Near ultraviolet light inactivation of dihydroxyacid dehydratase in Escherichia coli

The effects of near ultraviolet (NUV) light on a NUV chromophore-containing oxidant-sensitive enzyme, dihydroxyacid dehydratase (DHAD), were measured in seven strains of Escherichia coli. The strains differed in production of the oxidant-defense enzymes, superoxide dismutases (Fe-SOD and Mn-SOD), and catalases HPI and HPII. With the stress of aerobic growth but without NUV exposure, the strains lacking either Fe or Mn SOD or both SODs had 57%, 25%, and 12%, respectively, of the DHAD-specific activity of the parent (K12) strain. Under the same conditions, the catalase strains that were wild type, overproducing, and deficient had comparable DHAD-specific activities. When aerobic cultures were exposed for 30 min to NUV with a fluence of 216 J/m²/s at 310–400 nm, the percentage decreases in DHAD-specific activities were similar (ranging from 75% to 89%) in strains with none, either, or both SODs missing, and in the catalase-overproducing strain. However, the decreases were only 58% and 52% in the strain with catalase missing and in its parent, respectively. The NUV-induced loss of DHAD enzyme activity was not accompanied by any detectable loss of the DHAD protein as measured by polyclonal antibody to DHAD.     

High Resolution Proton NMR Spectroscopy of Multiple Sclerosis Lesions

Abstract: Tissue from postmortem multiple sclerosis and normal control brains was extracted with perchloric acid and analysed using proton NMR spectroscopy. The content of N -acetyl-derived groups (the sum of N -acetylaspartate, acetate, and N -acetylaspartylglutamate) was decreased in multiple sclerosis plaques compared with normal control white matter (mean, 4.36 vs. 6.64 µmol/g wet weight). In normal appearing white matter adjacent to plaques a corresponding decrease was seen, with no change in white matter distant from plaques. A decrease in the content of total creatine was observed in multiple sclerosis plaques in comparison with normal control white matter (mean, 4.64 vs. 6.56 µmol/g wet weight), which correlated strongly with the decrease in N -acetyl-derived groups. No changes in other metabolites such as total choline or myo -inositol were seen. The decreases in content of N -acetyl-derived groups are in agreement with observations from in vivo proton NMR spectroscopy in multiple sclerosis patients. The decrease in total creatine is in contrast to most of the observations made in vivo where total creatine is assumed to be unchanged and metabolite levels are often expressed as a total creatine ratio. The use of a total creatine ratio in vivo could lead to an underestimation of reductions in N -acetylaspartate and an apparent increase in other metabolites in the multiple sclerosis lesion.     

Force-induced melting of DNA—evidence for peeling and internal melting from force spectra on short synthetic duplex sequences

Overstretching of DNA occurs at about 60–70 pN when a torsionally unconstrained double-stranded DNA molecule is stretched by its ends. During the transition, the contour length increases by up to 70% without complete strand dissociation. Three mechanisms are thought to be involved: force-induced melting into single-stranded DNA where either one or both strands carry the tension, or a B-to-S transition into a longer, still base-paired conformation. We stretch sequence-designed oligonucleotides in an effort to isolate the three processes, focusing on force-induced melting. By introducing site-specific inter-strand cross-links in one or both ends of a 64 bp AT-rich duplex we could repeatedly follow the two melting processes at 5 mM and 1 M monovalent salt. We find that when one end is sealed the AT-rich sequence undergoes peeling exhibiting hysteresis at low and high salt. When both ends are sealed the AT sequence instead undergoes internal melting. Thirdly, the peeling melting is studied in a composite oligonucleotide where the same AT-rich sequence is concatenated to a GC-rich sequence known to undergo a B-to-S transition rather than melting. The construct then first melts in the AT-rich part followed at higher forces by a B-to-S transition in the GC-part, indicating that DNA overstretching modes are additive.     

