91 research outputs found
Time-Lapse Imaging of the Dynamics of CNS Glial-Axonal Interactions In Vitro and Ex Vivo
Myelination is an exquisite and dynamic example of heterologous cell-cell interaction, which consists of the concentric wrapping of multiple layers of oligodendrocyte membrane around neuronal axons. Understanding the mechanism by which oligodendrocytes ensheath axons may bring us closer to designing strategies to promote remyelination in demyelinating diseases. The main aim of this study was to follow glial-axonal interactions over time both in vitro and ex vivo to visualize the various stages of myelination.We took two approaches to follow myelination over time: i) time-lapse imaging of mixed CNS myelinating cultures generated from mouse spinal cord to which exogenous GFP-labelled murine cells were added, and ii) ex vivo imaging of the spinal cord of shiverer (Mbp mutant) mice, transplanted with GFP-labelled murine neurospheres. We demonstrate that oligodendrocyte-axonal interactions are dynamic events with continuous retraction and extension of oligodendroglial processes. Using cytoplasmic and membrane-GFP labelled cells to examine different components of the myelin-like sheath, we provide evidence from time-lapse fluorescence microscopy and confocal microscopy that the oligodendrocytes' cytoplasm-filled processes initially spiral around the axon in a corkscrew-like manner. This is followed subsequently by focal expansion of the corkscrew process to form short cuffs, which then extend longitudinally along the axons. We predict from this model that these spiral cuffs must extend over each other first before extending to form internodes of myelin.These experiments show the feasibility of visualizing the dynamics of glial-axonal interaction during myelination over time. Moreover, these approaches complement each other with the in vitro approach allowing visualization of an entire internodal length of myelin and the ex vivo approach validating the in vitro data
Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus
<p>Abstract</p> <p>Background</p> <p>Community acquired (CA) methicillin-resistant <it>Staphylococcus aureus </it>(MRSA) increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear.</p> <p>Results</p> <p>We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA), isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS) and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300-MRSA 2658 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb) plasmids were identified. USA300-MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid found in USA300-MRSA. Two regions found in US300-MRSA were absent in USA300-MSSA. One of these carried the arginine deiminase operon that appears to have been acquired from <it>S. epidermidis</it>. The USA300 sequence was aligned with other sequenced <it>S. aureus </it>genomes and regions unique to USA300 MRSA were identified.</p> <p>Conclusion</p> <p>USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens.</p
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Fine Structural Localization of Cholesterol-1,2-3h in Degenerating and Regenerating Mouse Sciatic Nerve
The localization of 3H-labeled cholesterol in nerves undergoing degeneration and regeneration was studied by radioautography at the electron microscope level. Two types of experiments were carried out: (a) Cholesterol-1,2-3H was injected intraperitoneally into suckling mice. 5 wk later, Wallerian degeneration was induced in the middle branch of the sciatic nerve, carefully preserving the collateral branches. The animals were then sacrificed at various times after the operation. During degeneration, radioactivity was found over myelin debris and fat droplets. In early stages of regeneration, radioactivity was found in myelin debris and regenerating myelin sheaths. Afterwards, radioactivity was found predominantly over the regenerated myelin sheaths. Radioactivity was also associated with the myelin sheaths of the unaltered fibers, (b) Wallerian degeneration was induced in the middle branch of the sciatic nerves of an adult mouse, preserving the collateral branches. Cholesterol-1,2-3H was injected 24 and 48 hr after the operation and the animal was sacrificed 6 wk later. Radioactivity was found in the myelin sheaths of the regenerated and unaltered fibers. The results from these experiments indicate that: (a) exogenous cholesterol incorporated into peripheral nerve during myelination remains within the nerve when it undergoes degeneration. Such cholesterol is kept in the myelin debris as an exchangeable pool from which it is reutilized for the formation of the newly regenerating fibers, especially myelin. (b) exogenous cholesterol incorporated into the nerves at the time that degeneration is beginning is also used in the formation of new myelin sheaths during regeneration, (c) mature myelin maintains its ability to incorporate cholesterol
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