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.     

Metabolic Enzyme Alterations and Astrocyte Dysfunction in a Murine Model of Alexander Disease With Severe Reactive Gliosis

Alexander disease (AxD) is a rare and fatal neurodegenerative disorder caused by mutations in the gene encoding glial fibrillary acidic protein (GFAP). In this report, a mouse model of AxD (GFAP^Tg;Gfap^+/R236H) was analyzed that contains a heterozygous R236H point mutation in murine Gfap as well as a transgene with a GFAP promoter to overexpress human GFAP. Using label-free quantitative proteomic comparisons of brain tissue from GFAP^Tg;Gfap^+/R236H versus wild-type mice confirmed upregulation of the glutathione metabolism pathway and indicated proteins were elevated in the peroxisome proliferator-activated receptor (PPAR) signaling pathway, which had not been reported previously in AxD. Relative protein-level differences were confirmed by a targeted proteomics assay, including proteins related to astrocytes and oligodendrocytes. Of particular interest was the decreased level of the oligodendrocyte protein, 2-hydroxyacylsphingosine 1-beta-galactosyltransferase (Ugt8), since Ugt8-deficient mice exhibit a phenotype similar to GFAP^Tg;Gfap^+/R236H mice (e.g., tremors, ataxia, hind-limb paralysis). In addition, decreased levels of myelin-associated proteins were found in the GFAP^Tg;Gfap^+/R236H mice, consistent with the role of Ugt8 in myelin synthesis. Fabp7 upregulation in GFAP^Tg;Gfap^+/R236H mice was also selected for further investigation due to its uncharacterized association to AxD, critical function in astrocyte proliferation, and functional ability to inhibit the anti-inflammatory PPAR signaling pathway in models of amyotrophic lateral sclerosis (ALS). Within Gfap⁺ astrocytes, Fabp7 was markedly increased in the hippocampus, a brain region subjected to extensive pathology and chronic reactive gliosis in GFAP^Tg;Gfap^+/R236H mice. Last, to determine whether the findings in GFAP^Tg;Gfap^+/R236H mice are present in the human condition, AxD patient and control samples were analyzed by Western blot, which indicated that Type I AxD patients have a significant fourfold upregulation of FABP7. However, immunohistochemistry analysis showed that UGT8 accumulates in AxD patient subpial brain regions where abundant amounts of Rosenthal fibers are located, which was not observed in the GFAP^Tg;Gfap^+/R236H mice.     

The influence of cell surface properties of thermophilic streptococci on attachment to stainlesssteel

The quality of milk products is threatened by the formation of biofilms of thermophilicstreptococci on the internal surfaces of plate heat exchangers used in milk processing. Althoughattachment to stainless steel surfaces is one of the first stages in the development of a biofilm, themechanisms involved in attachment have not been reported. The cell surface properties of 12strains of thermophilic streptococci were examined to determine their importance in attachment tostainless steel surfaces. Hydrophobicity, extracellular polysaccharide production and cell surfacecharge varied between the different strains but could not be related to numbers attaching. Treatingthe cells with sodium metaperiodate, lysozyme or trichloroacetic acid to disrupt cell surfacepolysaccharide had no effect on attachment. Treatment with trypsin or sodium dodecyl sulphate toremove cell surface proteins resulted in a 100-fold reduction in the number of bacteria attaching.This result suggests that the surface proteins of the thermophilic streptococci are important intheir attachment to stainless steel.     

Use of the Malthus conductance growth analyser to determine numbers of thermophilic streptococci on stainless steel

The use of the Malthus conductance growth analyser for the detection of Streptococcus bovis attached to stainless steel surfaces was evaluated. A comparison between the results from acridine orange epifluorescence direct counts, swab recovery viable count and conductance estimates of attached cell concentrations, based on calibrations for planktonic cells, showed that the conductance results were up to 2 log₁₀ greater than the epifluorescence results and the swab counts. The growth rates of planktonic and attached cells were similar over 16 h using the Malthus technique. This suggests that the Malthus technique detects more attached cells of Strep. bovis than epifluorescence microscopy or swab recovery.