Cargo properties play a critical role in myosin Va-driven cargo transport along actin filaments

High-resolution experiments revealed that a single myosin-Va motor can transport micron-sized cargo on actin filaments in a stepwise manner. However, intracellular cargo transport is mediated through the dense actin meshwork by a team of myosin Va motors. The mechanism of how motors interact mechanically to bring about efficient cargo transport is still poorly understood. This study describes a stochastic model where a quantitative understanding of the collective behaviors of myosin Va motors is developed based on cargo stiffness. To understand how cargo properties affect the overall cargo transport, we have designed a model in which two myosin Va motors were coupled by wormlike chain tethers with persistence length ranging from 10 to 80 nm and contour length from 100 to 200 nm, and predicted distributions of velocity, run length, and tether force. Our analysis showed that these parameters are sensitive to both the contour and persistence length of cargo. While the velocity of two couple motors is decreased compared to a single motor (from 531 ± 251 nm/s to as low as 318 ± 287 nm/s), the run length (716 ± 563 nm for a single motor) decreased for short, rigid tethers (to as low as 377 ± 187 μm) and increased for long, flexible tethers (to as high as 1.74 ± 1.50 μm). The sensitivity of processive properties to tether rigidity (persistence length) was greatest for short tethers, which caused the motors to exhibit close, yet anti-cooperative coordination. Motors coupled by longer tethers stepped more independently regardless of tether rigidity. Therefore, the properties of the cargo or linkage must play an essential role in motor-motor communication and cargo transport.     

Gas phase dimerization of neuropeptide head activator analogs useful for the noncovalent constraint of peptides

We have used electrospray mass spectrometry to examine the dimerization of EFLIVKS, a reversed sequence analog of part of the neuropeptide head activator, and other similar analogs. Observation of the EFLIVKS gas phase dimer is concentration-dependent, with a half-saturation concentration for relative dimer formation of 7.8 microM, similar to that of SKVILFE of 12 microM. The lowest energy conformers from quenched molecular dynamics simulations suggest EFLIVKS may dimerize in the gas phase by formation of multiple ion pairs across the dimer interface. Alanine-scan mutants also dimerize in the gas phase, with replacement of the interior residues FLIVK diminishing dimerization. The concentration-dependence of the EFLIVKS circular dichroism spectrum at pH 7.5 suggests the existence of different conformation states at different concentrations, but does not provide evidence supporting the saturable dimer formation in solution. Different analogs of EFLIVKS, when fused to each end of a 18mer unfolded peptide, induce solution structures with T(m)s of 42-50 degrees C. These peptides and analogs may thus be useful for the noncovalent constraint of peptides and peptide library members used in cellular screens.     

Infrared spectroscopy and differential scanning calorimetry studies of binary combinations of cis-6-octadecenoic acid and octadecanoic acid

Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) studies are reported for combinations of cis-6-octadecenoic acid (also termed petroselinic acid, PSA) and octadecanoic acid (also termed stearic acid, SA) across a wide range of binary mole ratio combinations. The data are then used to plot the phase diagram which is found to be montotectic with the PSA reducing the melting temperature of SA at all compositions. The relevance of these experiments to stratum corneum (SC) biophysical behavior, particularly the influence and potential mechanisms of PSA on dermal permeation, is discussed. The potential role of cis-6-octadecenoic acid as a permeation enhancer is discussed in the context of these studies of its interaction with saturated fatty acids.     

Application of saccharose as copper(II) ligand for electroless copper plating solutions

Saccharose, forming sufficiently stable complexes with copper(II) ions in alkaline solutions, was found to be a suitable ligand for copper(II) chelating in alkaline (pH>12) electroless copper deposition solutions. Reduction of copper(II)-saccharose complexes by hydrated formaldehyde was investigated and the copper deposits formed were characterized. The thickness of the compact copper coatings obtained under optimal operating conditions in 1h reaches ca. 2 microm at ambient temperature. The plating solutions were stable and no signs of Cu(II) reduction in the bulk solution were observed. Results were compared with those systems operating with other copper(II) ligands.     

