Knowledge of recommended antibiotic treatments for community-acquired infections in general medical practice: a cross-sectional study in Occitanie region, France.

To assess and analyse the knowledge of recommended antibiotic treatments, focusing on the appropriate drugs and treatment durations for the most common community-acquired infections in general medical practice in Occitanie region, France. A web-based survey was conducted over a 3-month period, from October, 2018 to January, 2019. All participants answered directly through the online platform. For the analysis of overtreatment risk, a score based system was adopted and two scores were produced: the duration score and the treatment score. 413 general practitioners completed the survey. The overall rate of concordance with guidelines in terms of both drug choice and treatment length was 2974/4956 (60%) answers. Diseases with at least 70% good answers included cystitis, group A streptococcal pharyngitis, and bacterial superficial skin infections. Diseases with fewer than 50% good answers included pyelonephritis, dog bite wounds, and community-acquired pneumonia in patients aged ≥ 65 years. Factors associated with the risk of overtreatment were age > 40 years, country setting and hospital employment. Knowledge of treatment durations is satisfactory with 60% of recommendations being met. However, varying levels were observed according to different diseases. This study highlighted a very high rate of adherence when recommendations were clear. In contrast, low levels of adherence were observed when recommendations were ambiguous or when conflicting guidelines existed.

Updated mortality and causes of death in 2020-2021 in people with HIV: a multicenter study in France.

OBJECTIVE: The aim of this study was to assess updated mortality and causes of death in people with HIV (PWH) in France. DESIGN AND METHODS: We analyzed all deaths in PWH followed up between January 1, 2020, and December 31, 2021, in 11 hospitals in the Paris region. We described the characteristics and causes of death among deceased PWH, and evaluated the incidence of mortality and associated risk factors using a multivariate logistic regression. RESULTS: Of the 12 942 patients followed in 2020--2021, 202 deaths occurred. Mean annual incidence of death [95% confidence interval (95% CI)] was 7.8 per 1000 PWH (6.3-9.5). Forty-seven patients (23%) died from non-AIDS nonviral hepatitis (NANH)-related malignancies, 38 (19%) from non-AIDS infections (including 21 cases of COVID-19), 20 (10%) from AIDS, 19 (9%) from cardiovascular diseases (CVD), 17 (8.4%) from other causes, six (3%) from liver diseases, and five (2.5%) from suicides/violent deaths. The cause of death was unknown in 50 (24.7%) patients. Risks factors for death were age [adjusted odds ratio (aOR) 1.93; 1.66-2.25 by additional decade), AIDS history (2.23; 1.61-3.09), low CD4 + cell count (1.95; 1.36-2.78 for 200-500 cells/µl and 5.76; 3.65-9.08 for ≤200 versus > 500 cells/µl), and viral load more than 50 copies/ml (2.03; 1.33-3.08), both at last visit. CONCLUSION: NANH malignancies remained in 2020-2021 the first cause of death. COVID-19 accounted for more than half of the mortality related to non-AIDS infections over the period. Aging, AIDS history, and a poorer viro-immunological control were associated with death.

Prescription of antibiotics by general practitioners for patients with a diagnosis of SARS- CoV-2 infection: Analysis of an electronic French health record.

OBJECTIVE: To define the factors associated with overprescription of antibiotics by general practitioners (GPs) for patients diagnosed with COVID-19 during the first wave of the pandemic. METHODS: Anonymised electronic prescribing records of 1370 GPs were analysed. Diagnosis and prescriptions were retrieved. The initiation rate by GP for 2020 was compared with 2017-2019. Prescribing habits of GPs who initiated antibiotics for > 10% of COVID-19 patients were compared with those who did not. Regional differences in prescribing habits of GPs who had consulted at least one COVID-19 patient were also analysed. RESULTS: For the March-April 2020 period, GPs who initiated antibiotics for > 10% of COVID-19 patients had more consultations than those who did not. They also more frequently prescribed antibiotics for non-COVID-19 patients consulting with rhinitis and broad-spectrum antibiotics for treating cystitis. Finally, GPs in the Île-de-France region saw more COVID-19 patients and more frequently initiated antibiotics. General practitioners in southern France had a higher but non-significant ratio of azithromycin initiation rate over total antibiotic initiation rate. CONCLUSION: This study identified a subset of GPs with overprescribing profiles for COVID-19 and other viral infections; they also tended to prescribe broad-spectrum antibiotics for a long duration. There were also regional differences concerning antibiotic initiation rates and the ratio of azithromycin prescribed. It will be necessary to evaluate the evolution of prescribing practices during subsequent waves.

