Reactivity of hydrogen sulfide with peroxynitrite and other oxidants of biological interest

Hydrogen sulfide (H(2)S) is an endogenously generated gas that can also be administered exogenously. It modulates physiological functions and has reported cytoprotective effects. To evaluate a possible antioxidant role, we investigated the reactivity of hydrogen sulfide with several one- and two-electron oxidants. The rate constant of the direct reaction with peroxynitrite was (4.8±1.4)×10(3)M(-1) s(-1) (pH 7.4, 37°C). At low hydrogen sulfide concentrations, oxidation by peroxynitrite led to oxygen consumption, consistent with a one-electron oxidation that initiated a radical chain reaction. Accordingly, pulse radiolysis studies indicated that hydrogen sulfide reacted with nitrogen dioxide at (3.0±0.3)×10(6)M(-1) s(-1) at pH 6 and (1.2±0.1)×10(7)M(-1) s(-1) at pH 7.5 (25°C). The reactions of hydrogen sulfide with hydrogen peroxide, hypochlorite, and taurine chloramine had rate constants of 0.73±0.03, (8±3)×10(7), and 303±27M(-1) s(-1), respectively (pH 7.4, 37°C). The reactivity of hydrogen sulfide was compared to that of low-molecular-weight thiols such as cysteine and glutathione. Considering the low tissue concentrations of endogenous hydrogen sulfide, direct reactions with oxidants probably cannot completely account for its protective effects.     

Deoxyribonucleic acid damage induced by doxorubicin in peripheral blood mononuclear cells: possible roles for the stress response and the deoxyribonucleic acid repair process

Doxorubicin is an antineoplastic drug widely used in cancer treatment. However, many tumors are intrinsically resistant to the drug or show drug resistance after an initial period of response. Among the different molecules implicated with doxorubicin resistance are the heat shock proteins (Hsps). At present we do not know with certainty the mechanism(s) involved in such resistance. In the present study, to advance our knowledge on the relationship between Hsps and drug resistance, we have used peripheral blood mononuclear cells obtained from healthy nonsmoker donors to evaluate the capacity of a preliminary heat shock to elicit the Hsp response and to establish the protection against the deoxyribonucleic acid (DNA) damage induced by doxorubicin. DNA damage and repair were determined using the alkaline comet assay. We also measured the expression of Hsp27, Hsp60, Hsp70, Hsp90, hMLH1, hMSH2, and proliferating cell nuclear antigen by immunocytochemistry. The damage induced by doxorubicin was more efficiently repaired when the cells were previously heat shocked followed by a resting period of 24 hours before drug exposure, as shown by (1) the increased number of undamaged cells (P < 0.05), (2) the increased DNA repair capacity (P < 0.05), and (3) the high expression of the mismatch repair (MMR) proteins hMLH1 and hMSH2 (P < 0.05). In addition, in the mentioned group of cells, we confirmed by Western blot high expression levels of Hsp27 and Hsp70. We also noted a nuclear translocation of Hsp27 and mainly of Hsp70. Furthermore, inducible Hsp70 was more expressed in the nucleus than Hsc70, showing a possible participation of Hsp70 in the DNA repair process mediated by the MMR system.     

Beneficial lactobacilli: effects on the vaginal tract in a murine experimental model

Vaginal probiotics containing lactic acid bacteria with activity towards pathogenic microorganisms that cause urogenital tract infections have been proposed as a valid strategy for their prophylaxis and therapy. A murine experimental model was set up to evaluate the colonization capability of beneficial human lactobacilli and their effects on the mouse vaginal mucosa and innate immune cells. Five Lactobacillus strains were intravaginally inoculated into previously estrogenized BALB/c mice. The significance of the effects observed in the vaginal tract was determined by analysis of variance using the general linear model. The numbers of viable vaginal lactobacilli were significantly higher at proestrous–estrous than those at the metaestrous–diestrous phase and decreased markedly on the days after inoculation. Lactobacilli inoculation did not cause cytological or histological modifications of the murine vaginal tract. Moreover, the intravaginal administration of Lactobacillus salivarius CRL (Centro de Referencia para Lactobacilos culture collection) 1328 and Lactobacillus gasseri CRL 1263 did not affect the amounts of granulocytes and macrophages present in vaginal washings. In conclusion, the results demonstrate that vaginal lactobacilli did not produce adverse effects on the murine vaginal tract. Therefore, they could be proposed as safe probiotic candidates to promote a balanced microbiota in the urogenital tract.     

