Mitochondrial Plasmids of the Gaeumannomyces-Phialophora Complex and Their Detection by Primed, in Situ Fluorescence Labeling

1995 ◽  
Vol 19 (4) ◽  
pp. 263-274 ◽  
Author(s):  
Joan M. Henson ◽  
The Can Caesar-TonThat
Keyword(s):  
Fluorescence Labeling ◽  
Mitochondrial Plasmids

scholarly journals Localization of ‘Candidatus Liberibacter solanacearum’ and Evidence for Surface Appendages in the Potato Psyllid Vector

2016 ◽  
Vol 106 (2) ◽  
pp. 142-154 ◽  
Author(s):  
J. M. Cicero ◽  
T. W. Fisher ◽  
J. K. Brown
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Fluorescence Labeling ◽  
Potato Psyllid ◽  
Bactericera Cockerelli ◽  
Visual Identification ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Identification Of Bacteria

The potato psyllid Bactericera cockerelli is implicated as the vector of the causal agent of zebra chip of potato and vein-greening of tomato diseases. Until now, visual identification of bacteria in the genus ‘Candidatus Liberibacter’ has relied on direct imaging by light and electron microscopy without labeling, or with whole-organ fluorescence labeling only. In this study, aldehyde fixative followed by a coagulant fixative, was used to process adult psyllids for transmission electron microscopy (TEM) colloidal gold in situ hybridization experiments. Results indicated that ‘Ca. Liberibacter solanacearum’ (CLso)-specific DNA probes annealed to a bacterium that formed extensive, monocultural biofilms on gut, salivary gland, and oral region tissues, confirming that it is one morphotype of potentially others, that is rod-shaped, approximately 2.5 µm in diameter and of variable length, and has a rough, granular cytosol. In addition, CLso, prepared from shredded midguts, and negatively stained for TEM, possessed pili- and flagella-like surface appendages. Genes implicating coding capacity for both types of surface structures are encoded in the CLso genome sequence. Neither type was seen for CLso associated with biofilms within or on digestive organs, suggesting that their production is stimulated only in certain environments, putatively, in the gut during adhesion leading to multiplication, and in hemolymph to afford systemic invasion.

scholarly journals The Isotope Array, a New Tool That Employs Substrate-Mediated Labeling of rRNA for Determination of Microbial Community Structure and Function

2003 ◽  
Vol 69 (11) ◽  
pp. 6875-6887 ◽  
Author(s):  
Justyna Adamczyk ◽  
Martin Hesselsoe ◽  
Niels Iversen ◽  
Matthias Horn ◽  
Angelika Lehner ◽  
...  
Keyword(s):  
Community Structure ◽  
Activated Sludge ◽  
Fluorescence Labeling ◽  
Specific Substrate ◽  
Ammonia Oxidizing Bacteria ◽  
Microarray Hybridization ◽  
Independent Manner ◽  
And Function

ABSTRACT A new microarray method, the isotope array approach, for identifying microorganisms which consume a 14C-labeled substrate within complex microbial communities was developed. Experiments were performed with a small microarray consisting of oligonucleotide probes targeting the 16S rRNA of ammonia-oxidizing bacteria (AOB). Total RNA was extracted from a pure culture of Nitrosomonas eutropha grown in the presence of [14C]bicarbonate. After fluorescence labeling of the RNA and microarray hybridization, scanning of all probe spots for fluorescence and radioactivity revealed that specific signals were obtained and that the incorporation of 14C into rRNA could be detected unambiguously. Subsequently, we were able to demonstrate the suitability of the isotope array approach for monitoring community composition and CO2 fixation activity of AOB in two nitrifying activated-sludge samples which were incubated with [14C]bicarbonate for up to 26 h. AOB community structure in the activated-sludge samples, as predicted by the microarray hybridization pattern, was confirmed by quantitative fluorescence in situ hybridization (FISH) and comparative amoA sequence analyses. CO2 fixation activities of the AOB populations within the complex activated-sludge communities were detectable on the microarray by 14C incorporation and were confirmed independently by combining FISH and microautoradiography. AOB rRNA from activated sludge incubated with radioactive bicarbonate in the presence of allylthiourea as an inhibitor of AOB activity showed no incorporation of 14C and thus was not detectable on the radioactivity scans of the microarray. These results suggest that the isotope array can be used in a PCR-independent manner to exploit the high parallelism and discriminatory power of microarrays for the direct identification of microorganisms which consume a specific substrate in the environment.