Structural insights into the interactions of glutathione transferases with a nitric oxide carrier and sodium nitroprusside.

Glutathione transferases are detoxification enzymes with multifaceted roles, including a role in the metabolism and scavenging of nitric oxide (NO) compounds in cells. Here, we explored the ability of Trametes versicolor glutathione transferases (GSTs) from the Omega class (TvGSTOs) to bind metal-nitrosyl compounds. TvGSTOs have been studied previously for their ligandin role and are interesting models to study protein‒ligand interactions. First, we determined the X-ray structure of the TvGSTO3S isoform bound to the dinitrosyl glutathionyl iron complex (DNGIC), a physiological compound involved in the storage of nitric oxide. Our results suggested a different binding mode compared to the one previously described in human GST Pi 1 (GSTP1). Then, we investigated the manner in which TvGSTO3S binds three nonphysiological metal-nitrosyl compounds with different metal cores (iron, ruthenium and osmium). We assayed sodium nitroprusside, a well-studied vasodilator used in cases of hypertensive crises or heart failure. Our results showed that the tested GST can bind metal-nitrosyls at two distinct binding sites. Thermal shift analysis with six isoforms of TvGSTOs identified TvGSTO6S as the best interactant. Using the Griess method, TvGSTO6S was found to improve the release of nitric oxide from sodium nitroprusside in vitro, whereas the effects of human GST alpha 1 (GSTA1) and GSTP1 were moderate. Our results open new structural perspectives for understanding the interactions of glutathione transferases with metal-nitrosyl compounds associated with the biochemical mechanisms of NO uptake/release in biological systems.

Same-day discharge to home is feasible and safe in up to 75% of unselected total hip and knee arthroplasty.

PURPOSE: Though numerous studies highlighted benefits of ambulatory total joint arthroplasty (TJA), most had selected patients with age and comorbidities thresholds. We aimed to report proportions of unselected TJAs that could be scheduled for and operated in ambulatory settings, and to determine factors that hinder same-day discharge (SDD). METHODS: We studied 1100 consecutive primary TJAs (644 THAs and 456 TKAs) that were prepared following a multidisciplinary protocol for patient education and logistical preparation. Data were stratified for THA vs TKA and for success vs failure of SDD to home and multivariable analysis was performed to determine factors associated with failure of scheduled SDD to home. RESULTS: In total, 860 (78.2%) were scheduled for ambulatory surgery, but only 819 (74.5%) achieved SDD to home; 240 (21.8%) were scheduled for non-ambulatory surgery, but 103 (9.3%) achieved SDD to rehabilitation centre. Re-operations were required in 9 (1.0%) ambulatory TJAs vs 2 (0.8%) non-ambulatory TJAs (p = 0.769), while revisions were required in 13 (1.5%) ambulatory TJAs vs 1 (0.4%) non-ambulatory TJAs (p = 0.181). Multivariable analysis confirmed that failure of SDD to home was greater for women (OR 2.59; p = 0.011) and THA (vs TKA, OR 2.41; p = 0.023). CONCLUSION: With appropriate education and preparation, 75% of unselected primary hip and knee arthroplasties achieved SDD to home without compromising risks of complications, re-operations, or revisions. A further 9% achieved SDD to rehabilitation centre, implying that 84% of patients did not require overnight stay. These findings suggest that ambulatory surgery is feasible and safe to implement in most unselected lower limb arthroplasties.

Intestinal absorption of S-nitrosothiols: Permeability and transport mechanisms.

S-Nitrosothiols, a class of NO donors, demonstrate potential benefits for cardiovascular diseases. Drugs for such chronic diseases require long term administration preferentially through the oral route. However, the absorption of S-nitrosothiols by the intestine, which is the first limiting barrier for their vascular bioavailability, is rarely evaluated. Using an in vitro model of intestinal barrier, based on human cells, the present work aimed at elucidating the mechanisms of intestinal transport (passive or active, paracellular or transcellular pathway) and at predicting the absorption site of three S-nitrosothiols: S-nitrosoglutathione (GSNO), S-nitroso-N-acetyl-l-cysteine (NACNO) and S-nitroso-N-acetyl-d-penicillamine (SNAP). These S-nitrosothiols include different skeletons carrying the nitroso group, which confer different physico-chemical characteristics and biological activities (antioxidant and anti-inflammatory). According to the values of apparent permeability coefficient, the three S-nitrosothiols belong to the medium class of permeability. The evaluation of the bidirectional apparent permeability demonstrated a passive diffusion of the three S-nitrosothiols. GSNO and NACNO preferentially cross the intestinal barrier though the transcellular pathway, while SNAP followed both the trans- and paracellular pathways. Finally, the permeability of NACNO was favoured at pH 6.4, which is close to the pH of the jejunal part of the intestine. Through this study, we determined the absorption mechanisms of S-nitrosothiols and postulated that they can be administrated through the oral route.