Effects of in vivo exposure to DEET on blood feeding behavior and fecundity in Anopheles quadrimaculatus (Diptera: Culicidae)

This study determined the effects of contact with DEET on guinea pig skin on mortality, probing time, blood feeding rate, engorgement time, and fecundity responses in female Anopheles quadrimaculatus Say. Exposure, in this manner, to 10% DEET (in ethanol) for 5 min, resulted in 98% mortality in mosquitoes after 24h. The median probing time (PT(50)) required by females, when exposed to 0.1%, 1.0%, and 10% DEET, was significantly (P<0.0001) longer (12.5, 12.1, and 19.1s, respectively) than the 6.8s required by females to probe ethanol-treated skin (control). Similarly, mean blood feeding rates in populations of females exposed to 1.0% DEET for < or = 5 min (14.4%) was 6x lower (P<0.001) (85.5%) than in females exposed to ethanol-treated skin, whereas the mean engorgement time on skin treated with 1.0% DEET (66.3s) was significantly shorter (P<0.0001) than for females feeding on the control guinea pigs (105.9s). The mean number of mature o?cytes per female (fecundity) in treatment (1.0% DEET) and control mosquitoes was not significantly different. The responses to DEET observed in this study suggest that repeated exposure of female A. quadrimaculatus populations to this repellent, in laboratory bioassays, could result in confounding of toxicant and repellent effects and inaccurate estimates of DEET repellency.     

Organismal differences in post-translational modifications in histones H3 and H4

Post-translational modifications (PTMs) of histones play an important role in many cellular processes, notably gene regulation. Using a combination of mass spectrometric and immunobiochemical approaches, we show that the PTM profile of histone H3 differs significantly among the various model organisms examined. Unicellular eukaryotes, such as Saccharomyces cerevisiae (yeast) and Tetrahymena thermophila (Tet), for example, contain more activation than silencing marks as compared with mammalian cells (mouse and human), which are generally enriched in PTMs more often associated with gene silencing. Close examination reveals that many of the better-known modified lysines (Lys) can be either methylated or acetylated and that the overall modification patterns become more complex from unicellular eukaryotes to mammals. Additionally, novel species-specific H3 PTMs from wild-type asynchronously grown cells are also detected by mass spectrometry. Our results suggest that some PTMs are more conserved than previously thought, including H3K9me1 and H4K20me2 in yeast and H3K27me1, -me2, and -me3 in Tet. On histone H4, methylation at Lys-20 showed a similar pattern as H3 methylation at Lys-9, with mammals containing more methylation than the unicellular organisms. Additionally, modification profiles of H4 acetylation were very similar among the organisms examined.     

Structure-based design, synthesis, evaluation, and crystal structures of transition state analogue inhibitors of inosine monophosphate cyclohydrolase

The inosine monophosphate cyclohydrolase (IMPCH) component (residues 1-199) of the bifunctional enzyme aminoimidazole-4-carboxamide ribonucleotide transformylase (AICAR Tfase, residues 200-593)/IMPCH (ATIC) catalyzes the final step in the de novo purine biosynthesis pathway that produces IMP. As a potential target for antineoplastic intervention, we designed IMPCH inhibitors, 1,5-dihydroimidazo[4,5-c][1,2,6]thiadiazin-4(3H)-one 2,2-dioxide (heterocycle, 1), the corresponding nucleoside (2), and the nucleoside monophosphate (nucleotide) (3), as mimics of the tetrahedral intermediate in the cyclization reaction. All compounds are competitive inhibitors against IMPCH (K(i) values = 0.13-0.23 microm) with the simple heterocycle 1 exhibiting the most potent inhibition (K(i) = 0.13 microm). Crystal structures of bifunctional ATIC in complex with nucleoside 2 and nucleotide 3 revealed IMPCH binding modes similar to that of the IMPCH feedback inhibitor, xanthosine 5'-monophosphate. Surprisingly, the simpler heterocycle 1 had a completely different IMPCH binding mode and was relocated to the phosphate binding pocket that was identified from previous xanthosine 5'-monophosphate structures. The aromatic imidazole ring interacts with a helix dipole, similar to the interaction with the phosphate moiety of 3. The crystal structures not only revealed the mechanism of inhibition of these compounds, but they now serve as a platform for future inhibitor improvements. Importantly, the nucleoside-complexed structure supports the notion that inhibitors lacking a negatively charged phosphate can still inhibit IMPCH activity with comparable potency to phosphate-containing inhibitors. Provocatively, the nucleotide inhibitor 3 also binds to the AICAR Tfase domain of ATIC, which now provides a lead compound for the design of inhibitors that simultaneously target both active sites of this bifunctional enzyme.