Yeast Pif1 Helicase Exhibits a One-base-pair Stepping Mechanism for Unwinding Duplex DNA

Kinetic analysis of the DNA unwinding and translocation activities of helicases is necessary for characterization of the biochemical mechanism(s) for this class of enzymes. Saccharomyces cerevisiae Pif1 helicase was characterized using presteady state kinetics to determine rates of DNA unwinding, displacement of streptavidin from biotinylated DNA, translocation on single-stranded DNA (ssDNA), and ATP hydrolysis activities. Unwinding of substrates containing varying duplex lengths was fit globally to a model for stepwise unwinding and resulted in an unwinding rate of ∼75 bp/s and a kinetic step size of 1 base pair. Pif1 is capable of displacing streptavidin from biotinylated oligonucleotides with a linear increase in the rates as the length of the oligonucleotides increased. The rate of translocation on ssDNA was determined by measuring dissociation from varying lengths of ssDNA and is essentially the same as the rate of unwinding of dsDNA, making Pif1 an active helicase. The ATPase activity of Pif1 on ssDNA was determined using fluorescently labeled phosphate-binding protein to measure the rate of phosphate release. The quantity of phosphate released corresponds to a chemical efficiency of 0.84 ATP/nucleotides translocated. Hence, when all of the kinetic data are considered, Pif1 appears to move along DNA in single nucleotide or base pair steps, powered by hydrolysis of 1 molecule of ATP.     

Involvement of the Global Regulator H-NS in the Survival of Escherichia coli in Stationary Phase

Long-term batch cultures of Escherichia coli grown in nutrient-rich medium accumulate mutations that provide a growth advantage in the stationary phase (GASP). We have examined the survivors of prolonged stationary phase to identify loci involved in conferring a growth advantage and show that a mutation in the hns gene causing reduced activity of the global regulator H-NS confers a GASP phenotype under specific conditions. The hns-66 allele bears a point mutation within the termination codon of the H-NS open reading frame, resulting in a longer protein that is partially functional. Although isolated from a long-term stationary-phase culture of the parent carrying the rpoS819 allele that results in reduced RpoS activity, the hns-66 survivor showed a growth disadvantage in the early stationary phase (24 to 48 h) when competed against the parent. The hns-66 mutant is also unstable and reverts at a high frequency in the early stationary phase by accumulating second-site suppressor mutations within the ssrA gene involved in targeting aberrant proteins for proteolysis. The mutant was more stable and showed a moderate growth advantage in combination with the rpoS819 allele when competed against a 21-day-old parent. These studies show that H-NS is a target for mutations conferring fitness gain that depends on the genetic background as well as on the stage of the stationary phase.     

Mutual and directional allogeneic cytotoxic reaction of hemocytes in the solitary ascidian Halocynthia roretzi revealed by one-step quantitative fluorimetric assay

A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls alloreactivity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with "effector cells" and those from the other animal of the pair with "target cells". In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.     

Consistent production of kojic acid from Aspergillus sojae SSC-3 isolated from rice husk

Molecular Biology Reports - A consistent kojic acid producing fungal strain has been isolated from rice husk using glucose-peptone medium. The isolate was identified as Aspergillus sojae SSC-3 on...     

Sequential ordinal modeling with applications to survival data

This paper considers the class of sequential ordinal models in relation to other models for ordinal response data. Markov chain Monte Carlo (MCMC) algorithms, based on the approach of Albert and Chib (1993, Journal of the American Statistical Association 88, 669-679), are developed for the fitting of these models. The ideas and methods are illustrated in detail with a real data example on the length of hospital stay for patients undergoing heart surgery. A notable aspect of this analysis is the comparison, based on marginal likelihoods and training sample priors, of several nonnested models, such as the sequential model, the cumulative ordinal model, and Weibull and log-logistic models.     

Neural mechanisms underlying paradoxical performance for monetary incentives are driven by loss aversion

Employers often make payment contingent on performance in order to motivate workers. We used fMRI with a novel incentivized skill task to examine the neural processes underlying behavioral responses to performance-based pay. We found that individuals' performance increased with increasing incentives; however, very high incentive levels led to the paradoxical consequence of worse performance. Between initial incentive presentation and task execution, striatal activity rapidly switched between activation and deactivation in response to?increasing incentives. Critically, decrements in performance and striatal deactivations were directly predicted by an independent measure of behavioral loss aversion. These results suggest that incentives associated with successful task performance are initially encoded as a potential gain; however, when actually performing a task, individuals encode the potential loss that would arise from failure.     

Psilostachyin, acetylated pseudoguaianolides and their analogues: preparation and evaluation of their anti-inflammatory potential

Naturally occurring acetylated pseudoguaianolides and psilostachyin including their analogues were synthesized. The structure of semi-synthetic psilostachyin was also confirmed by X-ray crystallography. The anti-inflammatory potential of all the derivatives has been evaluated through in vitro expression of TNF-α, IL-1β and IL-6 in murine neutrophils.     

Yeast Helicase Pif1 Unwinds RNA:DNA Hybrids with Higher Processivity than DNA:DNA Duplexes

Saccharomyces cerevisiae Pif1, an SF1B helicase, has been implicated in both mitochondrial and nuclear functions. Here we have characterized the preference of Pif1 for RNA:DNA heteroduplexes in vitro by investigating several kinetic parameters associated with unwinding. We show that the preferential unwinding of RNA:DNA hybrids is due to neither specific binding nor differences in the rate of strand separation. Instead, Pif1 is capable of unwinding RNA:DNA heteroduplexes with moderately greater processivity compared with its duplex DNA:DNA counterparts. This higher processivity of Pif1 is attributed to slower dissociation from RNA:DNA hybrids. Biologically, this preferential role of the helicase may contribute to its functions at both telomeric and nontelomeric sites.