Phenotypic Profiling of Scedosporium aurantiacum,an Opportunistic Pathogen Colonizing Human Lungs

Genotyping studies of Australian Scedosporium isolates have revealed the strong prevalence of a recently described species: Scedosporium aurantiacum. In addition to occurring in the environment, this fungus is also known to colonise the respiratory tracts of cystic fibrosis (CF) patients. A high throughput Phenotype Microarray (PM) analysis using 94 assorted substrates (sugars, amino acids, hexose-acids and carboxylic acids) was carried out for four isolates exhibiting different levels of virulence, determined using a Galleria mellonella infection model. A significant difference was observed in the substrate utilisation patterns of strains displaying differential virulence. For example, certain sugars such as sucrose (saccharose) were utilised only by low virulence strains whereas some sugar derivatives such as D-turanose promoted respiration only in the more virulent strains. Strains with a higher level of virulence also displayed flexibility and metabolic adaptability at two different temperature conditions tested (28 and 37°C). Phenotype microarray data were integrated with the whole-genome sequence data of S. aurantiacum to reconstruct a pathway map for the metabolism of selected substrates to further elucidate differences between the strains.     

High-Resolution Functional Profiling of the Norovirus Genome

Human noroviruses (HuNoV) are a major cause of nonbacterial gastroenteritis worldwide, yet details of the life cycle and replication of HuNoV are relatively unknown due to the lack of an efficient cell culture system. Studies with murine norovirus (MNV), which can be propagated in permissive cells, have begun to probe different aspects of the norovirus life cycle; however, our understanding of the specific functions of the viral proteins lags far behind that of other RNA viruses. Genome-wide functional profiling by insertional mutagenesis can reveal protein domains essential for replication and can lead to generation of tagged viruses, which has not yet been achieved for noroviruses. Here, transposon-mediated insertional mutagenesis was used to create 5 libraries of mutagenized MNV infectious clones, each containing a 15-nucleotide sequence randomly inserted within a defined region of the genome. Infectious virus was recovered from each library and was subsequently passaged in cell culture to determine the effect of each insertion by insertion-specific fluorescent PCR profiling. Genome-wide profiling of over 2,000 insertions revealed essential protein domains and confirmed known functional motifs. As validation, several insertion sites were introduced into a wild-type clone, successfully allowing the recovery of infectious virus. Screening of a number of reporter proteins and epitope tags led to the generation of the first infectious epitope-tagged noroviruses carrying the FLAG epitope tag in either NS4 or VP2. Subsequent work confirmed that epitope-tagged fully infectious noroviruses may be of use in the dissection of the molecular interactions that occur within the viral replication complex.     

Function of minor polypeptides in foot-and-mouth disease virus and poliovirus

Foot-and-mouth disease virus and poliovirus each contain several minor polypeptides, in addition to the four structural proteins. One of these, the viral RNA polymerase, can also act as a nuclease, hydrolysing the RNA and thus destroying viral infectivity. It is tightly bound to the RNA and may be the packaging signal for assembly of the particle.     

Capillary zone electrophoresis for the determination of atovaquone in serum

A rapid and simple capillary zone electrophoresis (CZE) method has been developed for the determination of atovaquone in serum. The drug was extracted from equine serum–chloroform (1:3, v/v) at greater than 80% recovery and assayed in buffer containing 25 mM sodium borate (pH 9.1) and 25% acetonitrile. A 100 μm I.D. fused-silica capillary was used and the detection was by UV-diode array at 254 nm; the migration time was approximately 8 min. Intra- and inter-assay variabilities were less than 7.8% and 5.8%, respectively, and the accuracy of the assay (expressed as % bias) ranged from 4.5 to −5.2%. The working assay range was from 2 to 100 μg/ml. This sensitivity could be increased by concentrating during the extraction procedure. Replacement of acetonitrile with 75 mM surfactant 3-(dimethyldodecylammonio)propanesulfonate gave similar sensitivity and provided an additional option to facilitate the separation of atovaquone on multiple-drug samples.