RESUMEN
Covalent modifications by reactive oxygen species can modulate the function and stability of proteins. Thermal unfolding experiments in solution are a standard tool for probing oxidation-induced stability changes. Complementary to such solution investigations, the stability of electrosprayed protein ions can be assessed in the gas phase by collision-induced unfolding (CIU) and ion-mobility spectrometry. A question that remains to be explored is whether oxidation-induced stability alterations in solution are mirrored by the CIU behavior of gaseous protein ions. Here, we address this question using chloramine-T-oxidized cytochrome c (CT-cyt c) as a model system. CT-cyt c comprises various proteoforms that have undergone MetO formation (+16 Da) and Lys carbonylation (LysCH2-NH2 â LysCHO, -1 Da). We found that CT-cyt c in solution was destabilized, with a â¼5 °C reduced melting temperature compared to unmodified controls. Surprisingly, CIU experiments revealed the opposite trend, i.e., a stabilization of CT-cyt c in the gas phase. To pinpoint the source of this effect, we performed proteoform-resolved CIU on CT-cyt c fractions that had been separated by cation exchange chromatography. In this way, it was possible to identify MetO formation at residue 80 as the key modification responsible for stabilization in the gas phase. Possibly, this effect is caused by newly formed contacts of the sulfoxide with aromatic residues in the protein core. Overall, our results demonstrate that oxidative modifications can affect protein stability in solution and in the gas phase very differently.
Asunto(s)
Citocromos c/química , Lisina/química , Cloraminas/química , Gases/química , Espectrometría de Movilidad Iónica , Oxidación-Reducción , Estabilidad Proteica , Desplegamiento Proteico , Soluciones/química , Espectrometría de Masa por Ionización de Electrospray , Termodinámica , Compuestos de Tosilo/químicaRESUMEN
The Amino acid Derivative Reactivity Assay (ADRA) is an in chemico alternative to animal testing for skin sensitization potential that uses two different nucleophilic reagents and it is known that ADRA hardly exhibts co-elution compared with the Direct Peptide Reactivity Assay (DPRA) based on the same scientific principles. In this study, we have analyzed the factors underlying why co-elution, which is sometimes an issue during DPRA testing, virtually never occurs during ADRA testing. Chloramine T and dimethyl isophthalate both exhibited co-elution during DPRA testing, but when quantified at both DPRA's 220â¯nm and ADRA's 281â¯nm, we found that when the later detection wavelength was used, these test chemicals produced extremely small peaks that did not interfere with quantification of the peptides. And although both salicylic acid and penicillin G exhibited co-elution during DPRA testing, when tested at a concentration just 1% of that used in DPRA, the very broad peak produced at the higher concentration was reduced significantly. However, both these test chemicals exhibited very sharp peaks when the pH of the injection sample was adjusted to be acidic. Based on these results, we were able to clarify that the reasons why nucleophlic reagents hardly co-elute with test chemicals during ADRA testing are depend on the following three major reasons: (1)differences in the detection wavelength, (2)differences in test chemical concentrations in the injection sample, (3)differences in composition of the injection solvent.
Asunto(s)
Alternativas a las Pruebas en Animales/métodos , Evaluación Preclínica de Medicamentos/métodos , Péptidos/química , Cloraminas/química , Cromatografía Líquida de Alta Presión , Indicadores y Reactivos/química , Ácido Salicílico/química , Piel/efectos de los fármacos , Solventes , Compuestos de Tosilo/químicaRESUMEN
Myeloperoxidase produces strong oxidants during the immune response to destroy invading pathogens. However, these oxidants can also cause tissue damage, which contributes to the development of numerous inflammatory diseases. Selenium containing compounds, including selenomethionine (SeMet) and 1,4-anhydro-5-seleno-D-talitol (SeTal), react rapidly with different MPO-derived oxidants to form the respective selenoxides (SeMetO and SeTalO). This study investigates the susceptibility of these selenoxides to undergo reduction back to the parent compounds by intracellular reducing systems, including glutathione (GSH) and the glutathione reductase and thioredoxin reductase systems. GSH is shown to reduce SeMetO and SeTalO, with consequent formation of GSSG with apparent second order rate constants, k2, in the range 103-104M-1s-1. Glutathione reductase reduces both SeMetO and SeTalO at the expense of NADPH via formation of GSSG, whereas thioredoxin reductase acts only on SeMetO. The presence of SeMet and SeTal also increased the rate at which NADPH was consumed by the glutathione reductase system in the presence of N-chloramines. In contrast, the presence of SeMet and SeTal reduced the rate of NADPH consumption by the thioredoxin reductase system after addition of N-chloramines, consistent with the rapid formation of selenoxides, but only slow reduction by thioredoxin reductase. These results support a potential role of seleno compounds to act as catalytic scavengers of MPO-derived oxidants, particularly in the presence of glutathione reductase and NADPH, assuming that sufficient plasma levels of the parent selenoether can be achieved in vivo following supplementation.
