Crystallization of mutant forms of the γ subunit of archaeal translation initiation factor 2

Archaeal translation initiation factor 2 (aIF2) is homologous to its eukaryotic counterpart (eIF2). It is a heterotrimeric protein consisting of α, β, and γ subunits. The protein e/aIF2 forms a ternary complex with guanosine 5′-triphosphate and the initiator methionyl-tRNA (Met-tRNA_i) and delivers the latter to the ribosome. In archaea, translation initiation factor 2 has an additional function. The γ subunit of aIF2 binds mRNAs with a triphosphate at the 5′-end and prevents 5′-to-3′ directional mRNA decay. To determine the mRNA-binding site on the surface of aIF2γ, mutations were introduced into the protein sequence at sites of possible interactions with mRNA. The crystals of the mutant forms of aIF2γ were obtained, and X-ray diffraction data sets suitable for structure determination at atomic resolution were collected.     

Fundamental groups of tangent conic-line arrangements with singularities up to order 6

We list all the possible fundamental groups of the complements of real conic-line arrangements with two conics which are tangent to each other at two points, with up to two additional lines. For the computations we use the topological local braid monodromies and the techniques of Moishezon–Teicher and van-Kampen. We also include some conjectures concerning the connection between the presentation of the fundamental group of the complements and the geometry of an interesting family of conic-line arrangements. M. Amram was partially supported by the Minerva Foundation of Germany, the DAAD fellowship (Germany), EAGER (EU Network, HPRN-CT-2009-00099) and the Golda Meir postdoctoral fellowship. D. Garber was partially supported by Lady Davis and Golda Meir postdoctoral fellowships     

Characterization of the mechanical properties of muskeg with special reference to vehicle mobility

Results of measurement of the load-sinkage and shearing characteristics of a muskeg in the Ottawa area, Ontario are presented. Mathematical models for the bearing capacity and shearing characteristics of muskeg are proposed. Methods for predicting the punching load, critical sinkage and shear strength of muskeg are suggested. Based on the characteristics of the mechanical properties of muskeg obtained, some aspects of the design of vehicles for use in organic terrain are discussed.     

Retail facility location under changing market conditions

In this paper we investigate the location of retail facilitiesunder changing market conditions when market conditions areexpected to change during the planning horizon. Three modelsare presented: (1) the minimax regret model where the objectivefunction is to minimize the maximum possible loss under differentmarket scenarios, (2) the Stackelberg equilibrium model wherebya future competitor enters the market and one wishes to maximizethe market share captured throughout the planning horizon incorporatingthe market share loss to the competitor, and (3) the thresholdmodel in which we consider a minimum threshold level such thatif the market share captured by the new facility fails to reachthat threshold, the facility will not survive. Our objectiveis to minimize the probability of failure.     

Efforts toward expansion of the genetic alphabet: structure and replication of unnatural base pairs

Expansion of the genetic alphabet has been a long-time goal of chemical biology. A third DNA base pair that is stable and replicable would have a great number of practical applications and would also lay the foundation for a semisynthetic organism. We have reported that DNA base pairs formed between deoxyribonucleotides with large aromatic, predominantly hydrophobic nucleobase analogues, such as propynylisocarbostyril (dPICS), are stable and efficiently synthesized by DNA polymerases. However, once incorporated into the primer, these analogues inhibit continued primer elongation. More recently, we have found that DNA base pairs formed between nucleobase analogues that have minimal aromatic surface area in addition to little or no hydrogen-bonding potential, such as 3-fluorobenzene (d3FB), are synthesized and extended by DNA polymerases with greatly increased efficiency. Here we show that the rate of synthesis and extension of the self-pair formed between two d3FB analogues is sufficient for in vitro DNA replication. To better understand the origins of efficient replication, we examined the structure of DNA duplexes containing either the d3FB or dPICS self-pairs. We find that the large aromatic rings of dPICS pair in an intercalative manner within duplex DNA, while the d3FB nucleobases interact in an edge-on manner, much closer in structure to natural base pairs. We also synthesized duplexes containing the 5-methyl-substituted derivatives of d3FB (d5Me3FB) paired opposite d3FB or the unsubstituted analogue (dBEN). In all, the data suggest that the structure, electrostatics, and dynamics can all contribute to the extension of unnatural primer termini. The results also help explain the replication properties of many previously examined unnatural base pairs and should help design unnatural base pairs that are better replicated.     

The stereochemical dependence of unimolecular dissociation of monosaccharide-glycolaldehyde anions in the gas phase: a basis for assignment of the stereochemistry and anomeric configuration of monosaccharides in oligosaccharides by mass spectrometry via a key discriminatory product ion of disaccharide fragmentation, m/z 221

Mass spectrometry of hexose-containing disaccharides often yields product ions of m/z 221 in the negative ion mode. Using a Paul trap, isolation and collision-induced dissociation of the m/z 221 anions yielded mass spectra that easily differentiated their stereochemistry and anomeric configuration, for all 16 stereochemical variants. The ions were shown to be glycopyranosyl-glycolaldehydes through chemical synthesis of their standards. The stereochemistry dramatically affected fragmentation which was dependent on four relative stereochemical arrangements: (1) the relationship between the hydroxyl group at position 2 and the anomeric configuration, (2) a cis relationship of the anomeric position and positions 2 and 3 (1,2,3-cis), (3) a 1,2 trans-2,3 cis relationship, and (4) the relationship between the hydroxyl group at position 4 and the anomeric configuration. After labeling the reducing carbonyl oxygen of a series of disaccharides with 18O to mass-discriminate between their monosaccharide components, it was demonstrated that m/z 221 anions are comprised of an intact nonreducing sugar glycosidically linked to a 2-carbon aglycon derived from the reducing sugar, irrespective of the linkage position between monosaccharides. This enabled the location of the intact sugar to be assigned to the nonreducing side of a glycosidic linkage. Detailed studies of experimental factors necessary for reproducibility demonstrated that the unique mass spectrum for each m/z 221 anion could be obtained from month-to-month through the use of an internal energy-input calibrant ion that ensured reproducible energy deposition into the ions. The counterparts to these ions for the 2-acetamido-2-deoxyhexoses were m/z 262 anions, and the anomeric configuration and stereochemistry of these anions could also be reproducibly discriminated for N-acetylglucosamine and N-acetylgalactosamine. The fragmentation patterns of m/z 221 anions provide a firm reproducible basis for assignment of sugar stereochemistries in the gas phase.     

