Microwave and Digital Imaging Technology Reduce Turnaround Times for Diagnostic Electron Microscopy

Giberson, Richard T.; Austin, Ronald L.; Charlesworth, Jon; Adamson, Grete N.; Herrera, Guillermo A.

doi:10.1080/01913120309937

Cited by 50 publications

(44 citation statements)

References 8 publications

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“…The HEp-2 cell pellets were next postfixed in 2% aqueous osmium tetroxide for 1 h. Dehydration was then performed in graded acetone, followed by embedding in epoxy resin. Osmium fixation, dehydration, and embedding were conducted in a Pelco Biowave microwave oven (Pelco International, Redding, CA) similar to the procedure described by Giberson et al (14). One-micrometer-thick sections were stained with toluidine blue, and ultrathin sections were stained with uranyl acetate and lead citrate.…”

Section: Methodsmentioning

confidence: 99%

Cholesterol-Enriched Membrane Microdomains Are Required for Inducing Host Cell Cytoskeleton Rearrangements in Response to Attaching-Effacing Escherichia coli

2005

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The diarrheal pathogens enterohemorrhagic Escherichia coli (EHEC) O157:H7 strain CL56 and enteropathogenic Escherichia coli (EPEC) O127:H6 strain E2348/69 adhere intimately to epithelial cells through attaching-effacing lesions, which are characterized by rearrangements of the host cytoskeleton, intimate adherence, and destruction of microvilli. These cytoskeletal responses require activation of host signal transduction pathways. Lipid rafts are signaling microdomains enriched in sphingolipid and cholesterol in the plasma membrane. The effect of perturbing plasma membrane cholesterol on bacterial intimate adherence was assessed. Infection of both HEp-2 cells and primary skin fibroblasts with strains CL56 and E2348/69 caused characteristic rearrangements of the cytoskeleton at sites of bacterial adhesion. CL56-and E2348/69-induced cytoskeletal rearrangements were inhibited following cholesterol depletion. Addition of exogenous cholesterol to depleted HEp-2 cells restored cholesterol levels and rescued bacterially induced ␣-actinin mobilization. Quantitative bacterial adherence assays showed that EPEC adherence to HEp-2 cells was dramatically reduced following cholesterol depletion, whereas the adherence of EHEC remained high. Cytoskeletal rearrangements on skin fibroblasts obtained from children with Niemann-Pick type C disease were markedly reduced. These findings indicate that host membrane cholesterol contained in lipid rafts is necessary for the cytoskeletal rearrangements following infection with attaching-effacing Escherichia coli. Differences in initial adherence indicate divergent roles for host membrane cholesterol in the pathogenesis of EHEC and EPEC infections.

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Section: Methodsmentioning

confidence: 99%

Cholesterol-Enriched Membrane Microdomains Are Required for Inducing Host Cell Cytoskeleton Rearrangements in Response to Attaching-Effacing Escherichia coli

2005

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“…The preparation of cyanobacterial cells for electron microscopy was based on the procedure described by Giberson et al (7). Samples were prepared using a scientific microwave (Ted Pella, Inc., Redding, Calif.) equipped with a variable wattage control and a PELCO Coldspot water recirculator to maintain constant temperature in the oven.…”

Section: Methodsmentioning

confidence: 99%

Pleiotropic Effect of a Histidine Kinase on Carbohydrate Metabolism in Synechocystis sp. Strain PCC 6803 and Its Requirement for Heterotrophic Growth

Singh

Sherman

2005

J Bacteriol

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The deletion of a gene coding for a histidine kinase (sll0750, Hik8) in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 resulted in a conditional lethal phenotype with a pleiotropic effect on the expression of genes involved in glucose metabolism. This mutant had comparable doubling times to wild type (WT) in continuous-light-grown photoautotrophic and mixotrophic cultures, whereas it grew poorly under mixotrophic conditions with different light and dark cycles. Growth was completely stopped, and cells eventually died, when the light duration was less than 6 h on a 24-h regimen. Northern blot analysis demonstrated that steady-state transcript levels of genes encoding key enzymes of glycolysis, gluconeogenesis, the oxidative pentose phosphate pathway, and glycogen metabolism were significantly altered in a strain with mutant hik8 (⌬hik8) grown with or without glucose. In some cases, differential expression was dependent on growth conditions (photoautotrophic versus mixotrophic). The enzyme activities of glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and phosphofructokinase were significantly reduced in ⌬hik8 compared to WT. Glycogen determination indicated that ⌬hik8 accumulated glycogen under mixotrophic conditions but was unable to utilize these reserves for heterotrophic growth. The results suggest that the loss of gap1 transcription in the absence of Hik8 was the key factor that rendered cells unable to catabolize glucose and grow heterotrophically. Additionally, the transcript levels of the phytochrome gene (cph1) and its cotranscribed response regulator gene (rcp1) were significantly reduced and its dark inducibility was lost in ⌬hik8. The results demonstrated that Hik8 plays an important role in glucose metabolism and is necessary for heterotrophic growth.

