SorLA in Glia: Shared Subcellular Distribution Patterns with Caveolin-1

SorLA is an established sorting and trafficking protein in neurons with demonstrated relevance to Alzheimer’s disease (AD). It shares these roles with the caveolins, markers of membrane rafts microdomains. To further our knowledge on sorLA’s expression and traffic, we studied sorLA expression in various cultured glia and its relation to caveolin-1 (cav-1), a caveolar microdomain marker. RT-PCR and immunoblots demonstrated sorLA expression in rat C6 glioma, primary cultures of rat astrocytes (PCRA), and human astrocytoma 1321N1 cells. PCRA were determined to express the highest levels of sorLA’s message. Induction of differentiation of C6 cells into an astrocyte-like phenotype led to a significant decrease in sorLA’s mRNA and protein expression. A set of complementary experimental approaches establish that sorLA and cav-1 directly or indirectly interact in glia: (1) co-fractionation in light-density membrane raft fractions of rat C6 glioma, PCRA, and human 1321N1 astrocytoma cells; (2) a subcellular co-localization distribution pattern in vesicular perinuclear compartments seen via confocal imaging in C6 and PCRA; (3) additional confocal analysis in C6 cells suggesting that the perinuclear compartments correspond to their co-localization in early endosomes and the trans-Golgi; and; (4) co-immunoprecipitation data strongly supporting their direct or indirect physical interaction. These findings further establish that sorLA is expressed in glia and that it shares its subcellular distribution pattern with cav-1. A direct or indirect cav-1/sorLA interaction could modify the trafficking and sorting functions of sorLA in glia and its proposed neuroprotective role in AD.     

The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain

Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections.     

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.     

Effects of modified cellulose nanocrystals on the barrier and migration properties of PLA nano-biocomposites

The aim of this paper is to report the impact of the addition of cellulose nanocrystals on the barrier properties and on the migration behaviour of poly(lactic acid), PLA, based nano-biocomposites prepared by the solvent casting method. Their microstructure, crystallinity, barrier and overall migration properties were investigated. Pristine (CNC) and surfactant-modified cellulose nanocrystals (s-CNC) were used, and the effect of the cellulose modification and content in the nano-biocomposites was investigated. The presence of surfactant on the nanocrystal surface favours the dispersion of CNC in the PLA matrix. Electron microscopy analysis shows the good dispersion of s-CNC in the nanoscale with well-defined single crystals indicating that the surfactant allowed a better interaction between the cellulose structures and the PLA matrix. Reductions of 34% in water permeability were obtained for the cast films containing 1wt.% of s-CNC while good oxygen barrier properties were detected for nano-biocomposites with both 1wt.% and 5wt.% of modified and un-modified cellulose nanocrystals, underlining the improvement provided by cellulose on the PLA films. Moreover, the migration level of the studied nano-biocomposites was below the overall migration limits required by the current normative for food packaging materials in both non-polar and polar simulants.     

Complete genome sequence of Bifidobacterium breve CECT 7263, a strain isolated from human milk

Bifidobacterium breve is an actinobacterium frequently isolated from colonic microbiota of breastfeeding babies. Here, we report the complete and annotated genome sequence of a B. breve strain isolated from human milk, B. breve CECT 7263. The genome sequence will provide new insights into the biology of this potential probiotic organism and will allow the characterization of genes related to beneficial properties.     

Role of active site histidines in the two half-reactions of the aryl-alcohol oxidase catalytic cycle

The crystal structure of aryl-alcohol oxidase (AAO), a flavoenzyme involved in lignin degradation, reveals two active-site histidines, whose role in the two enzyme half-reactions was investigated. The redox state of flavin during turnover of the variants obtained show a stronger histidine involvement in the reductive than in the oxidative half-reaction. This was confirmed by the k(cat)/K(m(Al)) and reduction constants that are 2-3 orders of magnitude decreased for the His546 variants and up to 5 orders for the His502 variants, while the corresponding O(2) constants only decreased up to 1 order of magnitude. These results confirm His502 as the catalytic base in the AAO reductive half-reaction. The solvent kinetic isotope effect (KIE) revealed that hydroxyl proton abstraction is partially limiting the reaction, while the α-deuterated alcohol KIE showed a stereoselective hydride transfer. Concerning the oxidative half-reaction, directed mutagenesis and computational simulations indicate that only His502 is involved. Quantum mechanical/molecular mechanical (QM/MM) reveals an initial partial electron transfer from the reduced FADH(-) to O(2), without formation of a flavin-hydroperoxide intermediate. Reaction follows with a nearly barrierless His502H(+) proton transfer that decreases the triplet/singlet gap. Spin inversion and second electron transfer, concomitant with a slower proton transfer from flavin N5, yields H(2)O(2). No solvent KIE was found for O(2) reduction confirming that the His502 proton transfer does not limit the oxidative half-reaction. However, the small KIE on k(cat)/K(m(Ox)), during steady-state oxidation of α-deuterated alcohol, suggests that the second proton transfer from N5H is partially limiting, as predicted by the QM/MM simulations.