Increased activin levels in cerebrospinal fluid of rabbits with bacterial meningitis are associated with activation of microglia

Activin, a member of the transforming growth factor superfamily, is upregulated in a number of inflammatory episodes such as septicemia and rheumatoid arthritis. In the CNS, activin has been predominantly assessed in terms of a neuroprotective role. In this report we characterized the activin response in the CNS in a rabbit model of meningitis. In normal animals, cerebrospinal fluid (CSF) activin levels were higher than those in serum, indicating an intracranial secretion of this cytokine. Following intracisternal inoculation with Streptococcus pneumoniae, activin in CSF was unchanged for the first 12 h and then rose progressively; levels were increased approximately 15-fold within 24 h. Activin levels were correlated positively with CSF protein content and with the number of apoptotic neurons in the dentate gyrus. No apparent correlation was observed between CSF activin concentrations and bacterial titer, lactate concentrations or leukocyte density. Using immunohistochemistry, activin staining was localized to epithelial cells of the choroid plexus, cortical neurons and the CA3 region of the hippocampus, with similar staining intensities in both normal and meningitic brains. However, in meningitic brains there was also strong staining in activated microglia and infiltrating macrophages. Taken together, these results demonstrate that activin forms part of the CNS response to immune challenge and may be an important mediator to modulate inflammatory processes in the brain.     

Classification of subcellular location by comparative proteomic analysis of native and density-shifted lysosomes

One approach to the functional characterization of the lysosome lies in the use of proteomic methods to identify proteins in subcellular fractions enriched for this organelle. However, distinguishing between true lysosomal residents and proteins from other cofractionating organelles is challenging. To this end, we implemented a quantitative mass spectrometry approach based on the selective decrease in the buoyant density of liver lysosomes that occurs when animals are treated with Triton-WR1339. Liver lysosome-enriched preparations from control and treated rats were fractionated by isopycnic sucrose density gradient centrifugation. Tryptic peptides derived from gradient fractions were reacted with isobaric tag for relative and absolute quantitation eight-plex labeling reagents and analyzed by two-dimensional liquid chromatography matrix-assisted laser desorption ionization time-of-flight MS. Reporter ion intensities were used to generate relative protein distribution profiles across both types of gradients. A distribution index was calculated for each identified protein and used to determine a probability of lysosomal residence by quadratic discriminant analysis. This analysis suggests that several proteins assigned to the lysosome in other proteomics studies are not true lysosomal residents. Conversely, results support lysosomal residency for other proteins that are either not or only tentatively assigned to this location. The density shift for two proteins, Cu/Zn superoxide dismutase and ATP-binding cassette subfamily B (MDR/TAP) member 6, was corroborated by quantitative Western blotting. Additional balance sheet analyses on differential centrifugation fractions revealed that Cu/Zn superoxide dismutase is predominantly cytosolic with a secondary lysosomal localization whereas ATP-binding cassette subfamily B (MDR/TAP) member 6 is predominantly lysosomal. These results establish a quantitative mass spectrometric/subcellular fractionation approach for identification of lysosomal proteins and underscore the necessity of balance sheet analysis for localization studies.     

Diversity surveys and evolutionary relationships of aoxB genes in aerobic arsenite-oxidizing bacteria

A new primer set was designed to specifically amplify ca. 1,100 bp of aoxB genes encoding the As(III) oxidase catalytic subunit from taxonomically diverse aerobic As(III)-oxidizing bacteria. Comparative analysis of AoxB protein sequences showed variable conservation levels and highlighted the conservation of essential amino acids and structural motifs. AoxB phylogeny of pure strains showed well-discriminated taxonomic groups and was similar to 16S rRNA phylogeny. Alphaproteobacteria-, Betaproteobacteria-, and Gammaproteobacteria-related sequences were retrieved from environmental surveys, demonstrating their prevalence in mesophilic As-contaminated soils. Our study underlines the usefulness of the aoxB gene as a functional marker of aerobic As(III) oxidizers.     

A Bacterial Ras-Like Small GTP-Binding Protein and Its Cognate GAP Establish a Dynamic Spatial Polarity Axis to Control Directed Motility

Regulated cell polarity is central to many cellular processes. We investigated the mechanisms that govern the rapid switching of cell polarity (reversals) during motility of the bacterium Myxococcus xanthus. Cellular reversals are mediated by pole-to-pole oscillations of motility proteins and the frequency of the oscillations is under the control of the Frz chemosensory system. However, the molecular mechanism that creates dynamic polarity remained to be characterized. In this work, we establish that polarization is regulated by the GTP cycle of a Ras-like GTPase, MglA. We initially sought an MglA regulator and purified a protein, MglB, which was found to activate GTP hydrolysis by MglA. Using live fluorescence microscopy, we show that MglA and MglB localize at opposite poles and oscillate oppositely when cells reverse. In absence of MglB, MglA-YFP accumulates at the lagging cell end, leading to a strikingly aberrant reversal cycle. Spatial control of MglA is achieved through the GAP activity of MglB because an MglA mutant that cannot hydrolyze GTP accumulates at the lagging cell end, despite the presence of MglB. Genetic and cell biological studies show that the MglA-GTP cycle controls dynamic polarity and the reversal switch. The study supports a model wherein a chemosensory signal transduction system (Frz) activates reversals by relieving a spatial inhibition at the back pole of the cells: reversals are allowed by Frz-activated switching of MglB to the opposite pole, allowing MglA-GTP to accumulate at the back of the cells and create the polarity switch. In summary, our results provide insight into how bacteria regulate their polarity dynamically, revealing unsuspected conserved regulations with eukaryots.     

Data standards, sense and stability: Scratchpads, the ICZN and ZooBank

The International Commission of Zoological Nomenclature has used the Scratchpads platform (currently being developed and maintained by ViBRANT) as the foundation for its redesigned website and as a platform for engaging with its users. The existing Scratchpad tools, with extensions to provide additional functions, have allowed for a major transformation in presentation of linked nomenclatural tools. Continued development of the new website will act as a springboard for the ICZN to participate more fully in the wider community of biodiversity informatics.     

The rare Chrysopidae (Neuroptera) of southwestern Europe

Quantitative surveys of the chrysopid fauna from southwestern Europe, namely the Iberian and Italian peninsulas, France south of 46° N, and the west-Mediterranean Islands, were analysed. A total of 56 species of Chrysopidae were reported, of which three species were abundant. These, Chrysoperla carnea (Stephens, 1836) sensu lato, Dichochrysa prasina (Burmeister, 1839) and D. flavifrons (Brauer, 1850), comprised a large percentage of the specimens. For the rarer species, comments are made on their distributions, the enhanced geographic range of exotic ones, and on levels of endemism and stenotopy.     

Ultrastructural detection of an unusual intranuclear bacterium in Pentastiridius leporinus (Hemiptera: Cixiidae)

Pentastiridius leporinus is an important vector of sugar beet pathogens in eastern France. An electron microscope survey on the insect-associated microflora revealed the occurrence of intranuclear prokaryotic cells in every internal organ analysed. These bacteria, which could also be found in the cytoplasm surrounding the nucleus, had a homogeneous coccoid (ca. 0.45 microm) or rod (0.45-1 microm) shape. The presence of three membrane layers was observed, the outermost forming a kind of vacuole containing generally a single microorganism. No cytopathological abnormalities were detected in the infected cells. To our knowledge, this is the first report of a hemipteran species infected by intranuclear bacteria. The possible identity of this microorganism is discussed.