Asunto(s)
Cloraminas/química , Glutatión Reductasa/metabolismo , Glutatión/metabolismo , Compuestos de Selenio/química , Compuestos de Sulfhidrilo/química , Reductasa de Tiorredoxina-Disulfuro/metabolismo , Catálisis , Hexosas/química , Cinética , NADP/química , Compuestos de Organoselenio/química , Oxidación-Reducción , Selenometionina/químicaRESUMEN
N-Chlorotaurine (NCT), a well-tolerated endogenous long-lived oxidant that can be applied topically as an antiseptic, was tested on its fungicidal activity against Scedosporium and Lomentospora, opportunistic fungi that cause severe infections with limited treatment options, mainly in immunocompromised patients. In quantitative killing assays, both hyphae and conidia of Scedosporium apiospermum, Scedosporium boydii, and Lomentospora prolificans (formerly Scedosporium prolificans) were killed by 55 mM (1.0%) NCT at pH 7.1 and 37°C, with a 1- to 4-log10 reduction in CFU after 4 h and a 4- to >6-log10 reduction after 24 h. The addition of ammonium chloride to NCT markedly increased this activity. LIVE/DEAD staining of conidia treated with 1.0% NCT for 0.5 to 3 h increased the permeability of the cell wall and membrane. Preincubation of the test fungi in 1.0% NCT for 10 to 60 min delayed the time to germination of conidia by 2 h to >12 h and reduced their germination rate by 10.0 to 100.0%. Larvae of Galleria mellonella infected with 1.0 × 10(7) conidia of S. apiospermum and S. boydii died at a rate of 90.0 to 100% after 8 to 12 days. The mortality rate was reduced to 20 to 50.0% if conidia were preincubated in 1.0% NCT for 0.5 h or if heat-inactivated conidia were used. Our study demonstrates the fungicidal activity of NCT against different Scedosporium and Lomentospora species. A postantifungal effect connected with a loss of virulence occurs after sublethal incubation times. The augmenting effect of ammonium chloride can be explained by the formation of monochloramine.
Asunto(s)
Cloruro de Amonio/farmacología , Antiinfecciosos Locales/farmacología , Antifúngicos/farmacología , Scedosporium/efectos de los fármacos , Taurina/análogos & derivados , Animales , Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Cloraminas/química , Cloraminas/farmacología , Hifa/efectos de los fármacos , Hifa/fisiología , Larva/microbiología , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas/microbiología , Scedosporium/fisiología , Esporas Fúngicas/efectos de los fármacos , Esporas Fúngicas/fisiología , Taurina/farmacologíaRESUMEN
The worldwide detection of pharmaceuticals and personal care products (PPCPs) in the aquatic environment and drinking water has been a cause for concern in recent years. The possibility for concurrent formation of nitrosamine DBPs (disinfection by-products) during chloramine disinfection has become another significant concern for delivered drinking water quality because of their potent carcinogenicity. This study demonstrates that a group of PPCPs containing amine groups can serve as nitrosamine precursors during chloramine disinfection. Molar yields higher than 1% are observed for eight pharmaceuticals, with ranitidine showing the strongest potential to form N-nitrosodimethylamine (NDMA). The molar conversion increases with the Cl(2):N mass ratio, suggesting that dichloramine is relevant to the formation of NDMA from these precursors. Although the trace level of PPCPs in the environment suggests that they may not account for the majority of nitrosamine precursors during the disinfection process, this study demonstrates a connection between the transformation of PPCPs and the formation of nitrosamines during chloramine disinfection. This both expands the pool of potential nitrosamine precursors, and provides a possible link between the presence of trace levels of certain PPCPs in drinking water sources and potential adverse health effects.
