scholarly journals Microscopic Photosensitization: A New Tool to Investigate the Role of Mitochondria in Cell Death

2002 ◽  
Vol 2 ◽  
pp. 1198-1208 ◽  
Author(s):  
May-Ghee Lum ◽  
Tetsuhiro Minamikawa ◽  
Phillip Nagley
Keyword(s):  
Cell Death ◽  
Laser Irradiation ◽  
Laser Scanning ◽  
Annexin V ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Rapid Loss ◽  
Active Involvement ◽  
Induction Of Apoptosis

Active involvement of mitochondria in cell death has been well-documented, but local apoptotic signaling between subsets of mitochondria has been poorly explored to date. Using mitochondrially localized CMXRos as a photosensitizer coupled to laser irradiation by confocal laser scanning microscopy, we demonstrate that partial irradiation of about half the mitochondria in human 143B TK–cells induces rapid loss of mitochondrial membrane potential (ΔΨm) in nonirradiated mitochondria. Cells so partially irradiated show apoptotic indications, including mobilization of cytochrome c and binding of annexin V within 2 h following irradiation. The loss of ΔΨm in nonirradiated mitochondria did not occur in cells photoirradiated in the absence of CMXRos. Increasing the proportion of irradiated mitochondria in each cell (up to about 50%) generated a correspondingly greater percentage of cells in which nonirradiated mitochondria lost ΔΨm and which also showed apoptotic indications. Only at the highest level of irradiation (global for all mitochondria in one cell) were signs of necrosis evident (judged by uptake of propidium iodide). Because laser irradiation is specific to the subpopulation of mitochondria targeted, the data imply that a signal emanating from irradiated mitochondria is processed by their nonirradiated counterparts. We conclude that intermitochondrial signaling occurs in the subcellular response to induction of apoptosis.

scholarly journals Potential role of nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase in apoptosis and oxidative stress

2001 ◽  
Vol 114 (9) ◽  
pp. 1643-1653 ◽  
Author(s):  
Z. Dastoor ◽  
J.L. Dreyer
Keyword(s):  
Oxidative Stress ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Apoptotic Cells ◽  
Transfected Cells ◽  
And Oxidative Stress

Recent studies indicating a role of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in apoptosis or oxidative stress has been reported. Using confocal laser-scanning microscopy, we have investigated the cellular distribution of GAPDH in central nervous system (CNS)-derived cells (neuroblastoma mNB41A3), in non-CNS derived cells (R6 fibroblast) and in an apoptosis-resistant Bcl2 overexpressing cell line (R6-Bcl2). Induction of apoptosis by staurosporine or MG132 and oxidative stress by H(2)O(2) or FeCN enhanced the nuclear translocation of endogenous GAPDH in all cell types, as detected by immunocytochemistry. In apoptotic cells, GAPDH expression is three times higher than in non-apoptotic cells. Consistent with a role for GAPDH in apoptosis, overexpression of a GAPDH-green fluorescent protein (GAPDH-GFP) hybrid increased nuclear import of GAPDH-GFP into transfected cells and the number of apoptotic cells, and made them more sensitive to agents that induce apoptosis. Bcl2 overexpression prevents nuclear translocation of GAPDH and apoptosis in untransfected cells, but not in transfected cells that overexpress GAPDH-GFP. Our observations indicate that nuclear translocation of GAPDH may play a role in apoptosis and oxidative stress, probably related to the activity of GAPDH as a DNA repair enzyme or as a nuclear carrier for pro-apoptotic molecules.

Detection and enumeration of microbial cells within highly porous calcareous reef sands

2006 ◽  
Vol 57 (4) ◽  
pp. 415 ◽  
Author(s):  
Christian Wild ◽  
Christian Laforsch ◽  
Markus Huettel
Keyword(s):  
Laser Scanning ◽  
Complex Surface ◽  
Porous Matrix ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Size Spectrum ◽  
Order Of Magnitude ◽  
Grain Surface ◽  
Highly Porous

In order to assess and to compare the abundances of prokaryotes in coral sands from three different areas in the Indo-Pacific, a technique was developed and evaluated for enumeration of prokaryotes living on and within calcareous grains. Propidium iodide labelling of prokaryotes and consecutive confocal laser scanning microscopy showed microbial colonisation within pores and small fissures of the coral sands. This embedded microbial colonisation required at least four extractions with weak acetic acid to dissolve the grain surface layer in order to detach 97% of the prokaryotic cells. Microbial enumeration based on this technique revealed that the abundance of prokaryotes in the carbonate sands were not significantly different among the three sites, but were about one order of magnitude higher than reported for silicate sands of a similar grain size spectrum. A possible reason for this high abundance of prokaryotes is the complex surface structure of the biogenic calcareous grains, their correspondingly highly porous matrix and the associated ability of prokaryotes to penetrate into carbonate grains. Our results highlight the role of calcareous reef sands as a substratum with a large specific surface area for prokaryotic colonisation and emphasise the contribution of calcium carbonate reef sands for element cycles in subtropical and tropical ecosystems.

