Chemical Probes for the Rapid Detection of Fatty-Acylated Proteins in Mammalian Cells

2007 ◽  
Vol 129 (10) ◽  
pp. 2744-2745 ◽  
Author(s):  
Howard C. Hang ◽  
Ernst-Jan Geutjes ◽  
Gijsbert Grotenbreg ◽  
Annette M. Pollington ◽  
Marie Jose Bijlmakers ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Mammalian Cells ◽  
Chemical Probes ◽  
Acylated Proteins

scholarly journals Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens

2020 ◽  
Vol 11 ◽  
Author(s):  
Luping Xu ◽  
Xingjian Bai ◽  
Shivendra Tenguria ◽  
Yi Liu ◽  
Rishi Drolia ◽  
...  
Keyword(s):  
Shelf Life ◽  
Mammalian Cell ◽  
Rapid Detection ◽  
Mammalian Cells ◽  
Host Cells ◽  
Detection Time ◽  
Primary Methods ◽  
Widespread Application ◽  
Lateral Flow Assays ◽  
Set Up

Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the Mammalian Cell-based ImmunoAssay (MaCIA) was validated with Salmonella enterica serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium, 11 other Salmonella serovars, and 14 non-Salmonella spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1–10 CFU/25 g was 16–21 h using a traditional enrichment set up but the detection time was shortened to 10–12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.

Rapid detection of apoptosis in mammalian cells by using intact cell MALDI mass spectrometry

The Analyst ◽  
2011 ◽  
Vol 136 (24) ◽  
pp. 5181 ◽  
Author(s):  
Hongjuan Dong ◽  
Wei Shen ◽  
Myra Ting Wai Cheung ◽  
Yimin Liang ◽  
Hon Yeung Cheung ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Rapid Detection ◽  
Mammalian Cells ◽  
Intact Cell ◽  
Maldi Mass Spectrometry

scholarly journals Molecular basis of fatty acid selectivity in the zDHHC family of S-acyltransferases revealed by click chemistry

2017 ◽  
Vol 114 (8) ◽  
pp. E1365-E1374 ◽  
Author(s):  
Jennifer Greaves ◽  
Kevin R. Munro ◽  
Stuart C. Davidson ◽  
Matthieu Riviere ◽  
Justyna Wojno ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Click Chemistry ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Acyl Chain ◽  
Fatty Acid Selectivity ◽  
Molecular Determinants ◽  
Chain Acyl ◽  
Domain 3 ◽  
Acylated Proteins

S-acylation is a major posttranslational modification, catalyzed by the zinc finger DHHC domain containing (zDHHC) enzyme family. S-acylated proteins can be modified by different fatty acids; however, very little is known about how zDHHC enzymes contribute to acyl chain heterogeneity. Here, we used fatty acid-azide/alkyne labeling of mammalian cells, showing their transformation into acyl-CoAs and subsequent click chemistry-based detection, to demonstrate that zDHHC enzymes have marked differences in their fatty acid selectivity. This difference in selectivity was apparent even for highly related enzymes, such as zDHHC3 and zDHHC7, which displayed a marked difference in their ability to use C18:0 acyl-CoA as a substrate. Furthermore, we identified isoleucine-182 in transmembrane domain 3 of zDHHC3 as a key determinant in limiting the use of longer chain acyl-CoAs by this enzyme. This study uncovered differences in the fatty acid selectivity profiles of cellular zDHHC enzymes and mapped molecular determinants governing this selectivity.

scholarly journals Proteomic Analysis of Fatty-acylated Proteins in Mammalian Cells with Chemical Reporters RevealsS-Acylation of Histone H3 Variants

2010 ◽  
Vol 10 (3) ◽  
pp. M110.001198 ◽  
Author(s):  
John P. Wilson ◽  
Anuradha S. Raghavan ◽  
Yu-Ying Yang ◽  
Guillaume Charron ◽  
Howard C. Hang
Keyword(s):  
Proteomic Analysis ◽  
Mammalian Cells ◽  
Histone H3 ◽  
Acylated Proteins

Rapid Detection of Apoptosis in Cultured Mammalian Cells

2017 ◽  
pp. 105-111 ◽  
Author(s):  
Igor Kudryavtsev ◽  
Maria Serebryakova ◽  
Liudmila Solovjeva ◽  
Maria Svetlova ◽  
Denis Firsanov
Keyword(s):  
Rapid Detection ◽  
Mammalian Cells

Rapid Detection of γ-H2AX by Flow Cytometry in Cultured Mammalian Cells

2017 ◽  
pp. 129-138
Author(s):  
Denis Firsanov ◽  
Liudmila Solovjeva ◽  
Olga Lublinskaya ◽  
Valeriy Zenin ◽  
Igor Kudryavtsev ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Rapid Detection ◽  
Mammalian Cells

Ultrastructural Alterations in Hela Cells Induced by Spider Venom

1967 ◽  
Vol 25 ◽  
pp. 20-21
Author(s):  
Dale E. McClendon ◽  
Paul N. Morgan ◽  
Bernard L. Soloff
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Carcinoma Cells ◽  
Venom Protein ◽  
Sterile Saline ◽  
Carbon Coated ◽  
Available Information ◽  
Loxosceles Reclusa ◽  
Essential Growth ◽  
Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

Use of Uranium-Labeled Antibodies for Electron Microscopic Localization of Various Antigens in Mammalian Cells

1967 ◽  
Vol 25 ◽  
pp. 136-137
Author(s):  
J. P. Petrali ◽  
E. J. Donati ◽  
L. A. Sternberger
Keyword(s):  
Endoplasmic Reticulum ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Specific Antiserum ◽  
Electron Microscopic ◽  
E Coli ◽  
Cytoplasmic Membranes ◽  
Viral Progeny ◽  
Ultrathin Sections ◽  
Electron Microscopic Localization

Specific contrast is conferred to subcellular antigen by applying purified antibodies, exhaustively labeled with uranium under immunospecific protection, to ultrathin sections. Use of Seligman’s principle of bridging osmium to metal via thiocarbohydrazide (TCH) intensifies specific contrast. Ultrathin sections of osmium-fixed materials were stained on the grid by application of 1) thiosemicarbazide (TSC), 2) unlabeled specific antiserum, 3) uranium-labeled anti-antibody and 4) TCH followed by reosmication. Antigens to be localized consisted of vaccinia antigen in infected HeLa cells, lysozyme in monocytes of patients with monocytic or monomyelocytic leukemia, and fibrinogen in the platelets of these leukemic patients. Control sections were stained with non-specific antiserum (E. coli).In the vaccinia-HeLa system, antigen was localized from 1 to 3 hours following infection, and was confined to degrading virus, the inner walls of numerous organelles, and other structures in cytoplasmic foci. Surrounding architecture and cellular mitochondria were unstained. 8 to 14 hours after infection, antigen was localized on the outer walls of the viral progeny, on cytoplasmic membranes, and free in the cytoplasm. Staining of endoplasmic reticulum was intense and focal early, and weak and diffuse late in infection.

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