernua at its southern periphery.     

Differential expression of duplicated LDH-A genes during temperature acclimation of weatherfish Misgurnus fossilis. Functional consequences for the enzyme

Temperature acclimation in poikilotherms entails metabolic rearrangements provided by variations in enzyme properties. However, in most cases the underlying molecular mechanisms that result in structural changes in the enzymes are obscure. This study reports that acclimation to low (5 degrees C) and high (18 degrees C) temperatures leads to differential expression of alternative forms of the LDH-A gene in white skeletal muscle of weatherfish, Misgurnus fossilis. Two isoforms of LDH-A mRNA were isolated and characterized: a short isoform (= 1332 bp) and a long isoform ( = 1550 bp), which both have 5'-UTRs and ORFs of the same length (333 amino acid residues), but differ in the length of the 3'-UTR. In addition, these two mRNAs have 44 nucleotide point mismatches of an irregular pattern along the complete sequence, resulting in three amino acid mismatches (Gly214Val; Val304Ile and Asp312Glu) between protein products from the short and long mRNA forms, correspondingly LDH-A(alpha) and LDH-A(beta) subunits. It is expected that the beta-subunit is more aliphatic due to the properties of the mismatched amino acids and therefore sterically more restricted. According to molecular modelling of M. fossilis LDH-A, the Val304Ile mismatch is located in the subunit contact area of the tetramer, whereas the remaining two mismatches surround the contact area; this is expected to manifest in the kinetic and thermodynamic properties of the assembled tetramer. In warm-acclimated fish the relative expression between alpha and beta isoforms of the LDH-A mRNA is around 5 : 1, whereas in cold-acclimated fish expression of is reduced almost to zero. This indicates that at low temperature the pool of total tetrameric LDH-A is more homogeneous in terms of alpha/beta-subunit composition. The temperature acclimation pattern of proportional pooling of subunits with different kinetic and thermodynamic properties of the tetrameric enzyme may result in fine-tuning of the properties of skeletal LDH-A, which is in line with previously observed kinetic and thermodynamic differences between 'cold' and 'warm' LDH-A purified from weatherfish. Also, an irregular pattern of nucleotide mismatches indicates that these mRNAs are the products of two independently evolving genes, i.e. paralogues. Karyotype analysis has confirmed that the experimental population of M. fossilis is tetraploid (2n = 100), therefore gene duplication, possibly through tetraploidy, may contribute to the adaptability towards temperature variation.     

Differentiation of species in Gonioloboceras

The type specimen ofGonioloboceras goniolobum (Meek), rediscovered by Spath in the British Museum, is the foundation for a more accurate comparative study of this and other species ofGonioloboceras.Gonioloboceras described asG. goniolobum byElias in 1938 is differentiated asGonioloboceras schmidti, new species. Suture sets (new term) for several growth stages inG. goniolobum (Meek),G. welleriSmith,G. schmidtiElias, G.eliasiMiller &Owen, andG. asiaticumLibrovitch are assembled and used for differentiation of the species.The Kazakhstan goniatite faunule containingG. asiaticum is considered of very late Pennsylvanian age.