scholarly journals Antibody response to the 45 kDa Candida albicans antigen in an animal model and potential role of the antigen in adherence

2008 ◽  
Vol 57 (12) ◽  
pp. 1466-1472 ◽  
Author(s):  
Helena Bujdáková ◽  
Ema Paulovičová ◽  
Silvia Borecká-Melkusová ◽  
Juraj Gašperík ◽  
Soňa Kucharíková ◽  
...  
Keyword(s):  
Animal Model ◽  
Candida Albicans ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Vaccine Development ◽  
Laser Scanning Microscopy ◽  
Model Systems ◽  
Confocal Laser

The Candida antigen CR3-RP (complement receptor 3-related protein) is supposed to be a ‘mimicry’ protein because of its ability to bind antibody directed against the α subunit of the mammalian CR3 (CD11b/CD18). This study aimed to (i) investigate the specific humoral isotypic response to immunization with CR3-RP in vivo in a rabbit animal model, and (ii) determine the role of CR3-RP in the adherence of Candida albicans in vitro using the model systems of buccal epithelial cells (BECs) and biofilm formation. The synthetic C. albicans peptide DINGGGATLPQ corresponding to 11 amino-acids of the CR3-RP sequence DINGGGATLPQALXQITGVIT, determined by N-terminal sequencing, was used for immunization of rabbits to obtain polyclonal anti-CR3-PR serum and for subsequent characterization of the humoral isotypic response of rabbits. A significant increase of IgG, IgA and IgM anti-CR3-RP specific antibodies was observed after the third (P<0.01) and the fourth (P<0.001) immunization doses. The elevation of IgA levels suggested peptide immunomodulation of the IgA1 subclass, presumably in coincidence with Candida epithelial adherence. Blocking CR3-RP with polyclonal anti-CR3-RP serum reduced the ability of Candida to adhere to BECs, in comparison with the control, by up to 35 % (P<0.001), and reduced biofilm formation by 28 % (P<0.001), including changes in biofilm thickness and integrity detected by confocal laser scanning microscopy. These properties of CR3-RP suggest that it has potential for future vaccine development.

Deletion of bem46 retards spore germination and may be related to the polar growth of Aspergillus fumigatus

2019 ◽  
Vol 58 (5) ◽  
pp. 690-697
Author(s):  
Yan Ma ◽  
Ying Ji ◽  
Jing Yang ◽  
Wen Li ◽  
Jiajuan Li ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Laser Scanning ◽  
Fluorescent Protein ◽  
Morphological Changes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Polarized Growth ◽  
Bud Emergence ◽  
Green Fluorescent

Abstract Bud emergence 46 (BEM46), a member of the α/β hydrolase superfamily, has been reported to be essential for polarized growth in Neurospora crassa. However, the role of BEM46 in aspergillus fumigatus (A. fumigatus) remains unclear. In this study, we constructed an A. fumigatus strain expressing BEM46 fused with enhanced green fluorescent protein, and a Δbem46 mutant, to explore the localization and the role of growth of BEM46 in A. fumigatus, respectively. Confocal laser scanning microscopy revealed that BEM46 was dominantly expressed in the sites where hyphae germinated from conidia in A. fumigatus. When compared with the control strain, the Δbem46 mutant exhibited insignificant morphological changes but delayed germination. No significant changes were found regarding the radial growth of both strains in response to various antifungal agents. These results suggest that BEM46 plays an essential role in timely germination in A. fumigatus. From the observation of fluorescence localization, we infer that that BEM46 might be involved in polarized growth in A. fumigatus.

scholarly journals High Adhesion and Increased Cell Death Contribute to Strong Biofilm Formation in Klebsiella pneumoniae

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 277
Author(s):  
Siddhi Desai ◽  
Kinjal Sanghrajka ◽  
Devarshi Gajjar
Keyword(s):  
Cell Death ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
High Adhesion ◽  
Biofilm Matrix

Klebsiella pneumoniae (Kp), is a frequent cause of hospital and community-acquired infections and WHO had declared it as a “priority pathogen”. Biofilm is a major virulence factor of Kp and yet the mechanism of strong biofilm formation in Kp is unclear. A key objective of the present study is to investigate the differences between strong and weak biofilms formed by clinical isolates of Kp on various catheters and in different media conditions and to identify constituents contributing to strong biofilm formation. Quantification of matrix components (extracellular DNA (eDNA), protein, exopolysaccharides (EPS), and bacterial cells), confocal laser scanning microscopy (CLSM), field emission gun scanning electron microscopy (FEG-SEM) and flow-cytometry analysis were performed to compare strong and weak biofilm matrix. Our results suggest increased biofilm formation on latex catheters compared to silicone and silicone-coated latex catheters. Higher amounts of eDNA, protein, EPS, and dead cells were observed in the strong biofilm of Kp. High adhesion capacity and cell death seem to play a major role in formation of strong Kp biofilms. The enhanced eDNA, EPS, and protein in the biofilm matrix appear as a consequence of increased cell death.