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“…After washing with buffer followed by water, cells were fixed again with reduced osmium (1% OsO 4 ϩ 1.5% K 3 Fe(CN) 6 in distilled water). Fixations were carried out in a Pella Model 3451 Scientific Microwave with variable wattage and a cold-spot attachment (Ted Pella, Inc. Redding, CA) at settings of 182 watts and 5 mm Hg vacuum using 40 s for each fixation step with 30 s rest (40). Samples were then dehydrated through a graded ethanol series and infiltrated through a graded ethanol/resin series followed by 3 changes of 100% LX-112 epoxy resin with accelerator (LADD Research Industries, Burlington, VT).…”

Section: Methodsmentioning

confidence: 99%

Recognition and Ubiquitination of Salmonella Type III Effector SopA by a Ubiquitin E3 Ligase, HsRMA1

Zhang¹,

Higashide²,

Dai

et al. 2005

Journal of Biological Chemistry

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Salmonella translocate bacterial effectors into host cells to confer bacterial entry and survival. It is not known how the host cells cope with the influx of these effectors. We report here that the Salmonella effector, SopA, interacts with host HsRMA1, a ubiquitin E3 ligase with a previously unknown function. SopA is ubiquitinated and degraded by the HsRMA1-mediated ubiquitination pathway. A sopA mutant escapes out of the Salmonella-containing vacuoles less frequently to the cytosol than wild type Salmonella in HeLa cells in a HsRMA1-dependent manner. Our data suggest that efficient bacterial escape into the cytosol of epithelial cells requires HsRMA1-mediated SopA ubiquitination and contributes to Salmonella-induced enteropathogenicity.Pathogenic Salmonella cause food poisoning, gastrointestinal inflammation, typhoid fever, and septicemia in humans. Salmonella enterica serovar typhimurium (S. typhimurium) encodes two Type III secretion systems within the Salmonella pathogenicity islands 1 and 2 (SPI-1 3 and SPI-2) that are required for Salmonella entry and subsequent survival inside the host, respectively (1-5). Among the secreted proteins in SPI-1, SipA, SipC, SopE, SopE2, and SopB (also known as SigD) were found to be responsible for promoting bacterial entry by modulating the host actin cytoskeleton (6 -11). SPI-2 effectors are responsible for subsequent Salmonella survival inside the host cells by modulating bacterial trafficking (12-16). In addition, SPI-1 effectors SipA, SopA, SopB, SopD, SopE, and SopE2 are largely responsible for inducing inflammation and diarrhea in animal models (17-19) through yet undefined mechanisms.Ubiquitination is the main protein degradation pathway that governs a variety of cellular processes including cell cycle, vesicle trafficking, and signal transduction (20). Ubiquitination involves a multienzyme cascade consisting of classes of enzymes known as E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3 (ubiquitin protein ligase) (20). Ubiquitinated proteins are either rapidly degraded by the 26 S proteasome (21) or targeted to various specific cellular compartments (20,22). The ubiquitin ligases (E3) play pivotal roles in defining the specificity of proteins targeted for ubiquitination.HsRMA1 (RING finger protein with membrane anchor, also named RNF5) is a newly identified membrane-bound ubiquitin E3 ligase belonging to the novel RING finger protein family (23-26). HsRMA1 is well conserved in higher eukaryotes but not present in the yeast genome. It has been reported that Arabidopsis thaliana AtRMA1 is able to complement the yeast temperature-sensitive, secretion-deficient sec15 mutation (23). One of the late-acting sec genes is sec15, which is involved in the vesicle trafficking from the Golgi to the plasma membrane (27, 28). Recently, HsRMA1 was reported to interact with paxillin to alter the localization of paxillin to regulate cell motility (29). The importance of ubiquitination in the secretory pathway and endocytosis has also been described (20). ...

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Microwave and Digital Imaging Technology Reduce Turnaround Times for Diagnostic Electron Microscopy

Cited by 50 publications

References 8 publications

Cholesterol-Enriched Membrane Microdomains Are Required for Inducing Host Cell Cytoskeleton Rearrangements in Response to Attaching-Effacing Escherichia coli

Cholesterol-Enriched Membrane Microdomains Are Required for Inducing Host Cell Cytoskeleton Rearrangements in Response to Attaching-Effacing Escherichia coli

Pleiotropic Effect of a Histidine Kinase on Carbohydrate Metabolism in Synechocystis sp. Strain PCC 6803 and Its Requirement for Heterotrophic Growth

Recognition and Ubiquitination of Salmonella Type III Effector SopA by a Ubiquitin E3 Ligase, HsRMA1

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