scholarly journals Enzymology and Ultrastructure of the in situ Pellicle in Caries-Active and Caries-Inactive Patients

2017 ◽  
Vol 51 (2) ◽  
pp. 109-118 ◽  
Author(s):  
Jasmin Kirsch ◽  
Christian Hannig ◽  
Sandra Pötschke ◽  
Sabine Basche ◽  
William H. Bowen ◽  
...  
Keyword(s):  
Exposure Time ◽  
Enzyme Activities ◽  
Fluorescence Labeling ◽  
Oral Exposure ◽  
Pellicle Formation ◽  
Caries Activity ◽  
Transmission Electron ◽  
High Uniformity ◽  
The Impact

Aim: The present study aimed to evaluate the impact of caries activity on the key enzymes and the ultrastructure of the in situ pellicle. Methods: Pellicle formation was performed on bovine enamel slabs. Intraoral exposure (3, 30, and 120 min) was accomplished by 14 caries-active (DMFS: 22.7 ± 12.1) and 13 caries-inactive (DMFS: 1.5 ± 1.8) individuals. The enzyme activities (lysozyme, peroxidase, α-amylase, glycosyltransferase [GTF]) in the in situ pellicle and resting saliva of all participants were analyzed directly after oral exposure. In addition, a simultaneous visualization of these enzymes, extracellular glucans, and adherent bacteria was carried out. Fluorescent patterns were analyzed with fluorescence labeling and 4′,6-diamidino-2-phenylindole/concanavalin A staining. In addition, the distribution of GTF B, C, and D and the ultrastructure of the pellicle were examined by gold immunolabeling and transmission electron microscopy with selected samples. Results: Enzyme activities of amylase, peroxidase, lysozyme, and GTF were detected on all enamel slabs in an active conformation. Neither exposure time nor caries activity had an impact on the enzyme activities. Gold immunolabeling indicated that the pellicle of caries-active subjects tends to more GTF D molecules. The pellicles of caries-inactive and -active individuals revealed a similar ultrastructural pattern. Conclusion: The enzyme activities as well as the pellicle's ultrastructure are of high similarity in caries-active and -inactive subjects. Thereby, oral exposure time has no significant influence. This reflects a high uniformity during the initial phase of bioadhesion (3-120 min) concerning enzymatic functions. However, there is a tendency towards more GTF D in caries-active individuals.

In Situ Live-Cell Nucleus Fluorescence Labeling with Bioinspired Fluorescent Probes

2017 ◽  
Vol 89 (15) ◽  
pp. 7861-7868 ◽  
Author(s):  
Pan Ding ◽  
Houyu Wang ◽  
Bin Song ◽  
Xiaoyuan Ji ◽  
Yuanyuan Su ◽  
...  
Keyword(s):  
Cell Nucleus ◽  
Fluorescent Probes ◽  
Live Cell ◽  
Fluorescence Labeling

Photo-clickable microRNA for in situ fluorescence labeling and imaging of microRNA in living cells

2017 ◽  
Vol 53 (48) ◽  
pp. 6452-6455 ◽  
Author(s):  
Lei Huang ◽  
Yingjie Chen ◽  
Lei Chen ◽  
Xiao Xiao ◽  
Xingxing Wang ◽  
...  
Keyword(s):  
Living Cells ◽  
Fluorescence Labeling ◽  
Spatiotemporal Resolution

A photo-clickable microRNA was constructed for in situ fluorescence labeling and imaging of microRNA in living cells with spatiotemporal resolution.

scholarly journals Three Optimized Methods for In Situ Quantification of Progenitor Cell Proliferation in Embryonic Kidneys Using BrdU, EdU, and PCNA