Aging and hypertension decrease endothelial NO-related dilating function and gamma-glutamyl transferase activity but not S-nitrosoglutathione-induced aortic vasodilation.

S-nitrosoglutathione (GSNO), which is involved in the transport and the storage of NO, induces vasorelaxation. It requires gamma-glutamyl transferase (GGT), an enzyme present on the endothelium, to transfer NO into the cell. We evaluated whether aging and hypertension, which induce NO-related dilating dysfunction, are associated with decreased vascular GGT activity and modify the vasorelaxant effect of GSNO. Thoracic aortic rings isolated from male spontaneous hypertensive rats (SHR) and Wistar-Kyoto rats (WKY) aged 20-22 (adult) or 57-60 weeks (mature) were preconstricted with phenylephrine, then submitted to concentration-vasorelaxant response curves (maximal response: Emax ; pD2 ) to GSNO and carbachol (the latter to measure NO-related dilating function). GGT activity was measured using chromogenic substrate. Both aging and hypertension lowered Emax values for carbachol (Emax -8% in adult SHR, -42% in mature SHR vs. age-matched WKY, page and phypertension  < 0.05) demonstrating NO-related dilating dysfunction. Aortic GGT activity also decreased with aging and hypertension (-22% in adult and -75%, reaching 3 nmol/min/g of tissue, in mature SHR vs. 12 in age-matched WKY and 23 in adult WKY, page and phypertension  < 0.05). The pD2 values of GSNO were similar in mature SHR and WKY but higher in adult SHR (pinteraction  < 0.05). Aging in hypertensive rats decreased NO-related vasorelaxant function and vascular GGT activity, but did not lower the vasorelaxant response to GSNO. This opens perspectives for GSNO-based therapeutics restoring nitric oxide bioavailability and vascular protection in a context of endothelial dysfunction.

In vivo and in silico evaluation of a new nitric oxide donor, S,S'-dinitrosobucillamine.

PURPOSE: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S'-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols. METHODS: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 µmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h. RESULTS: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO). CONCLUSION: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.

Regulation of protein function by S-nitrosation and S-glutathionylation: processes and targets in cardiovascular pathophysiology.

Decades of chemical, biochemical and pathophysiological research have established the relevance of post-translational protein modifications induced by processes related to oxidative stress, with critical reflections on cellular signal transduction pathways. A great deal of the so-called 'redox regulation' of cell function is in fact mediated through reactions promoted by reactive oxygen and nitrogen species on more or less specific aminoacid residues in proteins, at various levels within the cell machinery. Modifications involving cysteine residues have received most attention, due to the critical roles they play in determining the structure/function correlates in proteins. The peculiar reactivity of these residues results in two major classes of modifications, with incorporation of NO moieties (S-nitrosation, leading to formation of protein S-nitrosothiols) or binding of low molecular weight thiols (S-thionylation, i.e. in particular S-glutathionylation, S-cysteinylglycinylation and S-cysteinylation). A wide array of proteins have been thus analyzed in detail as far as their susceptibility to either modification or both, and the resulting functional changes have been described in a number of experimental settings. The present review aims to provide an update of available knowledge in the field, with a special focus on the respective (sometimes competing and antagonistic) roles played by protein S-nitrosations and S-thionylations in biochemical and cellular processes specifically pertaining to pathogenesis of cardiovascular diseases.

One-week in vivo sustained release of a peptide formulated into in situ forming implants.

The LR12 peptide has been reported to reduce the size of infarct and improve both cardiac function and survival in myocardial infarction in murine models, after daily repeated intraperitoneal injections. In order to protect peptide from degrading and to prolong its release, in situ implants based on biocompatible biodegradable polymers were prepared and both in vitro and in vivo releases were evaluated after subcutaneous administration to Wistar rats. A progressive and complete release was obtained in vitro in 3 weeks. In vivo, a 7-day sustained release was demonstrated after administrating the formulation once; bioavailability was improved by protecting the peptide against the degradation identified as a dimerization through disulfide bond formation. As a conclusion, in situ forming formulations are a suitable alternative for the therapeutic use of this peptide.

