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1.
Chem Commun (Camb) ; 59(85): 12707-12710, 2023 Oct 24.
Article in English | MEDLINE | ID: mdl-37801331

ABSTRACT

The production of ß-lactamases by bacterial pathogens endangers antimicrobial therapy, and new inhibitors for ß-lactamases are urgently needed. We report the development of a luminescent-based biosensor that quantifies ß-lactamase inhibition in a cellular context, based on the activation of transcriptional factor AmpR following the exposure of bacterial cells to ß-lactams. This rapid method can account for factors like membrane permeability and can be employed to identify new ß-lactamase inhibitors.


Subject(s)
Anti-Bacterial Agents , beta-Lactamase Inhibitors , beta-Lactamase Inhibitors/pharmacology , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , beta-Lactams/pharmacology , beta-Lactamases , Bacteria
2.
J Vis Exp ; (170)2021 04 09.
Article in English | MEDLINE | ID: mdl-33900285

ABSTRACT

Fatty acylation, the covalent addition of saturated fatty acids to protein substrates, is important in regulating a myriad of cellular functions in addition to its implications in cancer and neurodegenerative diseases. Recent developments in fatty acylation detection methods have enabled efficient and non-hazardous detection of fatty acylated proteins, particularly through the use of click chemistry with bio-orthogonal labeling. However, click chemistry detection can be limited by the poor solubility and potential toxic effects of adding long chain fatty acids to cell culture. Described here is a labeling approach with optimized delivery using saponified fatty acids in combination with fatty-acid free BSA, as well as delipidated media, which can improve detection of hard to detect fatty acylated proteins. This effect was most pronounced with the alkynyl-stearate analog, 17-ODYA, which has been the most commonly used fatty acid analog in click chemistry detection of acylated proteins. This modification will improve cellular incorporation and increase sensitivity to acylated protein detection. In addition, this approach can be applied in a variety of cell types and combined with other assays such as pulse-chase analysis, stable isotope labeling with amino acids in cell culture, and mass spectrometry for quantitative profiling of fatty acylated proteins.


Subject(s)
Click Chemistry/methods , Fatty Acids/metabolism , Serum Albumin, Bovine/metabolism , Acylation , Fatty Acids/chemistry , HEK293 Cells , Humans , Mass Spectrometry , Serum Albumin, Bovine/chemistry
3.
AIDS Res Hum Retroviruses ; 22(10): 1031-5, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17067274

ABSTRACT

NeuroAIDS, the neurological, motor, and cognitive impairments that occur in acquired immunodeficiency syndrome (AIDS) patients, is characterized by compromised function in frontal cortical and subcortical brain regions including impairments in motor control, reaction time, and executive functions. Executive function is a cognitive domain involving the regulation of behavior, including inhibitory control. The present study evaluated the effects of simian immunodeficiency virus (SIV) infection on the object retrieval detour (ORD) task to assess inhibitory control. The ORD task measures the ability to inhibit the prepotent response of reaching directly toward a food reinforcer placed in a transparent box. The box has one open side, and the animal must inhibit the initial reaching response and look to see which side is open. Subjects were 12 experimentally naive pigtailed macaques; six monkeys were infected with SIV. Baseline performance was compared to performance under "terminal" conditions (the week prior to the scheduled euthanasia) to determine if progression of SIV disease led to decreased ORD performance. SIV-infected monkeys acquired ORD performance at the same levels as uninfected control monkeys, and had similar latencies and error rates. However, in the terminal week there was a significant difference between the groups in the number of barrier reach errors (touching the side of the transparent box). Three individual SIV-infected monkeys were impaired on ORD performance both in terms of errors and speed of performance. Given the sensitivity of ORD performance to dopaminergic dysfunction, these results further implicate dopaminergic dysfunction as a mechanism of cognitive and motor impairments in NeuroAIDS.


Subject(s)
AIDS Dementia Complex/psychology , Psychomotor Performance , Simian Acquired Immunodeficiency Syndrome/psychology , Animals , Learning , Macaca nemestrina , Male
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