Bacterial mesosomes: Method dependent artifacts

Ebersold, Hans Rudolf; Cordier, J.-L.; Lüthy, Peter

doi:10.1007/bf00527066

Cited by 87 publications

(48 citation statements)

References 14 publications

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“…Although mesosomes are artifactual structures resulting from chemical fixation, they are only inducible under certain physiological conditions (5,6). Their constant occurrence after 60 min of incubation with sublethal NCT concentrations provides strong indirect evidence for alterations related to the bacterial cell membrane.…”

Section: Discussionmentioning

confidence: 99%

Bactericidal Activity of Micromolar N -Chlorotaurine: Evidence for Its Antimicrobial Function in the Human Defense System

Nagl¹,

Hess

Pfaller

et al. 2000

Antimicrob Agents Chemother

104

108

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N-Chlorotaurine, the main representative of long-lived oxidants found in the supernatant of stimulated granulocytes, has been investigated systematically with regard to its antibacterial activity at different physiological concentrations for the first time. N-Chlorotaurine (12.5 to 50 M) demonstrated a bactericidal effect i.e., a 2 to 4 log 10 reduction in viable counts, after incubation at 37°C for 6 to 9 h at pH 7.0, which effect was significantly enhanced in an acidic milieu (at pH 5.0), with a 3 to 4 log 10 reduction after 2 to 3 h. Moreover, bacteria were attenuated after being incubated in N-chlorotaurine for a sublethal time, as demonstrated with the mouse peritonitis model. The supernatant of stimulated granulocytes exhibited similar activity. Transmission electron microscopy revealed changes in the bacterial cell membrane and cytoplasmic disintegration with both reacting systems, even in the case of a mere attenuation. The results of this study suggest a significant bactericidal function of N-chlorotaurine and other chloramines during inflammation.

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Section: Discussionmentioning

confidence: 99%

Bactericidal Activity of Micromolar N -Chlorotaurine: Evidence for Its Antimicrobial Function in the Human Defense System

Nagl¹,

Hess

Pfaller

et al. 2000

Antimicrob Agents Chemother

104

108

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“…Variations in fixation regimens have also been reported to dramatically alter the appearance of procaryotes (1,7,15). The most exhaustive study to date relied on extraction of lipids from Acholeplasma laidlawii, an atypical bacterium that lacks a cell wall.…”

Section: * Corresponding Authormentioning

confidence: 99%

Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria

1990

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Freeze-substitution and more conventional embedding protocols were evaluated for their accurate preservation of eubacterial ultrastructure. Radioisotopes were specifically incorporated into the RNA, DNA, peptidoglycan, and lipopolysaccharide of two isogenic derivatives of Escherichia coli K-12 as representative gram-negative eubacteria and into the RNA and peptidoglycan of Bacillus subtilis strains 168 and W23 as representative gram-positive eubacteria. Radiolabeled bacteria were processed for electron microscopy by conventional methods with glutaraldehyde fixation, osmium tetroxide postfixation, dehydration in either a graded acetone or ethanol series, and infiltration in either Spurr or Epon 812 resin. A second set of cells were simultaneously freeze-substituted by plunge-freezing in liquid propane, substituting in anhydrous acetone containing 2% (wt/vol) osmium tetroxide, and 2% (wt/vol) uranyl acetate, and infiltrating in Epon 812. Extraction of radiolabeled cell components was monitored by liquid scintillation counting at all stages of processing to indicate retention of cell labels. Electron microscopy was also used to visually confirm ultrastructural integrity. Radiolabeled nucleic acids and wail components were extracted by both methods. In conventionally embedded specimens, dehydration was particularly damaging, with ethanol-dehydrated cells losing significantly more radiolabeled material during dehydration and subsequent infiltration than acetonetreated cells. For freeze-substituted specimens, postsubstitution washes in acetone were the most deleterious step for gram-negative cells, while infiltration was more damaging for gram-positive cells. Autoradiographs of specimens collected during freeze-substitution were scanned with an optical densitometer to provide an indication of freezing damage; the majority of label lost from freeze-substituted cells was a result of poor freezing to approximately one-half of the cell population, thus accounting for the relatively high levels of radiolabel detected in the processing fluids. These experiments revealed that gram-positive and gram-negative cells respond differently to freezing; these differences are discussed with reference to wall structure. It was apparent that the cells frozen first (i.e., the first to contact the cryogen) retained the highest percentage of all radioisotopes, and the highest level of cellular infrastructure, indicative of better preservation. The preservation of these select cells was far superior to that obtained by more conventional techniques.Although freeze-substitution yields images which are strikingly different from those obtained by more conventional techniques and which have profoundly affected current views of bacterial ultrastructure and the native distribution of cellular constituents (11), freeze-substitution has been used only infrequently in the study of procaryotic structure. Several factors are responsible for the limited use of freeze-substitution. The technique requires expertise in ultrarapid freezing, the necessary e...

