Independent cerebral vasoconstrictive effects of hyperoxia and accompanying arterial hypocapnia at 1 ATA.

Breathing 100% O2 at 1 atmosphere absolute (ATA) is known to be associated with a decrease in cerebral blood flow (CBF). It is also accompanied by a fall in arterial Pco2 leading to uncertainty as to whether the cerebral vasoconstriction is totally or only in part caused by arterial hypocapnia. We tested the hypothesis that the increase in arterial Po2 while O2 was breathed at 1.0 ATA decreases CBF independently of a concurrent fall in arterial Pco2. CBF was measured in seven healthy men aged 21-62 yr by using noninvasive continuous arterial spin-labeled-perfusion MRI. The tracer in this technique, magnetically labeled protons in blood, has a half-life of seconds, allowing repetitive measurements over short time frames without contamination. CBF and arterial blood gases were measured while breathing air, 100% O2, and 4 and 6% CO2 in air and O2 backgrounds. Arterial Po2 increased from 91.7 +/- 6.8 Torr in air to 576.7 +/- 18.9 Torr in O2. Arterial Pco2 fell from 43.3 +/- 1.8 Torr in air to 40.2 +/- 3.3 Torr in O2. CBF-arterial Pco2 response curves for the air and hyperoxic runs were nearly parallel and separated by a distance representing a 28.7-32.6% decrement in CBF. Regression analysis confirmed the independent cerebral vasoconstrictive effect of increased arterial Po2. The present results also demonstrate that the magnitude of this effect at 1.0 ATA is greater than previously measured.     

Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

Identification of the Wzx flippase,Wzy polymerase and sugar-modifying enzymes for spore coat polysaccharide biosynthesis in Myxococcus xanthus

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N-acetylgalactosamine-1-P to undecaprenyl-phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export.