HIV-1 tat Expression and Sulphamethoxazole Hydroxylamine Mediated Oxidative Stress Alter the Disulfide Proteome in Jurkat T Cells

Background Adverse drug reactions (ADRs) are a significant problem for HIV patients, with the risk of developing ADRs increasing as the infection progresses to AIDS. However, the pathophysiology underlying ADRs remains unknown. Sulphamethoxazole (SMX) via its active metabolite SMX-hydroxlyamine, when used prophylactically for pneumocystis pneumonia in HIV-positive individuals, is responsible for a high incidence of ADRs. We previously demonstrated that the HIV infection and, more specifically, that the HIV-1 Tat protein can exacerbate SMX-HA-mediated ADRs. In the current study, Jurkat T cell lines expressing Tat and its deletion mutants were used to determine the effect of Tat on the thiol proteome in the presence and absence of SMX-HA revealing drug-dependent changes in the disulfide proteome in HIV infected cells. Protein lysates from HIV infected Jurkat T cells and Jurkat T cells stably transfected with HIV Tat and Tat deletion mutants were subjected to quantitative slot blot analysis, western blot analysis and redox 2 dimensional (2D) gel electrophoresis to analyze the effects of SMX-HA on the thiol proteome. Results Redox 2D gel electrophoresis demonstrated that untreated, Tat-expressing cells contain a number of proteins with oxidized thiols. The most prominent of these protein thiols was identified as peroxiredoxin. The untreated, Tat-expressing cell lines had lower levels of peroxiredoxin compared to the parental Jurkat E6.1 T cell line. Conversely, incubation with SMX-HA led to a 2- to 3-fold increase in thiol protein oxidation as well as a significant reduction in the level of peroxiredoxin in all the cell lines, particularly in the Tat-expressing cell lines. Conclusion SMX-HA is an oxidant capable of inducing the oxidation of reactive protein cysteine thiols, the majority of which formed intermolecular protein bonds. The HIV Tat-expressing cell lines showed greater levels of oxidative stress than the Jurkat E6.1 cell line when treated with SMX-HA. Therefore, the combination of HIV Tat and SMX-HA appears to alter the activity of cellular proteins required for redox homeostasis and thereby accentuate the cytopathic effects associated with HIV infection of T cells that sets the stage for the initiation of an ADR

THE METABOLISM OF STYRENE OXIDE IN THE ISOLATED PERFUSED RAT LIVER Identification and Quantitation of Major Metabolites

ABSTRACT Isolated perfused rat livers rapidly metabolized '4C-styrene oxide

Attenuation of oxidative stress in HEK 293 cells by the TCM constituents schisanhenol, baicalein, resveratrol or crocetin and two defined mixtures

PURPOSE: Our working hypothesis is that single bioactive phytochemicals with antioxidant properties that are important constituents of Traditional Chinese Medicine (TCM) and their defined mixtures have potential as chemoprotective agents for chronic conditions characterized by oxidative and nitrosative stress, including Alzheimer’s. Here we evaluate the ability of baicalein, crocetin, trans-resveratrol or schisanhenol and two defined mixtures of these TCM phytochemicals to attenuate the toxicity resulting from exposure to cell permeant t-butyl hydroperoxide (tBPH) in wild-type and bioengineered (to express choline acetyltransferase) HEK 293 cells. METHODS: Endpoints of tBHP-initiated oxidative and nitrosative stress in both types of HEK 293 cells and its attenuation by TCM constituents and mixtures included cytotoxicity (LDH release); depletion of intracellular glutathione (GSH); formation of S-glutathionylated proteins; oxidative changes to the disulfide proteome; and real-time changes in intracellular redox status. RESULTS: At low µM concentrations, each of the TCM constituents and mixtures effectively attenuated intracellular toxicity due to exposure of HEK 293 cells to 50 or 250 µM tBHP for 30 min to 3 h. Confocal microscopy of HEK 293 cells transfected with mutated green fluorescent protein (roGFP2) showed effective attenuation of tBHP oxidation by baicalein in real time. Three redox-regulated proteins prominent in the disulfide proteome of HEK 293 cells were identified by MALDI-TOF mass spectrometry. CONCLUSIONS: We conclude that single TCM chemicals and their simple mixtures have potential for use in adjunct chemoprotective therapy. Advantages of mixtures compared to single TCM constituents include the ability to combine compounds with varying molecular mechanisms of cytoprotection for enhanced biological activity; and to combine chemicals with complementary pharmacokinetic properties to increase half-life and prolong activity in vivo