2019 ◽  
Vol 6 ◽  
pp. 205435811987193
Author(s):  
Rosalie E. O’Hara ◽  
Michel G. Arsenault ◽  
Blanca P. Esparza Gonzalez ◽  
Ashley Patriquen ◽  
Sunny Hartwig
Keyword(s):  
Cell Proliferation ◽  
Progenitor Cells ◽  
Kidney Development ◽  
Fluorescence Labeling ◽  
Nuclear Antigen ◽  
Ureteric Bud ◽  
Proliferating Cells ◽  
Advantages And Disadvantages ◽  
Nephron Progenitor

Background: Nephron progenitor cells derived from the metanephric mesenchyme undergo a complex balance of self-renewal and differentiation throughout kidney development to give rise to the mature nephron. Cell proliferation is an important index of progenitor population dynamics. However, accurate and reproducible in situ quantification of cell proliferation within progenitor populations can be technically difficult to achieve due to the complexity and harsh tissue treatment required of certain protocols. Objective: To optimize and compare the performance of the 3 most accurate S phase–specific labeling methods used for in situ detection and quantification of nephron progenitor and ureteric bud cell proliferation in the developing kidney, namely, 5-bromo-2’-deoxyuridine (BrdU), 5-ethynyl-2’-deoxyuridine (EdU), and proliferating cell nuclear antigen (PCNA). Methods: Protocols for BrdU, EdU, and PCNA were optimized for fluorescence labeling on paraformaldehyde-fixed, paraffin-embedded mouse kidney tissue sections, with co-labeling of nephron progenitor cells and ureteric bud with Six2 and E-cadherin antibodies, respectively. Image processing and analysis, including quantification of proliferating cells, were carried out using free ImageJ software. Results: All 3 methods detect similar ratios of nephron progenitor and ureteric bud proliferating cells. The BrdU staining protocol is the lengthiest and most complex protocol to perform, requires tissue denaturation, and is most subject to interexperimental signal variability. In contrast, bound PCNA and EdU protocols are relatively more straightforward, consistently yield clear results, and far more easily lend themselves to co-staining; however, the bound PCNA protocol requires substantive additional postexperimental analysis to distinguish the punctate nuclear PCNA staining pattern characteristic of proliferating cells. Conclusions: All 3 markers exhibit distinct advantages and disadvantages in quantifying cell proliferation in kidney progenitor populations, with EdU and PCNA protocols being favored due to greater technical ease and reproducibility of results associated with these methods.

scholarly journals A versatile toolbox for semi-automatic cell-by-cell object-based colocalization analysis

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Anders Lunde ◽  
Joel C. Glover
Keyword(s):  
3D Visualization ◽  
Image Data ◽  
Cell Types ◽  
Cellular Heterogeneity ◽  
Cell Segmentation ◽  
Fluorescence Labeling ◽  
Data Sets ◽  
Object Based ◽  
Imagej Plugin

Abstract Differential fluorescence labeling and multi-fluorescence imaging followed by colocalization analysis is commonly used to investigate cellular heterogeneity in situ. This is particularly important when investigating the biology of tissues with diverse cell types. Object-based colocalization analysis (OBCA) tools can employ automatic approaches, which are sensitive to errors in cell segmentation, or manual approaches, which can be impractical and tedious. Here, we present a novel set of tools for OBCA using a semi-automatic approach, consisting of two ImageJ plugins, a Microsoft Excel macro, and a MATLAB script. One ImageJ plugin enables customizable processing of multichannel 3D images for enhanced visualization of features relevant to OBCA, and another enables semi-automatic colocalization quantification. The Excel macro and the MATLAB script enable data organization and 3D visualization of object data across image series. The tools are well suited for experiments involving complex and large image data sets, and can be used in combination or as individual components, allowing flexible, efficient and accurate OBCA. Here we demonstrate their utility in immunohistochemical analyses of the developing central nervous system, which is characterized by complexity in the number and distribution of cell types, and by high cell packing densities, which can both create challenging situations for OBCA.