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“…In secondary tegumentation and envelopment, very few homologies are known, e.g., in the innermost tegument and the glycoproteins (10). Most models of herpesvirus morphogenesis rely on transmission electron microscopy (TEM) data acquired by chemical fixation of cell cultures, which has been shown to induce several artifacts (2,14,22). In recent years, cryofixation methods have been established for routine use and now represent a state of the art of biological specimen preparation for a Localization of nuclear budding events and primary enveloped particles.…”

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confidence: 99%

Cytomegalovirus Primary Envelopment Occurs at Large Infoldings of the Inner Nuclear Membrane

Buser

Walther

Mertens

et al. 2007

J Virol

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We have investigated the morphogenesis of human and murine cytomegalovirus by transmission electron microscopy after high-pressure freezing, freeze substitution, and plastic embedding. We observed large tubular infoldings of the inner nuclear membrane that were free of lamina and active in primary envelopment and subsequent transport of capsids to the nuclear periphery. Semiquantitative determinations of the enlarged inner nuclear membrane area and the location of the primary envelopment of nucleocapsids demonstrated that this structure represents a virus-induced specialized membrane domain at which the particles are preferentially enveloped. This is a previously undescribed structural element relevant in cytomegalovirus morphogenesis.Murine cytomegalovirus (MCMV) and human CMV (HCMV) are members of the Betaherpesvirinae. Both viruses encode more than 200 open reading frames (15). The morphogenesis of herpesviruses is a complex process involving multiple interactions between viral and cellular components, especially membranes, and thus is of high interest for both virology and cell biology. The stepwise assembly of the virion has been studied extensively in alphaherpesviruses (10,11,18,20). However, much less is known about the morphogenesis of CMVs (3,12). When the findings from alphaherpesviruses are compared with those from cytomegaloviruses, it has to be kept in mind that the sequence homology is only partial and that the latter viruses have a larger coding capacity. Since the size of all herpesvirus capsids prevents their transport into the cytoplasm through the nuclear pore complex, nuclear egress requires the penetration of the nuclear membranes and the nuclear lamina, probably through an envelopment/de-envelopment process, which is still under debate (11,24). This may also be different for CMVs, since the involved alphaherpesviral kinase US3 is not conserved in betaherpesviruses (7, 16). For MCMV, it has been shown that the viral protein M50 inserts into the inner nuclear membrane and is aggregated by a second viral protein, M53, to form the putative capsid docking site (13). M50 then recruits cellular protein kinases for phosphorylation and dissolution of the nuclear lamina. Additionally, it has been shown for HCMV that pUL97 in concert with the cellular p32 acts by the redistribution of lamina components (9). While the list of viral proteins involved in this process is growing, little ultrastructural information on nuclear egress is available. After release to the cytoplasm, CMV capsids are tegumented and enveloped at Golgi apparatus-derived cisternae (6) by a wrapping process and released by fusion with the plasma membrane.We have investigated cytomegalovirus nuclear egress by electron microscopy using high-pressure freezing, freeze substitution, and plastic embedding. Fibroblast cell monolayers (3T3 murine fibroblasts for MCMV or human foreskin fibroblasts for HCMV) were grown on carbon-coated sapphire discs (3-mm diameter; Engineering Office M. Wohlwend GmbH, Switzerland) and infected at a multiplic...

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Bacterial mesosomes: Method dependent artifacts

Cited by 87 publications

References 14 publications

Bactericidal Activity of Micromolar N -Chlorotaurine: Evidence for Its Antimicrobial Function in the Human Defense System

Bactericidal Activity of Micromolar N -Chlorotaurine: Evidence for Its Antimicrobial Function in the Human Defense System

Evaluation of freeze-substitution and conventional embedding protocols for routine electron microscopic processing of eubacteria

Cytomegalovirus Primary Envelopment Occurs at Large Infoldings of the Inner Nuclear Membrane

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