Potential complementary therapy for adverse drug reactions to sulfonamides: Chemoprotection against oxidative and nitrosative stress by TCM constituents and defined mixtures

PURPOSE: Our working hypothesis is that bioactive phytochemicals that are important constituents of Traditional Chinese Medicine and their defined mixtures have potential as complementary therapy for chemoprotection against adverse drug reactions whose toxicity is not related to the pharmacological action of the drug but where oxidative and nitrosative stress are causative factors. METHODS: In this investigation we measured cytotoxicity, lipid peroxidation, protein carbonylation and ROS/NOS-mediated changes in the disulfide proteome of Jurkat E6.1 cells resulting from exposure to sulfamethoxazole N-hydroxylamine with or without pretreatment with low µM concentrations of baicalein, crocetin, resveratrol and schisanhenol alone and in defined mixtures to compare the ability of these treatment regimens to protect against ROS/RNS toxicity to Jurkat E6.1 cells in culture. RESULTS: Each of the Traditional Chinese Medicine constituents and defined mixtures tested had significant chemoprotective effects against the toxicity of ROS/RNS formed by exposure of Jurkat E6.1 cells to reactive metabolites of sulfamethoxazole implicated as the causative factors in adverse drug reactions to sulfa drugs used for therapy. At equimolar concentrations, the defined mixtures tended to be more effective chemoprotectants overall than any of the single constituents against ROS/RNs toxicity in this context. CONCLUSIONS: At low µM concentrations, defined mixtures of TCM constituents that contain ingredients with varied structures and multiple mechanisms for chemoprotection have excellent potential for complementary therapy with sulfa drugs to attenuate adverse effects caused by oxidative/nitrosative stress. Typically, such mixtures will have a combination of immediate activity due to short in vivo half-lives of some ingredients cleared rapidly following metabolism by phase 2 conjugation enzymes; and some ingredients with more prolonged halflives and activity reliant on phase 1 oxidation enzymes for their metabolic clearance

Novel diagnostic DNA methylation episignatures expand and refine the epigenetic landscapes of Mendelian disorders

Overlapping clinical phenotypes and an expanding breadth and complexity of genomic associations are a growing challenge in the diagnosis and clinical management of Mendelian disorders. The functional consequences and clinical impacts of genomic variation may involve unique, disorder-specific, genomic DNA methylation episignatures. In this study, we describe 19 novel episignature disorders and compare the findings alongside 38 previously established episignatures for a total of 57 episignatures associated with 65 genetic syndromes. We demonstrate increasing resolution and specificity ranging from protein complex, gene, sub-gene, protein domain, and even single nucleotide-level Mendelian episignatures. We show the power of multiclass modeling to develop highly accurate and disease-specific diagnostic classifiers. This study significantly expands the number and spectrum of disorders with detectable DNA methylation episignatures, improves the clinical diagnostic capabilities through the resolution of unsolved cases and the reclassification of variants of unknown clinical significance, and provides further insight into the molecular etiology of Mendelian conditions

Novel diagnostic DNA methylation episignatures expand and refine the epigenetic landscapes of Mendelian disorders.

Overlapping clinical phenotypes and an expanding breadth and complexity of genomic associations are a growing challenge in the diagnosis and clinical management of Mendelian disorders. The functional consequences and clinical impacts of genomic variation may involve unique, disorder-specific, genomic DNA methylation episignatures. In this study, we describe 19 novel episignature disorders and compare the findings alongside 38 previously established episignatures for a total of 57 episignatures associated with 65 genetic syndromes. We demonstrate increasing resolution and specificity ranging from protein complex, gene, sub-gene, protein domain, and even single nucleotide-level Mendelian episignatures. We show the power of multiclass modeling to develop highly accurate and disease-specific diagnostic classifiers. This study significantly expands the number and spectrum of disorders with detectable DNA methylation episignatures, improves the clinical diagnostic capabilities through the resolution of unsolved cases and the reclassification of variants of unknown clinical significance, and provides further insight into the molecular etiology of Mendelian conditions

Histone H3.3 beyond cancer: Germline mutations in Histone 3 Family 3A and 3B cause a previously unidentified neurodegenerative disorder in 46 patients

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A (H3F3A) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation

R.: Prediction of pulmonary benzo(a)pyrene 4,5oxide clearance: A pharmacokinetic analysis of epoxide metabolizing enzymes in rabbit

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