Neuron model system: Correlative confocal laser scanned microscopy and membrane physiology

1990 ◽  
Vol 48 (3) ◽  
pp. 170-171
Author(s):  
Donald H. Szarowski ◽  
Michael Fejtl ◽  
Paul McCauley ◽  
David O. Carpenter ◽  
James N. Turner
Keyword(s):  
Laser Scanning ◽  
Model System ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Patch Clamping ◽  
Surface Geometry ◽  
Receptor Stimulation ◽  
Membrane Physiology ◽  
Conventional Light Microscopy

Confocal laser scanning microscopy (CLSM) has been used to correlate morphology and membrane physiology in cultured neurons, providing a model system for studying physiologic and pathologic conditions. Ion channels are studied by patch-clamp methods as a function of receptor stimulation and toxic excitatory amino acids, including those implicated in Alzheimer’s dementia. Glial cells are often closely associated with the neurons, and are difficult to detect in living cultures due to the relative sizes of glia and neurons (5-20 μm versus 125 μm), compounded with the fact that they are thick phase objects. Groups of glia can also be confused with neurons. Thus it is difficult to select appropriate cells and/or cell regions for patch-clamping. We are correlating physiology and conventional light microscopy with CLSM to determine the role of glia, and neuron surface geometry on the ability to establish Gigaohm membrane-micropipette seals. Morphology of the system as observed by CLSM is presented here.

Time-resolved study of biofilm architecture and transport processes using experimental and simulation techniques: the role of EPS

2001 ◽  
Vol 43 (6) ◽  
pp. 143-151 ◽  
Author(s):  
M. Kuehn ◽  
M. Mehl ◽  
M. Hausner ◽  
H.-J. Bungartz ◽  
S. Wuertz
Keyword(s):  
Solute Transport ◽  
Confocal Laser Scanning Microscopy ◽  
Laser Scanning ◽  
Extracellular Polymeric Substances ◽  
Transport Processes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Scanning Microscopy ◽  
Confocal Laser

Cellular material and extracellular polymeric substances are the basic structural elements in biofilm systems. The structure and role of EPS for biofilm development and metabolic processes have not been precisely determined and, therefore, have not yet been included as a necessary element in modelling and simulation studies. This is due to the difficulty of experimentally detecting the extracellular polymeric substances in situ and differentiating them from cellular material on the one hand, and to the subsequent uncertainty about appropriate models - e.g. rigid hindrances, porous microstructure or visco-elastic structure - on the other hand. In this work, we report on the use of confocal laser scanning microscopy to monitor the development of a monoculture biofilm of Sphingomonas sp. grown in a flow cell. The bacterial strain was genetically labelled resulting in strong constitutive expression of the green fluorescent protein. The development of extracellular polymeric substances was followed bybinding of the lectin concavalin A to cell exopolysaccharides. The growth of the resulting strain was digitally recorded by automated confocal laser scanning microscopy. In addition, local velocity profiles of fluorescent carboxylate-modified microspheres were observed on pathlines throughout the biofilm. The CLSM image stacks were used as direct input for the explicit modelling and three-dimensional numerical simulation of flow fields and solute transport processes based on the conservation laws of continuum mechanics. At present, a strongly simplifying EPS-model is applied for numerical simulations. The EPSs are preliminarily assumed to behave like a rigid and dense hindrance with diffusive-reactive solute transport.

scholarly journals Pili and other surface proteins influence the structure and the nanomechanical properties of Lactococcus lactis biofilms

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ibrahima Drame ◽  
Christine Lafforgue ◽  
Cecile Formosa-Dague ◽  
Marie-Pierre Chapot-Chartier ◽  
Jean-Christophe Piard ◽  
...  
Keyword(s):  
Lactococcus Lactis ◽  
Laser Scanning ◽  
Binding Proteins ◽  
Binding Protein ◽  
Young Modulus ◽  
Surface Proteins ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
The Impact