Sulfur Denitrosylation by an Engineered Trx-like DsbG Enzyme Identifies Nucleophilic Cysteine Hydrogen Bonds as Key Functional Determinant.

Exposure of bacteria to NO results in the nitrosylation of cysteine thiols in proteins and low molecular weight thiols such as GSH. The cells possess enzymatic systems that catalyze the denitrosylation of these modified sulfurs. An important player in these systems is thioredoxin (Trx), a ubiquitous, cytoplasmic oxidoreductase that can denitrosylate proteins in vivo and S-nitrosoglutathione (GSNO) in vitro However, a periplasmic or extracellular denitrosylase has not been identified, raising the question of how extracytoplasmic proteins are repaired after nitrosative damage. In this study, we tested whether DsbG and DsbC, two Trx family proteins that function in reducing pathways in the Escherichia coli periplasm, also possess denitrosylating activity. Both DsbG and DsbC are poorly reactive toward GSNO. Moreover, DsbG is unable to denitrosylate its specific substrate protein, YbiS. Remarkably, by borrowing the CGPC active site of E. coli Trx-1 in combination with a T200M point mutation, we transformed DsbG into an enzyme highly reactive toward GSNO and YbiS. The pKa of the nucleophilic cysteine, as well as the redox and thermodynamic properties of the engineered DsbG are dramatically changed and become similar to those of E. coli Trx-1. X-ray structural insights suggest that this results from a loss of two direct hydrogen bonds to the nucleophilic cysteine sulfur in the DsbG mutant. Our results highlight the plasticity of the Trx structural fold and reveal that the subtle change of the number of hydrogen bonds in the active site of Trx-like proteins is the key factor that thermodynamically controls reactivity toward nitrosylated compounds.

Albumin as a carrier for NO delivery: preparation, physicochemical characterization, and interaction with gold nanoparticles.

BACKGROUND: Nitric oxide (NO) is a gaseous transmitter playing numerous physiological roles and characterized by a short half-life. Its binding to endogenous thiols increases its stability, facilitating its storage and transport. The purpose of this study was to investigate the nitrosated serum albumin (SA-SNO) and to provide a reference for its easy preparation for further use in in vitro studies. METHODS: Serum albumin (SA) was S-nitrosated by reacting with (i) NaNO2 in acidic medium; (ii) different low-molecular weight S-nitrosothiols (RSNO) (S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), and S,S'-dinitrosobucillamine (Buc(NO)2)); and (iii) diethylamine NONOate (DEA/NO). SA-SNO was purified by size exclusion chromatography and the S-nitrosation site and the rate were studied by mass spectrometry and Griess-Saville assay, respectively. Then, SA-SNO was characterized by spectrofluorimetry, dynamic light scattering, and circular dichroism. Finally, SA-SNO reactivity with citrate stabilized gold nanoparticles (AuNP-citrate) was investigated via determination of NO release. RESULTS: S-nitrosation rates of SA were 90.1 ± 3.3, 76.8 ± 2.7, 80.3 ± 3.2, 84.8 ± 5.0, and 15.4 ± 1.9% (n = 5), when SA was reacted with acidified NaNO2, CysNO, GSNO, Buc(NO)2, and DEA/NO, respectively. The physicochemical characterization indicated that the resulting product corresponded to a mono-S-nitrosothiol (on cysteine-34), and the conformational construction remained unchanged. Stability studies showed that the NO content was preserved over 1 week. AuNP-citrate reacted with SA-SNO with increase of its hydrodynamic diameter but preservation of SNO bond. CONCLUSIONS: SA-SNO prepared and stored under the reported conditions affords a well-defined reference suitable for in vitro studies.

S,S'-dinitrosobucillamine, a new nitric oxide donor, induces a better vasorelaxation than other S-nitrosothiols.

