Trace elemental content of biological materials

The advantages and disadvantages of neutron activation analysis (NAA) and inductively coupled plasma-source mass spectrometry (ICP-MS) for the analysis of biological materials is reviewed. Comparison is made between NAA (instrumental) and ICP-MS (conventional pneumatic solution nebulization and laser ablation) analysis of the biological reference material National Bureau of Standards (NBS) SRM 1577 Bovine Liver. Relatively good agreement is achieved between the results for the 18 elements analyzed by both techniques and those either certified or reported in the literature. Elemental concentrations for Li, Mg, Al, Ca, Cr, Mn, Fe, Cu, Zn, Br, Rb, and Cs are also reported for IAEA Mixed Human Diet (H9), NBS SRM 909 Human Serum, and NBS SRM 1577a Bovine Liver, analyzed by solution nebulization ICP-MS.     

Novel mutation in hexokinase 2 confers resistance to 2-deoxyglucose by altering protein dynamics

Glucose is central to many biological processes, serving as an energy source and a building block for biosynthesis. After glucose enters the cell, hexokinases convert it to glucose-6-phosphate (Glc-6P) for use in anaerobic fermentation, aerobic oxidative phosphorylation, and the pentose-phosphate pathway. We here describe a genetic screen in Saccharomyces cerevisiae that generated a novel spontaneous mutation in hexokinase-2, hxk2^G238V, that confers resistance to the toxic glucose analog 2-deoxyglucose (2DG). Wild-type hexokinases convert 2DG to 2-deoxyglucose-6-phosphate (2DG-6P), but 2DG-6P cannot support downstream glycolysis, resulting in a cellular starvation-like response. Curiously, though the hxk2^G238V mutation encodes a loss-of-function allele, the affected amino acid does not interact directly with bound glucose, 2DG, or ATP. Molecular dynamics simulations suggest that Hxk2^G238V impedes sugar binding by altering the protein dynamics of the glucose-binding cleft, as well as the large-scale domain-closure motions required for catalysis. These findings shed new light on Hxk2 dynamics and highlight how allosteric changes can influence catalysis, providing new structural insights into this critical regulator of carbohydrate metabolism. Given that hexokinases are upregulated in some cancers and that 2DG and its derivatives have been studied in anti-cancer trials, the present work also provides insights that may apply to cancer biology and drug resistance.     

New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds

We have developed a new approach to create microsatellite primer sets that have high utility across a wide range of species. The success of this method was demonstrated using birds. We selected 35 avian EST microsatellite loci that had a high degree of sequence homology between the zebra finch Taeniopygia guttata and the chicken Gallus gallus and designed primer sets in which the primer bind sites were identical in both species. For 33 conserved primer sets, on average, 100% of loci amplified in each of 17 passerine species and 99% of loci in five non-passerine species. The genotyping of four individuals per species revealed that 24-76% (mean 48%) of loci were polymorphic in the passerines and 18-26% (mean 21%) in the non-passerines. When at least 17 individuals were genotyped per species for four Fringillidae finch species, 71-85% of loci were polymorphic, observed heterozygosity was above 0.50 for most loci and no locus deviated significantly from Hardy-Weinberg proportions. This new set of microsatellite markers is of higher cross-species utility than any set previously designed. The loci described are suitable for a range of applications that require polymorphic avian markers, including paternity and population studies. They will facilitate comparisons of bird genome organization, including genome mapping and studies of recombination, and allow comparisons of genetic variability between species whilst avoiding ascertainment bias. The costs and time to develop new loci can now be avoided for many applications in numerous species. Furthermore, our method can be readily used to develop microsatellite markers of high utility across other taxa.     

Exploration of the structure-activity relationship of the diaryl anilide class of ligands for translocator protein-potential novel positron emitting tomography imaging agents

A series of novel ligands based on the diaryl anilide (DAA) class of translocator protein (TSPO) ligands was synthesised and evaluated as potential positron emitting tomography (PET) ligands for imaging TPSO in vivo. Fluorine-18 labelling of the molecules was achieved using direct radiolabelling or synthon based labelling approaches. Several of the ligands prepared have promising profiles as potential TSPO PET imaging ligands and will be evaluated further as potential clinical imaging agents.     