AbstractLactic acid bacteria, in particular Lactococcus lactis, are widely used in the food industry, for the control and/or the protection of the manufacturing processes of fermented food. While L. lactis has been reported to form compact and uniform biofilms it was recently shown that certain strains able to display pili at their surface form more complex biofilms exhibiting heterogeneous and aerial structures. As the impact of those biofilm structures on the biomechanical properties of the biofilms is poorly understood, these were investigated using AFM force spectroscopy and imaging. Three types of strains were used i.e., a control strain devoid of pili and surface mucus-binding protein, a strain displaying pili but no mucus-binding proteins and a strain displaying both pili and a mucus-binding protein. To identify potential correlations between the nanomechanical measurements and the biofilm architecture, 24-h old biofilms were characterized by confocal laser scanning microscopy. Globally the strains devoid of pili displayed smoother and stiffer biofilms (Young Modulus of 4–100 kPa) than those of piliated strains (Young Modulus around 0.04–0.1 kPa). Additional display of a mucus-binding protein did not affect the biofilm stiffness but made the biofilm smoother and more compact. Finally, we demonstrated the role of pili in the biofilm cohesiveness by monitoring the homotypic adhesion of bacteria to the biofilm surface. These results will help to understand the role of pili and mucus-binding proteins withstanding external forces.

scholarly journals Role of Exopolysaccharides in Pseudomonas aeruginosa Biofilm Formation and Architecture

2011 ◽  
Vol 77 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Aamir Ghafoor ◽  
Iain D. Hay ◽  
Bernd H. A. Rehm
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Bacterial Biofilm ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Content Type ◽  
Biofilm Architecture

ABSTRACTPseudomonas aeruginosais an opportunistic human pathogen and has been established as a model organism to study bacterial biofilm formation. At least three exopolysaccharides (alginate, Psl, and Pel) contribute to the formation of biofilms in this organism. Here mutants deficient in the production of one or more of these polysaccharides were generated to investigate how these polymers interactively contribute to biofilm formation. Confocal laser scanning microscopy of biofilms formed in flow chambers showed that mutants deficient in alginate biosynthesis developed biofilms with a decreased proportion of viable cells than alginate-producing strains, indicating a role of alginate in viability of cells in biofilms. Alginate-deficient mutants showed enhanced extracellular DNA (eDNA)-containing surface structures impacting the biofilm architecture. PAO1 ΔpslAΔalg8overproduced Pel, and eDNA showing meshwork-like structures presumably based on an interaction between both polymers were observed. The formation of characteristic mushroom-like structures required both Psl and alginate, whereas Pel appeared to play a role in biofilm cell density and/or the compactness of the biofilm. Mutants producing only alginate, i.e., mutants deficient in both Psl and Pel production, lost their ability to form biofilms. A lack of Psl enhanced the production of Pel, and the absence of Pel enhanced the production of alginate. The function of Psl in attachment was independent of alginate and Pel. A 30% decrease in Psl promoter activity in the alginate-overproducing MucA-negative mutant PDO300 suggested inverse regulation of both biosynthesis operons. Overall, this study demonstrated that the various exopolysaccharides and eDNA interactively contribute to the biofilm architecture ofP. aeruginosa.

scholarly journals The Barley Apoptosis Suppressor Homologue Bax Inhibitor-1 Compromises Nonhost Penetration Resistance of Barley to the Inappropriate Pathogen Blumeria graminis f. sp. tritici

2004 ◽  
Vol 17 (5) ◽  
pp. 484-490 ◽  
Author(s):  
Ruth Eichmann ◽  
Holger Schultheiss ◽  
Karl-Heinz Kogel ◽  
Ralph Hückelhoven
Keyword(s):  
Cell Death ◽  
Laser Scanning ◽  
Penetration Resistance ◽  
Suppressor Gene ◽  
Blumeria Graminis ◽  
Epidermal Cells ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Over Expression ◽  
Enhanced Expression

BAX inhibitor-1 (BI-1) proteins have been characterized as suppressors of programmed cell death in mammals and plants. The barley BI-1 is a suppressor of nonspecific background resistance and mlo-mediated penetration resistance to the biotrophic fungal pathogen Blumeria graminis f. sp. hordei when overexpressed in epidermal cells of barley. We report here that BI-1 expression is also slightly up-regulated during interaction with the inappropriate wheat pathogen Blumeria graminis f. sp. tritici. Significantly, over-expression of BI-1 in single epidermal cells of barley by microprojectile-mediated transformation rendered cells susceptible to penetration by inappropriate B. graminis f. sp. tritici. The degree of transgene-induced accessibility to B. graminis f. sp. tritici was thereby similar to the effect achieved by overexpression of the defense suppressor gene Mlo and could not be further enhanced by double expression of both BI-1 and Mlo. Confocal laser scanning microscopy was used to locate a functional green fluorescing GFP:BI-1 fusion protein in endomembranes and the nuclear envelope of barley epidermal cells. Together, enhanced expression of barley BI-1 suppresses penetration resistance to B. graminis f. sp. tritici, linking barley nonhost resistance with cell death regulation.

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