A Rapid and Inexpensive Bioassay to Evaluate the Decontamination of Organophosphates

An inexpensive and rapid bioassay using adult red flour beetles was developed for use in assessing the decontamination of environments containing organophosphates and related chemicals. A decontamination protocol was developed which demonstrated that 2 to 3 applications of 5% bleach solution were required to obtain nearly complete decontamination of malathion. The bioassay was also used to screen common household cleaners as potential decontaminating agents, but only 5% bleach was effective at improving survival of insects on steel plates treated with 25% malathion. A toxic degradation product (malaoxon) was detected using gas chromatography/ mass spectrophotometry; this toxin affected the decontamination efficacy and resulted in continued toxicity to the beetles until subsequent decontaminations. The bioassay provides evidence to support the use of red flour beetles as a sensitive, less expensive method for determining safety levels of environments contaminated with malathion and other toxins, and may have application in the study of chemical warfare agents

Human Mediator Subunit MED26 Functions as a Docking Site for Transcription Elongation Factors

SummaryPromoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription

Intrinsic Capability of Budding Yeast Cofilin to Promote Turnover of Tropomyosin-Bound Actin Filaments

The ability of actin filaments to function in cell morphogenesis and motility is closely coupled to their dynamic properties. Yeast cells contain two prominent actin structures, cables and patches, both of which are rapidly assembled and disassembled. Although genetic studies have shown that rapid actin turnover in patches and cables depends on cofilin, how cofilin might control cable disassembly remains unclear, because tropomyosin, a component of actin cables, is thought to protect actin filaments against the depolymerizing activity of ADF/cofilin. We have identified cofilin as a yeast tropomyosin (Tpm1) binding protein through Tpm1 affinity column and mass spectrometry. Using a variety of assays, we show that yeast cofilin can efficiently depolymerize and sever yeast actin filaments decorated with either Tpm1 or mouse tropomyosins TM1 and TM4. Our results suggest that yeast cofilin has the intrinsic ability to promote actin cable turnover, and that the severing activity may rely on its ability to bind Tpm1

High-scoring binders of Tpm1 affinity purifications identified by MudPIT.

a<p>Proteins were first ranked based on their frequency of detection in the Tmp1 affinity purifications and their absence (or presence at much lower levels based on NSAF values) in the BSA negative controls. Proteins were then ranked based on their NSAF values averaged across the three Tmp1 experiments.</p>b<p>NSAF (normalized spectral abundance factor) values measured in the Urea or KCl eluted samples and negative controls, as well as NSAF values calculated when spectral counts and proteins from all four runs were merged (ALL_NSAF).</p>c<p>All_P, All_sS, and All_uS are respectively the peptide, shared spectral, and unique spectral counts when the four runs were merged, while All_SC is the final sequence coverage.</p

Yeast Cofilin binds Tpm1.

<p>A) Tpm1 binds directly to wild-type Cof1 and mutant Cof1-5 but not Cof1-22. BSA beads were used as a control. Beads coated with Tpm1 were incubated with 50 nM Cof1, Cof1-5 or Cof1-22. Bound cofilin was visualized by immunoblotting using yeast cofilin antibody <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#pone.0003641-Moon1" target="_blank">[39]</a>. B) Determination of dissociation constant K_d between Cof1 and Tpm1. Beads coated with 0 to 8 µM Tpm1 were incubated with 50 nM Cof1. Bound cofilin was visualized as in (A). C) Bound and free Tpm1 from (B) were quantified and plotted. The calculated K_d was 3.34±0.12 µM (mean±SD).</p

Severing of Tpm1-bound yeast F-actin by Cof1 but not Cof1-22.

<p>A) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with Tpm1 (lower panels), after incubation with 50 nM Cof1 for lengths of time as indicated (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#s4" target="_blank">Materials and Methods</a> for more details). B) Representative confocal images of F-actin (5 µM), assembled without (upper panels) or with 5 µM Tpm1 (lower panels), after incubation with 50 nM Cof1-5 (left panels) or Cof1-22 (right panels) for 2 min. C) Measurements of actin filaments length from images recorded in experiments in (A) and (B). Shown are averages of filament length measurements from three fields per sample with error bars representing standard deviations. D) Representative confocal images of F-actin (5 µM), assembled with 5 µM Tpm1, after incubation with 50 nM Cof1-22 for lengths of time as indicated.</p

Effects of tropomyosin on actin depolymerization and actin binding by yeast Cof1 or mouse cofilin 1.

<p>A) Actin filament depolymerization was followed for 4 min at 25°C by the decrease in light-scattering at 400 nm. 5 µM yeast F-actin assembled in the presence or absence of Tpm1 was diluted to reactions containing final concentrations of 0.5 µM Cof1 or Cof1-22 and 0.5 µM F-actin. The spontaneous depolymerization of F-actin (no cofilin), with or without Tpm1 bound was monitored in parallel. B) Decrease in pyrene actin fluorescence was followed for 4 min at 25°C after dilution of F-actin (5 µM with 8% pyrene-labelled, assembled in the presence or absence of Tpm1) to a final concentration 0.5 µM F-actin with or without of 0.5 µM Cof1, Cof1-5 or Cof1-22. C) The same experiment as in (B) was carried out with muscle F-actin assembled with or without TM1, and in the presence or absence of mouse cofilin 1.</p

Yeast Cof1 mutants Cof1-5 and Cof1-22 can depolymerize F-actin with or without tropomyosin bound.

<p>A, B, C) F-actin (10 µM) polymerized without Tpm1p was incubated with 0, 10, 20 µM Cof1, Cof1-5 and Cof1-22 respectively and with 10 µM Tpm1p was incubated with 0, 2.5, 5, 10 and 20 µM Cof1, Cof1-5 and Cof1-22 respectively. The supernatants and pellets after ultracentrifugation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#s4" target="_blank">Materials and Methods</a>) were analyzed on an SDS-PAGE gel. D) The cofilin/actin molar ratios in the pellet from two independent co-sedimentation experiments using 10 µM actin and 10 µM cofilin. Ratios were determined by densitometry of Coomassie-stained SDS-gels shown in (A), (B) and (C).</p

Yeast cofilin but not mouse cofilin 1 depolymerizes F-actin decorated with tropomyosin.

<p>A) Yeast F-actin (10 µM) polymerized with or without 10 µM tropomyosin was incubated with 0, 10 or 20 µM Cof1 (Tpm1-containing sample), or with 0 or 10 µM Cof1 (TM1 or TM4-containing sample). The supernatants and pellets after ultracentrifugation (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003641#s4" target="_blank">Materials and Methods</a>) were analyzed on an SDS-PAGE gel. B) F-actin (10 µM) polymerized with or without 10 µM Tpm1p was incubated with 0, 2.5, 5, 10, 20 and 40 µM Cof1. Subsequent analysis was done as in (A). C) Rabbit muscle actin (10 µM) polymerized with or without 10 µM TM1 was incubated with 0, 5, 10, 20 and 40 µM mouse cofilin 1. Subsequent analysis was done as in (A). D, E) Quantification by densitometry of actin in pellet fractions (as % of the total actin) from experiments in (B) and (C), respectively.</p