5 research outputs found
Kinetics of the DNA polymerase \u3ci\u3epyrococcus kodakaraensis\u3c/i\u3e
The polymerase chain reaction is one of the most important reactions in molecular biology. Single stranded DNA is copied in a complex series of steps, at the core of which lies the action of the DNA polymerase. At each nucleotide along the template, the polymerase screens the dNTP pool until it finds the complementary dNTP. The insertion of each dNMP is a balance between high fidelity and rapid elongation. In this study the kinetics of the Ī² type polymerase pyrococcus kodakaraensis (KOD) is analyzed. The kinetics is influenced by reaction conditions such as the dNTP pool composition and temperature. In a previous study by Viljoen et al. [2005, A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide selection. Computational Biology and Chemistry 29, 101ā110], a macroscopic kinetics expression of the polymerase chain reaction has been derived. The model contains four parameters that are intrinsic to a specific polymerase. The experiments to measure the temperature- dependence of the parameters for KOD DNA polymerase are reported. The results indicate that the optimal temperature for an equimolar dNTP pool is 72.5 Ā°C and the optimum temperature shifts to lower temperatures when the dNTP pool composition is biased
Regulation of [Ā³H]d-aspartate release by the 5-F(2t)-isoprostane and its 5-epimer in isolated bovine retina.
International audienceWe have evidence that 15-Fā-isoprostanes (15-Fā-IsoPs) regulate excitatory neurotransmitter release in ocular tissues. Although 5-Fā-IsoPs are abundantly produced in mammals, their pharmacological actions on neurotransmitter release remain unknown. In the present study, we compared the effect of the 5-Fā-IsoP epimer pair, 5-F(2t)-IsoP (C5-OH in Ī²-position) and 5-epi-5-F(2t)-IsoP (C5-OH in Ī±-position), on Kāŗ-evoked [Ā³H]D-aspartate release in isolated bovine retina. We further examined the role of prostanoid receptors on the inhibitory action of 5-epi-5-F(2t)-IsoP on [Ā³H]D-aspartate overflow. Isolated bovine retina were prepared for studies of Kāŗ-evoked release of [Ā³H]D-aspartate using the superfusion method. 5-epi-5-F(2t)-IsoP (0.01 nM to 1 Ī¼M), attenuated Kāŗ-evoked [Ā³H]D-aspartate release in a concentration-dependent manner, with the inhibitory effect of 26.9% (P < 0.001; ICāā
= 0.2 Ī¼M) being achieved at 1 Ī¼M concentration. Its 5-(S)-OH-epimer, 5-F(2t)-IsoP (0.1 nM-1 Ī¼M), exhibited an inhibitory biphasic action, yielding a maximal response of 35.7% (P < 0.001) at 10 nM concentration of the drug (ICāā
value of 3 nM). Although the prostanoid-receptor antagonists, AH 6809 (10 Ī¼M; EPāāā/DP) and BAY-u3405 (10 Ī¼M; DP/Tx) exhibited no effect on 5-epi-5-F(2t)-IsoP (10 nM-1 Ī¼M)-mediated inhibition, SC-19220 (1 Ī¼M; EPā) completely reversed 5-epi-5-F(2t)-IsoP (0.1 Ī¼M and 1 Ī¼M)-induced attenuation of Kāŗ-evoked [Ā³H]D-aspartate release. Similarly, both SC-51322 (10 Ī¼M; EPā and AH 23848 (1 Ī¼M; EPā) reversed the inhibitory action elicited by 5-epi-5-F(2t)-IsoP (0.1 Ī¼M) on the neurotransmitter release. We conclude that the 5-Fā-IsoP epimer pair, 5-F(2t)-IsoP and 5-epi-5-F(2t)-IsoP, attenuate Kāŗ-induced [Ā³H]D-aspartate release in isolated bovine retina presumably via prostanoid receptor dependent mechanisms. The trans-orientation of the allylic hydroxyl group at position C5 accounts for the apparent biphasic response exhibited by 5-F(2t)-IsoP on excitatory neurotransmitter release
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Functional Knowledge Transfer for High-accuracy Prediction of Under-studied Biological Processes
A key challenge in genetics is identifying the functional roles of genes in pathways. Numerous functional genomics
techniques (e.g. machine learning) that predict protein function have been developed to address this question. These
methods generally build from existing annotations of genes to pathways and thus are often unable to identify additional
genes participating in processes that are not already well studied. Many of these processes are well studied in some
organism, but not necessarily in an investigatorās organism of interest. Sequence-based search methods (e.g. BLAST) have
been used to transfer such annotation information between organisms. We demonstrate that functional genomics can
complement traditional sequence similarity to improve the transfer of gene annotations between organisms. Our method
transfers annotations only when functionally appropriate as determined by genomic data and can be used with any
prediction algorithm to combine transferred gene function knowledge with organism-specific high-throughput data to
enable accurate function prediction. We show that diverse state-of-art machine learning algorithms leveraging functional
knowledge transfer (FKT) dramatically improve their accuracy in predicting gene-pathway membership, particularly for
processes with little experimental knowledge in an organism. We also show that our method compares favorably to
annotation transfer by sequence similarity. Next, we deploy FKT with state-of-the-art SVM classifier to predict novel genes to
11,000 biological processes across six diverse organisms and expand the coverage of accurate function predictions to
processes that are often ignored because of a dearth of annotated genes in an organism. Finally, we perform in vivo
experimental investigation in Danio rerio and confirm the regulatory role of our top predicted novel gene, wnt5b, in leftward
cell migration during heart development. FKT is immediately applicable to many bioinformatics techniques and will help
biologists systematically integrate prior knowledge from diverse systems to direct targeted experiments in their organism of
study