Transmission Electron Microscopy

Burghardt, Robert C.; Droleskey, Robert E.

doi:10.1002/9780471729259.mc02b01s03

Cited by 37 publications

(27 citation statements)

References 13 publications

(13 reference statements)

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“…Cultured Ube3a KD and scrambled control Clone 9 cells were grown on chambered Thermanox slides (Nunc) and fixed, osmicated, stained en bloc , and dehydrated as described (Burghardt and Droleskey, 2006). Infiltration was carried out with Epon diluted in 100% ethanol on the chambered slide.…”

Section: Methodsmentioning

confidence: 99%

The Angelman Syndrome Protein Ube3a/E6AP Is Required for Golgi Acidification and Surface Protein Sialylation

Condon

Robinson

et al. 2013

J. Neurosci.

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Angelman syndrome (AS) is a severe disorder of postnatal brain development caused by neuron-specific loss of the HECT (homologous to E6AP carboxy terminus) domain E3 ubiquitin ligase Ube3a/E6AP. The cellular role of Ube3a remains enigmatic despite recent descriptions of synaptic and behavioral deficits in AS mouse models. Although neuron-specific imprinting is thought to limit the disease to the brain, Ube3a is expressed ubiquitously, suggesting a broader role in cellular function. In the current study, we demonstrate a profound structural disruption and cisternal swelling of the Golgi apparatus (GA) in the cortex of AS (UBE3Am−/p+) mice. In Ube3a knockdown cell lines and UBE3Am−/p+ cortical neurons, the GA is severely under-acidified, leading to osmotic swelling. Both in vitro and in vivo, the loss of Ube3a and corresponding elevated pH of the GA is associated with a marked reduction in protein sialylation, a process highly dependent on intralumenal Golgi pH. Altered ion homeostasis of the GA may provide a common cellular pathophysiology underlying the diverse plasticity and neurodevelopmental deficits associated with AS.

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Section: Methodsmentioning

confidence: 99%

The Angelman Syndrome Protein Ube3a/E6AP Is Required for Golgi Acidification and Surface Protein Sialylation

Condon

Robinson

et al. 2013

J. Neurosci.

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“…The grids were then dried and viewed using a Philips CM100 BioTWIN transmission electron microscope (Philips/FEI Corporation, Eindhoven, Netherlands). Images were captured with a MegaView III digital camera (Olympus Soft Imaging Solutions GmbH, Muenster, Germany) (20). The number of bacterial cells with a flagellum was counted per 100 cells.…”

Section: Extended Transcriptional Analysis Measured By Reverse Transcmentioning

confidence: 99%

Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

Lawley

Sims

Tannock

2013

Appl Environ Microbiol

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“…Immunogold localization was done as described earlier [18] at the Transmission Electron Microscopy Facility, Advanced Instrumentation Research Facility, JNU, New Delhi. Briefly , cells from log-phase cultures of M. bovis and M. smegmatis strains were harvested and washed with 0.1 M phosphate buffer.…”

Section: Methodsmentioning

confidence: 99%

Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovisis regulated in response to nitrogen availability

2013

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BackgroundThe cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis.ResultsWe observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain.ConclusionsThe physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1 was more efficient in biofilm formation than the wild type strain. This indicates that PLG layer favors biofilm formation. This study demonstrate that the nitrogen availability not only regulates GS expression and activity in M. bovis but also affects cell surface properties by modulating synthesis of PLG.

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Transmission Electron Microscopy

Cited by 37 publications

References 13 publications

The Angelman Syndrome Protein Ube3a/E6AP Is Required for Golgi Acidification and Surface Protein Sialylation

The Angelman Syndrome Protein Ube3a/E6AP Is Required for Golgi Acidification and Surface Protein Sialylation

Whole-Transcriptome Shotgun Sequencing (RNA-seq) Screen Reveals Upregulation of Cellobiose and Motility Operons of Lactobacillus ruminis L5 during Growth on Tetrasaccharides Derived from Barley β-Glucan

Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovisis regulated in response to nitrogen availability

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