scholarly journals Identification of FourEntamoeba histolyticaOrganellar DNA Polymerases of the Family B and Cellular Localization of theEhodp1Gene and EhODP1 Protein

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
María Esther Herrera-Aguirre ◽  
Juan Pedro Luna-Arias ◽  
María Luisa Labra-Barrios ◽  
Esther Orozco
Keyword(s):  
Cellular Localization ◽  
Polyclonal Antibodies ◽  
Dna Polymerases ◽  
Protozoan Parasite ◽  
Mitochondrial Plasmids ◽  
Cytoplasmic Dna ◽  
The Family ◽  
Cytoplasmic Structures ◽  
Active Genes

We report the identification of a family of four active genes (Ehodp1,Ehodp2,Ehodp3,andEhodp4) encoding putative DNA polymerases inEntamoeba histolytica, the protozoan parasite responsible of human amoebiasis. The fourEhodpgenes show similarity to DNA polymerases encoded in fungi and plant mitochondrial plasmids. EhODP polypeptides conserve the3′-5′exonuclease II and5′-3′polymerization domains, and they have the I, II, and III conserved boxes that characterize them as DNA polymerases of family B. Furthermore, we found in EhODP polymerases two novel A and B boxes, present also in DNA polymerases encoded in fungi mitochondrial plasmids. By in situ PCR,Ehodp1gene was located in nuclei and in DNA-containing cytoplasmic structures. Additionally, using polyclonal antibodies against a recombinant rEhODP1-168 polypeptide, and confocal microscopy, EhODP1 was located in cytoplasmic DNA-containing structures.

scholarly journals Correlative live and super-resolution imaging reveals the dynamic structure of replication domains

2018 ◽  
Vol 217 (6) ◽  
pp. 1973-1984 ◽  
Author(s):  
Wanqing Xiang ◽  
M. Julia Roberti ◽  
Jean-Karim Hériché ◽  
Sébastien Huet ◽  
Stephanie Alexander ◽  
...  
Keyword(s):  
Genome Mapping ◽  
Super Resolution ◽  
Dynamic Structure ◽  
Fluorescence Labeling ◽  
Biophysical Parameters ◽  
Mechanical Coupling ◽  
Resolution Imaging ◽  
Dynamic Description ◽  
Super Resolution Microscopy

Chromosome organization in higher eukaryotes controls gene expression, DNA replication, and DNA repair. Genome mapping has revealed the functional units of chromatin at the submegabase scale as self-interacting regions called topologically associating domains (TADs) and showed they correspond to replication domains (RDs). A quantitative structural and dynamic description of RD behavior in the nucleus is, however, missing because visualization of dynamic subdiffraction-sized RDs remains challenging. Using fluorescence labeling of RDs combined with correlative live and super-resolution microscopy in situ, we determined biophysical parameters to characterize the internal organization, spacing, and mechanical coupling of RDs. We found that RDs are typically 150 nm in size and contain four co-replicating regions spaced 60 nm apart. Spatially neighboring RDs are spaced 300 nm apart and connected by highly flexible linker regions that couple their motion only <550 nm. Our pipeline allows a robust quantitative characterization of chromosome structure in situ and provides important biophysical parameters to understand general principles of chromatin organization.

scholarly journals Protein-AMPylierungs-Identifikation in lebenden Zellen

BIOspektrum ◽  
2020 ◽  
Vol 26 (7) ◽  
pp. 743-746
Author(s):  
Tobias Becker ◽  
Pavel Kielkowski
Keyword(s):  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Neural Development ◽  
Click Reaction ◽  
Living Cells ◽  
Fluorescence Labeling ◽  
Post Translational Modification ◽  
Design Synthesis ◽  
Stress Regulation

AbstractProtein AMPylation is a prevalent protein post-translational modification in human cells involved in endoplasmic reticulum stress regulation and neural development. In this article we describe the design, synthesis and application of a pronucleotide probe suitable for in situ fluorescence imaging and chemical protemics profiling of AMPylated proteins. Our probe utilizes straightforward strain-promoted azidealkyne click reaction for fluorescence labeling in living cells.

Sign in / Sign up

Export Citation Format

Share Document