Asunto(s)
Cloraminas/química , Desinfección/métodos , Nitrosaminas/síntesis química , Preparaciones Farmacéuticas/química , Contaminantes Químicos del Agua/química , Cosméticos/efectos adversos , Cosméticos/química , Suplementos Dietéticos/efectos adversos , Dimetilnitrosamina/síntesis química , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Preparaciones para el Cabello/efectos adversos , Preparaciones para el Cabello/química , Purificación del AguaRESUMEN
Fibrinolysis consists of a plasmatic part and a cellular part. A rapid global assay for plasmatic fibrinolysis is the fibrinolysis parameters assay (FIPA). Cellular fibrinolysis is measured by testing the clot lysis capacity using the microtitre plate clot lysis assay with polymorphonuclear neutrophils (CLA-PMN). Individual citrated plasma or pooled normal plasma (50 microl) of 232 patients was recalcified, incubated for 90 min at 37 degrees C, oxidized with 0 or 1.5 mmol/l (final concentration) chloramine-T, and supplemented with 50 microl respective polymorphonuclear neutrophil plasma. The turbidity of the clots was measured at 405 nm after 12 h and 60 h (37 degrees C). Plasma (50 microl) was also incubated with 5 microl of 100 IU/ml urokinase, 6 mmol/l tranexamic acid, 6% human albumin for 10 min (37 degrees C). Then 100 microl of 0.5 mmol/l Val-Leu-Lys-pNA in 2.45 mol/l arginine, pH 8.6, was added and the increase in absorbance with time was measured. The different CLA-PMN assay versions correlated with each other with r = 0.543-0.782. Cellular fibrinolysis (34 +/- 30% lysis; normal: 25 +/- 10%) did not correlate with the FIPA (72 +/- 27%; normal: 100 +/- 15%), prothrombin time, activated partial thromboplastin time, fibrinogen, C-reactive protein, or the blood counts of thrombocytes, leukocytes, or polymorphonuclear neutrophils. Chloramine (1.5 mmol/l) oxidation of the microclots favours their fibrinolytic breakdown, especially if lysis-resistant microclots are oxidized. The FIPA and CLA-PMN are new economical tests for the fibrinolytic state in patient blood.
Asunto(s)
Pruebas de Coagulación Sanguínea , Fibrinólisis , Pruebas de Coagulación Sanguínea/economía , Pruebas de Coagulación Sanguínea/normas , Proteínas Sanguíneas/química , Cloraminas/química , Humanos , Recuento de Leucocitos , Oxidación-Reducción , Recuento de Plaquetas , Compuestos de Tosilo/químicaRESUMEN
OBJECTIVES: To evaluate cytotoxicity and bactericidal effects of chloramine-T. METHODS: In vitro study of various concentrations and exposure times to preparations containing human fibroblasts or 1.5 x 10 colony forming units per milliliter (CFU/mL) of 3 gram-positive bacteria-Staphylococcus aureus, methicillin-resistant S aureus, and vancomycin-resistant Enterococcus faecalis-and 2 gram-negative bacteria-Escherichia coli and Pseudomonas aeruginosa-with and without fetal bovine serum present. MAIN OUTCOME MEASURES: Percentage reduction of bacterial growth and percentage of viable fibroblasts 48 hours after exposure. RESULTS: All gram-positive growth was reduced by 95% to 100%, regardless of dose, with or without serum. E coli (gram-negative; with/without serum) was reduced 94% to 100% at antiseptic concentrations of 300 and 400 ppm. At 200 ppm, E coli growth was fully inhibited without serum present and by 50% with serum. P aeruginosa (gram-negative) was not significantly affected under any conditions. At 100 and 200 ppm, cell viability remained greater than 90% under all experimental conditions. A 300-ppm, 3-minute exposure to chloramine-T resulted in cell viability of up to 70%, with longer exposures producing lower viabilities. Serum did not affect cell viability in any condition. CONCLUSIONS: In vitro, chloramine-T at 200 ppm for 5 to 20 minutes was effective against 3 virulent gram-positive bacteria without fibroblast damage. At 300 ppm and 3 and 5 minutes, 30% of fibroblasts were damaged and 95% to 100 % of E coli were inhibited, respectively.