Assessing corn stover composition and sources of variability via NIRS

Corn stover, the above-ground, non-grain portion of the crop, is a large, currently available source of biomass that potentially could be collected as a biofuels feedstock. Biomass conversion process economics are directly affected by the overall biochemical conversion yield, which is assumed to be proportional to the carbohydrate content of the feedstock materials used in the process. Variability in the feedstock carbohydrate levels affects the maximum theoretical biofuels yield and may influence the optimum pretreatment or saccharification conditions. The aim of this study is to assess the extent to which commercial hybrid corn stover composition varies and begin to partition the variation among genetic, environmental, or annual influences. A rapid compositional analysis method using near-infrared spectroscopy/partial least squares multivariate modeling (NIR/PLS) was used to evaluate compositional variation among 508 commercial hybrid corn stover samples collected from 47 sites in eight Corn Belt states after the 2001, 2002, and 2003 harvests. The major components of the corn stover, reported as average (standard deviation) % dry weight, whole biomass basis, were glucan 31.9 (2.0), xylan 18.9 (1.3), solubles composite 17.9 (4.1), and lignin (corrected for protein) 13.3 (1.1). We observed wide variability in the major corn stover components. Much of the variation observed in the structural components (on a whole biomass basis) is due to the large variation found in the soluble components. Analysis of variance (ANOVA) showed that the harvest year had the strongest effect on corn stover compositional variation, followed by location and then variety. The NIR/PLS rapid analysis method used here is well suited to testing large numbers of samples, as tested in this study, and will support feedstock improvement and biofuels process research.     

Enzyme production, growth, and adaptation of T. reesei strains QM9414, L-27, RL-P37, and rut C-30 to conditioned yellow poplar sawdust hydrolysate

National Renewable Energy Laboratory (NREL) has developed a conditioning process that decreases acetic acid levels in pretreated yellow poplar hydrolysate. Trichoderma reesei is sensitive to acetic acid and this conditioning method has enabled applied cellulase production with hardwoods. T. reesei strains QM9414, L-27, RL-P37, and Rut C-30 were screened for growth on conditioned hydrolysate liquor. Tolerance to hydrolysate was found to be strain-dependent. Strain QM9414 was adapted to grow in 80% (v/v) conditioned hydrolysate (40 g/L of soluble sugars and 1.6 g/L acetic acid from pretreated poplar). However, enzyme production was highest at 20% (v/v) hydrolysateusing strain L-27. Cellulasetiters of 2–3 International Filter Paper Units (IFPU)/mL were achieved using pretreated yellow poplar liquors and solids as the sole carbon sources.     

MRI gradient fields increase brain mannitol space

Following nephrectomy and intravenous injection of tritiated mannitol, adult male rats were exposed to magnetic resonance imaging (MRI) procedures at 1.5 T, 0.5 T, and 0.3 T. Compared to rats similarly handled but not exposed to MRI procedures, brain mannitol concentration, expressed as a percentage of mean body concentration, was significantly increased at 0.3 T and 0.5 T but not at 1.5 T. At 0.3 T, exposure to gradient-field fluctuations used for imaging increased brain mannitol concentration, but exposures to static main field and pulsed radiofrequency energies did not. Increased brain mannitol associated with gradient-field flux may reflect increased blood-brain barrier permeability or blood volume in brain. MRI effects on brain mannitol space are of uncertain clinical significance, but are consistent with prior evidence of an MRI-induced increase of brain capillary endothelial cell transport observed with horseradish peroxidase. Further studies are needed to confirm these findings and to explore the processes underlying changes in mannitol distribution related to MRI.     

Photoactive Analogs of Farnesyl Pyrophosphate Containing Benzoylbenzoate Esters: Synthesis and Application to Photoaffinity Labeling of Yeast Protein Farnesyltransferase

Farnesyl pyrophosphate (FPP) is involved in a large number of cellular processes including the prenylation of transforming mutants of Ras proteins implicated in cancer. Photoactive analogs could provide useful information about enzyme active sites that bind farnesyl pyrophosphate; however, the availability of such compounds is extremely limited. Molecules that incorporate benzophenone moieties are attractive photoaffinity labeling reagents because of their useful photochemical properties. Here, the syntheses of two compounds, 3a and 3b, containing para- and meta-substituted benzoylbenzoates are described. Compounds 3a and 3b are competitive inhibitors (with respect to FPP) of yeast protein farnesyltransferase (PFTase) with K(i) values of 910 and 380 nM, respectively. Both compounds inactivate PFTase upon photolysis, resulting in as much as 44% inactivation of enzyme activity. Photolysis of PFTase in the presence of [(32)P]3a or of [(32)P]3b results in preferential labeling of the beta subunit, suggesting that this subunit is involved in prenyl group recognition. These compounds should be valuable tools for studying enzymes that utilize FPP as a substrate.