Cellular Localization of Chemical Carcinogens Studied by Fluorescence Microscopy

Oncology ◽  
1980 ◽  
Vol 37 (1) ◽  
pp. 20-22 ◽  
Author(s):  
Claude Stora
Keyword(s):  
Fluorescence Microscopy ◽  
Cellular Localization ◽  
Chemical Carcinogens

Intracellular Localization of Two Betaine Lipids by Cell Fractionation and Immunomicroscopy

1997 ◽  
Vol 52 (7-8) ◽  
pp. 487-495 ◽  
Author(s):  
K. Department of Chemistry and Biochem ◽  
W. Department of Chemistry and Biochem ◽  
A. Faculty of Biology, University of B
Keyword(s):  
Fluorescence Microscopy ◽  
Cellular Localization ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Rabbit Serum ◽  
Cell Fractionation ◽  
Thin Sections ◽  
Ochromonas Danica ◽  
Betaine Lipids ◽  
Cyt C

Abstract The cellular localization of the betaine lipids diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) was investi­ gated by a) chemical analysis of subcellular fractions and b) immunochemical methods using specific antisera and either fluorescence microscopy or electron microscopy for detection of the label. A homogenate of Lycopodium annotinum (Pteridophyta) was fractionated by differential and density gradient centrifugation. The particulate fractions obtained were analyzed for chlorophyll, cyt c oxidase, NADH-cyt c reductase and DGTS. Non-plastidial fractions were enriched in DGTS and only minor amounts of this lipid could be attributed to chloroplasts. Anti-DGTS and anti-DGTA sera were produced by immunization of rabbits. The monospecificity of the antisera was examined with cells of Chlamydomonas reinhardtii (Chlorophyceae) containing DGTS, Pavlova lutheri (Haptophyceae) containing DGTA and Ochromonas danica (Chrysophyceae) containing both DGTS and DGTA. Euglena gracilis which is free of betaine lipids, was used as a control. For the test, a FITC-coupled goat anti-rabbit antibody was used and detected by fluorescence microscopy. Thin sections of Ochromonas and Pavlova were incubated first with the anti-lipid sera and subsequently with a gold-coupled anti-rabbit serum and then examined in the electron microscope. With O chro­ monas, anti-DGTS as well as anti-DGTA sera gave an accumulation of gold label in the cytoplasmic space but not in the chloroplasts. Similar results were obtained with Pavlova using anti-DGTA serum. These results describe for the first time the cytochemical localiza­ tion of DGTS and DGTA strongly suggesting both these lipids to be associated mainly with non-plastidial structures.

scholarly journals The Cellular Localization of the p42 and p46 Oligoadenylate Synthetase 1 Isoforms and Their Impact on Mitochondrial Respiration

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1122 ◽  
Author(s):  
Stig Skrivergaard ◽  
Monica Skou Jensen ◽  
Tine Breckling Rolander ◽  
Tram Bao Ngoc Nguyen ◽  
Amanda Bundgaard ◽  
...  
Keyword(s):  
High Resolution ◽  
Fluorescence Microscopy ◽  
Cellular Localization ◽  
Immunoblot Analysis ◽  
Rnase L ◽  
Oligoadenylate Synthetase ◽  
Innate Response ◽  
Viral Pathogens ◽  
The Difference ◽  
Caax Motif

The importance of the IFN-induced oligoadenylate synthetase (OAS) proteins and the OAS/RNase L pathway in the innate response against viral pathogens is well-established, however the observed differences in anti-viral activity between the human OAS1 p46 and p42 isoforms are not fully understood. The protein expression of these isoforms is determined by the SNP rs10774671, either being an A or a G allele resulting in expression of either the p42 or the p46 isoform. Using fluorescence microscopy and immunoblot analysis of fractionated cell samples, we show here that the CaaX motif is of key importance to the cellular localization. The OAS1 p42 isoform is mainly located in the cytosol, whereas the p46 isoform with a C-terminal CaaX motif is translocated to membranous organelles, like the mitochondria. We furthermore observed differences between p42 and p46 in their effect on mitochondrial physiology using high resolution respirometry and fluorometry. Overexpression of OAS1 p42 and IFN-β treatment of HeLa cells (AA genotype) resulted in significantly increased respiration, which was not seen with p46 overexpression. The difference in subcellular localization and mitochondrial effect of these two OAS1 isoforms might help to explain the anti-viral mechanisms that differentiate these proteins.

scholarly journals Using fluorescence microscopy to shed light on the mechanisms of antimicrobial peptides