An algorithm for analyzing linkages affected by heterozygous translocations: QuadMap

Heterozygous chromosome rearrangements such as reciprocal translocations are most accurately displayed as two-dimensional linkage maps. Standard linkage mapping software packages, such as MapMaker, generate only one-dimensional maps and so reciprocal translocations appear as clusters of markers, even though they originate from two nonhomologous chromosomes. To more accurately map these regions, researchers have developed statistical methods that use the variance in map distance to distinguish among the four segments (two translocation, two interstitial) of the translocation. In this study, we describe modifications to one of these protocols, that proposed by Livingstone et al. (2000). We also introduce QuadMap, a new software application for dissecting heterozygous translocation-affected linkage maps.     

Microaerophilic–aerobic sequential decolourization/biodegradation of textile azo dyes by a facultative Klebsiella sp. strain VN-31

Four different azo dyes were decolourized and biodegraded in a sequential microaerophilic–aerobic treatment by a facultative Klebsiella sp. strain VN-31, a bacterium isolated from activated sludge process of the textile industry. Dye decolourization was performed under microaerophilic conditions until no colour was observed (decolourization percentage >94%). The medium was then aerated to promote the biodegradation of the amines produced. The presence of aromatic amine in the microaerophilic stage and its absence in the aerobic stage demonstrate azo bond reduction and an oxidative biodegradation process, respectively. Total Organic Carbon (TOC) reduction for the growth medium plus dyes was ～50% in the microaerophilic stage and ～80% in the aerobic stage. The degradation products were also characterized by FT-IR and UV–vis techniques and their toxicity measured using Daphnia magna. The results provide evidence that the successive microaerophilic/aerobic stages, using a single Klebsiella sp. strain VN-31 in the same bioreactor, were able to form aromatic amines by the reductive break down of the azo bond and to oxidize them into non-toxic metabolites.     

An atomic level model for the interactions of molybdenum nitrogenase with carbon monoxide, acetylene, and ethylene

A combination of density functional theory and molecular mechanics calculations has been used to study the possible interactions of CO, C(2)H(2), and C(2)H(4) with the central Fe and terminal Mo sites of the iron-molybdenum cofactor of nitrogenase. The most favorable binding mode for CO on the central section of the FeMoco appears to be end-on to a single Fe and results in a change from high to low spin for the ligating Fe atom. If a coordination site for CO is available on the Mo, this becomes the preferred CO binding site. Calculated nu(CO) infrared frequencies are compared with the experimental values given in the literature. C(2)H(2) binds weakly in a side-on orientation to a single Fe site; addition of a single H(+)/e(-) couple to the substrate results in spontaneous migration of the resulting -CH=CH(2) group from Fe to a central S atom of the cofactor. Further reduction liberates C(2)H(4) or alternatively can give an S=CHCH(3) intermediate, which then goes on to produce C(2)H(6). A model for C(2)H(2) reduction by nitrogenase is proposed, based on the results of the calculations and the extensive literature on this process.     

An atomic-level mechanism for molybdenum nitrogenase. Part 2. Proton reduction,inhibition of dinitrogen reduction by dihydrogen,and the HD formation reaction

Quantum calculations have been used to examine the energetics of the reactions of diazene and isodiazene with H(2) and the properties of the Fe and Mo sites of the nitrogenase iron-molybdenum cofactor with respect to the binding of H and H(2). The results have been used to extend the model for N(2) reduction by nitrogenase given in the preceding paper to describe the formation of HD from D(2). The proposed mechanism for HD formation invokes a combination of two well-established chemical reactions, namely, competitive protonation of metal N(2) species at either the metal or at N(2), followed by scrambling of D(2) at a metal hydride. The model is evaluated against the available biochemical data for the nitrogenase HD formation reaction and extended to account for H(2) inhibition of N(2) reduction and the reduction of H(+) in the absence of other substrates.