S-nitrosothiols (RSNO) are considered as potential drugs for delivering nitric oxide (NO) or related species in cardiovascular disorders associated with decrease in NO bioavailability. We have synthesized a new RSNO, i.e. S,S'-dinitrosobucillamine (BUC(NO)2), which combines in its structure two S-mononitrosothiols, S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO). Synthesized BUC(NO)2 was structurally characterized using high-performance liquid chromatography/mass spectrometry (HPLC/MS), (1)H nuclear magnetic resonance ((1)H NMR), infrared (IR) and UV-visible spectroscopies, and thermal analysis; resulting data are consistent with the expected structure. The vasorelaxant effect of BUC(NO)2 was evaluated using isolated rat aortic rings and compared to SNAP, NACNO, and to an equimolar mixture of NACNO plus SNAP in order to mimic the number of NO contained in a BUC(NO)2 molecule. BUC(NO)2 (pD2=7.8±0.1) was more potent in vasorelaxation than NACNO (pD2=6.4±0.2), SNAP (pD2=6.7±0.1) and the mixture of SNAP plus NACNO (pD2=6.7±0.2). The release of NO from BUC(NO)2 was 6-fold that of the basal value and significantly higher than the release of NO from the SNAP plus NACNO mixture (4-fold increase versus basal value). Finally, the role of protein disulfide isomerase (PDI) in BUC(NO)2 metabolism was investigated. Vasorelaxant effect (pD2=6.8±0.2) and NO release decreased in the presence of a PDI inhibitor (both P<0.05 versus BUC(NO)2). In conclusion, BUC(NO)2 releases a larger amount of NO into the aorta, partially through PDI activation, and induces vasorelaxation at lower concentrations than other RSNO previously reported.

Pitfalls of assays devoted to evaluation of oxidative stress induced by inorganic nanoparticles.

During the last years, there has been a remarkable increase in the use of inorganic nanoparticles (NP) in different applications, including consumer and medical products. Despite these promising applications, the extremely small size of NP allows them to penetrate cells, in which they can interact with intracellular structures causing serious side effects. A number of studies showed that NP cause adverse effects predominantly via induction of an oxidative stress - an imbalance between damaging oxidants and protective antioxidants - resulting in inflammation, immune response, cell damages, genotoxicity, etc … Most of the in vitro methods used for measurement of oxidative stress biomarkers were designed and standardized for conventional organic, inorganic and biochemical compounds. More recently, these methods have been adapted to studies related to various nanomaterials. Thus, this review is an attempt to highlight some current methods employed in and to provide a critical analysis of the major challenges and issues faced in this emerging field.

S-nitrosation/denitrosation in cardiovascular pathologies: facts and concepts for the rational design of S-nitrosothiols.

Nitric oxide (•NO) is a physiological mediator of vasorelaxation constitutively synthesized by endothelial nitric oxide synthase. Because •NO has a short half-life, it is stored by proteins through S-nitrosation reactions. S-nitrosation was recently defined as a post-translational modification of proteins for cellular signalling, as important as glycosylation and phosphorylation. Disulfide forming/ isomerizing enzymes like thioredoxin (Trx), protein disulfide isomerase (PDI), which are chaperone proteins, are implicated into transnitrosation reactions, which are the transfer of •NO from one cysteine residue to another one. Furthermore, Trx has been shown to denitrosate S-nitrosoproteins depending on its redox status. S-nitrosation of Trx on Cys residues apart from active site, under nitrosative or oxidative stresses, enhances its activity, thereby reducing intracellular reactive oxygen species. Trx and PDI have therefore an essential role for cell signalling control which leads, among other actions, to cardio and vasculo-protection. The diminution of either •NO synthesis or bioavailability is implicated into a large number of cardiovascular pathologies associated to hypoxia or vasoconstriction like, endothelial dysfunction, arterial hypertension and atherosclerosis. In order to mimic the physiological storage of •NO as S-nitrosothiols, the development of •NO donors should be based on the covalent S-NO bond. The chemical stabilisation of the S-NO bond and protection against enzymatically active proteins such as PDI//Trx are major points for the design of stable compounds. S-nitrosothiols entrapment in innovative formulations (films, gels, microparticles, nanoparticles) is an emerging field in order to stabilise and protect them, and to deliver •NO under a sustained release at the targeted site.

Endothelial γ-glutamyltransferase contributes to the vasorelaxant effect of S-nitrosoglutathione in rat aorta.