2019 ◽  
Vol 11 (18) ◽  
pp. 2445-2458
Author(s):  
Anne K Buck ◽  
Donald E Elmore ◽  
Louise EO Darling
Keyword(s):  
Fluorescence Microscopy ◽  
Antimicrobial Peptides ◽  
Structure Function ◽  
Temporal Resolution ◽  
Cellular Localization ◽  
Bacterial Resistance ◽  
Mechanisms Of Action ◽  
Membrane Interaction ◽  
Research Questions ◽  
Shed Light

Antimicrobial peptides (AMPs) are promising in the fight against increasing bacterial resistance, but the development of AMPs with enhanced activity requires a thorough understanding of their mechanisms of action. Fluorescence microscopy is one of the most flexible and effective tools to characterize AMPs, particularly in its ability to measure the membrane interactions and cellular localization of peptides. Recent advances have increased the scope of research questions that can be addressed via microscopy through improving spatial and temporal resolution. Unique combinations of fluorescent labels and dyes can simultaneously consider different aspects of peptide–membrane interaction mechanisms. This review emphasizes the central role that fluorescence microscopy will continue to play in the interrogation of AMP structure–function relationships and the engineering of more potent peptides.

scholarly journals Type II Secretion System Secretin PulD Localizes in Clusters in the Escherichia coli Outer Membrane

2008 ◽  
Vol 191 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Martin Krehenbrink ◽  
Frédéric Pecorari ◽  
Anthony P. Pugsley
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscopy ◽  
Outer Membrane ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Sodium Dodecyl ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Ii Secretion System

ABSTRACT The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

scholarly journals CELLULAR LOCALIZATION OF GLUCAGON BY FLUORESCENCE MICROSCOPY REACTION OF NH2-TERMINAL HISTIDINE WITH o-PHTHALDIALDEHYDE

1973 ◽  
Vol 21 (1) ◽  
pp. 13-16 ◽  
Author(s):  
M. J. BRODY ◽  
R. HAKANSON ◽  
I. LUNDQUIST ◽  
C. OWMAN ◽  
F. SUNDLER
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescence Intensity ◽  
Guinea Pigs ◽  
Cellular Localization ◽  
Cobalt Chloride ◽  
Spectral Characteristics ◽  
Histidine Residue ◽  
Histochemical Detection ◽  
Intense Fluorescence ◽  
Concomitant Reduction

Exposure to o-phthaldialdehyde (OPT) causes intense fluorescence in A2-cells in sections from pancreas. Alloxan treatment of mice was followed by a marked increase in the number and fluorescence intensity of the OPT-reactive A2-cells, whereas cobalt chloride given to guinea pigs induced an almost total depletion of the cytoplasmic fluorescence with concomitant reduction of their argyrophilia. Microspectrofluorometric analysis of the fluorophore revealed excitation maxima at 370 and 420 mµ; the corresponding emission maxima were 430 and 490 mµ. This closely resembled the spectral characteristics of the OPT-induced fluorescence of authentic glucagon. It is concluded that OPT forms fluorphores wth glucagon in the A2-cells and that the NH2-terminal histidine residue of the polypeptide is responsible for the formation of the fluorophore. It is proposed that OPT is a useful reagent for the histochemical detection not only of histamine but also of histidyl peptides.

scholarly journals Epstein-Barr Virus-Encoded RK-BARF0 Protein Expression

1999 ◽  
Vol 73 (10) ◽  
pp. 8902-8906 ◽  
Author(s):  
Norbert Kienzle ◽  
Marion Buck ◽  
Sonia Greco ◽  
Kenia Krauer ◽  
Tom B. Sculley
Keyword(s):  
Fluorescence Microscopy ◽  
Cellular Localization ◽  
Epstein Barr Virus ◽  
Cellular Proteins ◽  
Rabbit Serum ◽  
Differential Splicing ◽  
Barr Virus ◽  
Associated Proteins ◽  
Epstein Barr ◽  
Nuclear Structures

ABSTRACT The cellular localization of the Epstein-Barr virus-encoded RK-BARF0 protein was analyzed by fluorescence microscopy and immunoblotting. The recombinant RK-BARF0 protein was found to be tightly bound to nuclear structures, whereas 16- to 20-kDa RK-BARF0 derivatives, generated by differential splicing of the RK-BARF0 transcript, were present throughout the cell. Moreover, a previously generated anti-RK-BARF0 rabbit serum was found to cross-react with cellular proteins, showing that the previously identified 30- to 35-kDa membrane-associated proteins do not represent RK-BARF0.

Potentiation of trastuzumab emtansine (T-DM1)-driven antitumor activity by pertuzumab in a HER2-positive gastric cancer model.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e13502-e13502
Author(s):  
Yoriko Yamashita-Kashima ◽  
Sei Shu ◽  
Naoki Harada ◽  
Kaori Fujimoto-Ouchi
Keyword(s):  
Gastric Cancer ◽  
Antitumor Activity ◽  
Fluorescence Microscopy ◽  
Cell Surface ◽  
Cellular Localization ◽  
Phase Iii ◽  
Human Gastric Cancer ◽  
Concomitant Treatment ◽  
Tumor Cell Death ◽  
Her2 Positive

e13502 Background: The efficacy and safety of T-DM1 combined with pertuzumab in HER2-positive breast cancer is currently being investigated in a phase III clinical trial (MARIANNE). Anti-HER2 therapy with T-DM1 combined with pertuzumab in HER2-positive gastric cancer is of great interest. In a previous study in a HER2-positive gastric cancer xenograft model, T-DM1 in combination with pertuzumab demonstrated enhanced antitumor activity compared with each agent alone, and potentiated ADCC and apoptosis activity (AACR, 2011). In this study, we investigated the mechanisms underlying the enhanced activity observed with these two drugs. Methods: The binding of T-DM1 to cell surface HER2 was measured in a HER2-positive human gastric cancer cell line (NCI-N87) by flow cytometry using Alexa Fluor 647-labeled T-DM1. The phosphorylation status of HER signaling molecules was assessed by Western blot, and cellular localization of T-DM1 was determined by fluorescence microscopy using labeled T-DM1 in the presence/absence of pertuzumab. Results: Binding of T-DM1 to cell surface HER2 was significantly increased in the presence of pertuzumab. Concomitant treatment with T-DM1 and pertuzumab led to a reduction of phosphorylated (p)EGFR or pHER3 in EGF or heregulin-stimulated cells, respectively, resulting in suppression of downstream pERK or pAkt pathways. Fluorescence microscopy revealed that pertuzumab facilitated the internalization of HER2-bound T-DM1 from the cell surface into the cytoplasm. Conclusions: Results suggest that the enhanced antitumor activity observed with the combination of T-DM1 and pertuzumab may be attributed to 1) increased binding of T-DM1 to HER2, which could potentially augment ADCC and diminish downstream HER2-signaling, and 2) increased T-DM1 internalization into the cytoplasm, which may potentiate tumor cell death caused by DM1. These results suggest that T-DM1 in combination with pertuzumab may provide clinical benefit for patients with HER2-positive gastric cancer.

scholarly journals Alternative splicing of the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1) uncovers a novel mitochondrial protein maturation mechanism

2020 ◽  
Vol 295 (10) ◽  
pp. 3029-3039 ◽  
Author(s):  
Simon J. Mayr ◽  
Juliane Röper ◽  
Guenter Schwarz
Keyword(s):  
Alternative Splicing ◽  
Fluorescence Microscopy ◽  
Cellular Localization ◽  
Mitochondrial Protein ◽  
Molybdenum Cofactor ◽  
Type I ◽  
Cell Fractionation ◽  
Mitochondrial Matrix ◽  
Protein Maturation ◽  
Exon 1

Molybdenum cofactor (Moco) biosynthesis is a highly conserved multistep pathway. The first step, the conversion of GTP to cyclic pyranopterin monophosphate (cPMP), requires the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1). Alternative splicing of MOCS1 within exons 1 and 9 produces four different N-terminal and three different C-terminal products (type I–III). Type I splicing results in bicistronic transcripts with two open reading frames, of which only the first, MOCS1A, is translated, whereas type II/III splicing produces MOCS1AB proteins. Here, we first report the cellular localization of alternatively spliced human MOCS1 proteins. Using fluorescence microscopy, fluorescence spectroscopy, and cell fractionation experiments, we found that depending on the alternative splicing of exon 1, type I splice variants (MOCS1A) either localize to the mitochondrial matrix (exon 1a) or remain cytosolic (exon 1b). MOCS1A proteins required exon 1a for mitochondrial translocation, but fluorescence microscopy of MOCS1AB variants (types II and III) revealed that they were targeted to mitochondria independently of exon 1 splicing. In the latter case, cell fractionation experiments displayed that mitochondrial matrix import was facilitated via an internal motif overriding the N-terminal targeting signal. Within mitochondria, MOCS1AB underwent proteolytic cleavage resulting in mitochondrial matrix localization of the MOCS1B domain. In conclusion, MOCS1 produces two functional proteins, MOCS1A and MOCS1B, which follow different translocation routes before mitochondrial matrix import for cPMP biosynthesis involving both proteins. MOCS1 protein maturation provides a novel alternative splicing mechanism that ensures the coordinated mitochondrial targeting of two functionally related proteins encoded by a single gene.

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