S-nitrosoglutathione (GSNO) involved in storage and transport of nitric oxide ((•)NO) plays an important role in vascular homeostasis. Breakdown of GSNO can be catalyzed by γ-glutamyltransferase (GGT). We investigated whether vascular GGT influences the vasorelaxant effect of GSNO in isolated rat aorta. Histochemical localization of GGT and measurement of its activity were performed by using chromogenic substrates in sections and in aorta homogenates, respectively. The role of GGT in GSNO metabolism was evaluated by measuring GSNO consumption rate (absorbance decay at 334 nm), (•)NO release was visualized and quantified with the fluorescent probe 4,5-diaminofluorescein diacetate. The vasorelaxant effect of GSNO was assayed using isolated rat aortic rings (in the presence or absence of endothelium). The role of GGT was assessed by stimulating enzyme activity with cosubstrate glycylglycine, as well as using two independent inhibitors, competitive serine borate complex and non-competitive acivicin. Specific GGT activity was histochemically localized in the endothelium. Consumption of GSNO and release of free (•)NO decreased and increased in presence of serine borate complex and glycylglycine, respectively. In vasorelaxation experiments with endothelium-intact aorta, the half maximal effective concentration of GSNO (EC50 = 3.2 ± 0.5.10(-7) M) increased in the presence of the two distinct GGT inhibitors, serine borate complex (1.6 ± 0.2.10(-6) M) and acivicin (8.3 ± 0.6.10(-7) M), while it decreased with glycylglycine (4.7 ± 0.9.10(-8) M). In endothelium-denuded aorta, EC(50) for GSNO alone increased to 2.3 ± 0.3.10(-6) M, with no change in the presence of serine borate complex. These data demonstrate the important role of endothelial GGT activity in mediating the vasorelaxant effect of GSNO in rat aorta under physiological conditions. Because therapeutic treatments based on GSNO are presently under development, this endothelium-dependent mechanism involved in the vascular effects of GSNO should be taken into account in a pharmacological perspective.

Accurate measurement of reduced glutathione in gamma-glutamyltransferase-rich brain microvessel fractions.

Investigation of the redox status in the cerebral circulation is of great importance in order to evaluate intensity of oxidative stress-related diseases and the corresponding therapeutic effects. Changes in levels of reduced glutathione (GSH) are a major indicator of oxidative stress conditions. However, an important limitation for measurement of GSH as a biomarker is the possible presence in samples of gamma-glutamyltransferase (GGT) activity, i.e., the enzyme catalysing GSH breakdown. An accurate assay for the measurement of GSH in rat brain microvessels was developed, taking into account the high GGT activity expressed in this tissue compartment. Based on a sensitive fluorescence-based microtiter plate method using 2,3-naphthalenedicarboxyaldehyde as GSH-selective fluorogenic probe, the assay was applied to brain microvessels isolated from individual male Wistar rats. Pooling of microvessel fractions from several animals, as required by other procedures, could thus be avoided. In order to prevent GSH consumption via GGT activity, serine-boric acid complex (SBC) was added as inhibitor all along the microvessels isolation process. In the absence of GGT inhibition GSH in isolated brain microvessels was below the limit of quantification. Addition of SBC almost completely suppressed GGT activity, thus allowing GSH quantification (4.4±1.6 nmol.mg(-1) protein, n=3). Following the administration of a GSH depletor (diethyl maleate, 1g.kg(-1), i.p.), decreased GSH levels were measured in liver, brain tissue and brain microvessels as well, thus confirming the reliability of the method for safe GSH measurements in small-sized, individual samples presenting high GGT activity.

Nanoparticles as tools for evaluation of cellular redox state.

Signaling pathways in living cells which regulate cell cycle and activate defense mechanisms against environmental stresses are mainly controlled through cell redox potential status. The main cellular redox buffer is constituted by oxidized/reduced glutathione system (GSSG/2GSH). Physiological functions and pathological situations resulting from oxidative stress are driven by glutathione homeostasis and its dysfunction, respectively. Many methodologies devoted to the investigation of cellular redox potential relying on the determination of reduced and oxidized glutathione concentrations measured in various cellular compartments, tissues and organs have been reported. Most of them are till now based on separative and non-separative techniques and include extractive step from biological samples. They are time-consuming and tedious in order to preserve reduced to oxidized glutathione ratio, thus introducing bias in its evaluation. New trends are focused on in situ evaluation of redox state especially through the monitoring of glutathione pool. This review reports recent developments in fluorescence imaging of cellular redox state, especially those involving nanotechnologies.

Role of glutathione metabolism status in the definition of some cellular parameters and oxidative stress tolerance of Saccharomyces cerevisiae cells growing as biofilms.

The resistance of Saccharomyces cerevisiae to oxidative stress (H(2)O(2) and Cd(2+)) was compared in biofilms and planktonic cells, with the help of yeast mutants deleted of genes related to glutathione metabolism and oxidative stress. Biofilm-forming cells were found predominantly in the G1 stage of the cell cycle. This might explain their higher tolerance to oxidative stress and the young replicative age of these cells in an old culture. The reduced glutathione status of S. cerevisiae was affected by the growth phase and apparently plays an important role in oxidative stress tolerance in cells growing as a biofilm.