Nonspecific depolarization of the plasma membrane potential induces cytoskeletal modifications of bovine corneal endothelial cells in culture

Modifications in the cell membrane potential have been suggested to affect signaling mechanisms participating in diverse cellular processes, many of which involve structural cellular alterations. In order to contribute some evidence in this respect, we explored the effects of several depolarizing procedures on the structure and monolayer organization of bovine corneal endothelial cells in culture. Visually confluent cell monolayers were incubated with or without the depolarizing agent, either in a saline solution or in culture medium for up to 30 min. Membrane potential was monitored by fluorescence microscopy using oxonol V. Fluorescent probes were employed for F-actin, microtubules, and vinculin. Depolarization of the plasma membrane, achieved via the incorporation of gramicidin D into confluent endothelial cells or by modifications of the extracellular saline composition, provoked an increment of oxonol fluorescence and changes in cell morphology, consisting mainly of modifications in the cytoskeletal organization. In some areas, noticeable intercellular spaces appear. The cytoskeleton modifications mainly consist of a marked redistribution of F-actin and microtubules, with accompanying changes in vinculin localization. The results suggest that the depolarization of the plasma membrane potential may participate in mechanisms involved in cytoskeleton organization and monolayer continuity in corneal endothelial cells in culture.     

Reactions of desferrioxamine with peroxynitrite-derived carbonate and nitrogen dioxide radicals

The iron chelating agent desferrioxamine inhibits peroxynitrite-mediated oxidations and attenuates nitric oxide and oxygen radical-dependent oxidative damage both in vitro and in vivo. The mechanism of protection is independent of iron chelation and has remained elusive over the past decade. Herein, stopped-flow studies revealed that desferrioxamine does not react directly with peroxynitrite. However, addition of peroxynitrite to desferrioxamine in both the absence and the presence of physiological concentrations of CO2 and under excess nitrite led to the formation of a one-electron oxidation product, the desferrioxamine nitroxide radical, consistent with desferrioxamine reacting with the peroxynitrite-derived species carbonate (CO3*-) and nitrogen dioxide (*NO2) radicals. Desferrioxamine inhibited peroxynitrite-dependent free radical-mediated processes, including tyrosine dimerization and nitration, oxyhemoglobin oxidation in the presence of CO2, and peroxynitrite plus carbonate-dependent chemiluminescence. The direct two-electron oxidation of glutathione by peroxynitrite was unaffected by desferrioxamine. The reactions of desferrioxamine with CO3*- and *NO2 were unambiguously confirmed by pulse radiolysis studies, which yielded second-order rate constants of 1.7 x 10(9) and 7.6 x 10(6) M(-1) s(-1), respectively. Desferrioxamine also reacts with tyrosyl radicals with k = 6.3 x 10(6) M(-1) s(-1). However, radical/radical combination reactions between tyrosyl radicals or of tyrosyl radical with *NO2 outcompete the reaction with desferrioxamine and computer-assisted simulations indicate that the inhibition of tyrosine oxidation can be fully explained by scavenging of the peroxynitrite-derived radicals. The results shown herein provide an alternative mechanism to account for some of the biochemical and pharmacological actions of desferrioxamine via reactions with CO3*- and *NO2 radicals.     

Thyroid hormone receptor α<Subscript>1</Subscript>–β<Subscript>1</Subscript> expression in epididymal epithelium from euthyroid and hypothyroid rats

The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express TRα₁–β₁ isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In consonance to the immunocytochemical analysis, the expression of TRα₁–β₁ isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions. Furthermore, TR expression of both α₁ and β₁ isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.     

Prevention of rectal SHIV transmission in macaques by daily or intermittent prophylaxis with emtricitabine and tenofovir

Background

In the absence of an effective vaccine, HIV continues to spread globally, emphasizing the need for novel strategies to limit its transmission. Pre-exposure prophylaxis (PrEP) with antiretroviral drugs could prove to be an effective intervention strategy if highly efficacious and cost-effective PrEP modalities are identified. We evaluated daily and intermittent PrEP regimens of increasing antiviral activity in a macaque model that closely resembles human transmission.

Methods and Findings

We used a repeat-exposure macaque model with 14 weekly rectal virus challenges. Three drug treatments were given once daily, each to a different group of six rhesus macaques. Group 1 was treated subcutaneously with a human-equivalent dose of emtricitabine (FTC), group 2 received orally the human-equivalent dosing of both FTC and tenofovir-disoproxil fumarate (TDF), and group 3 received subcutaneously a similar dosing of FTC and a higher dose of tenofovir. A fourth group of six rhesus macaques (group 4) received intermittently a PrEP regimen similar to group 3 only 2 h before and 24 h after each weekly virus challenge. Results were compared to 18 control macaques that did not receive any drug treatment. The risk of infection in macaques treated in groups 1 and 2 was 3.8- and 7.8-fold lower than in untreated macaques (p = 0.02 and p = 0.008, respectively). All six macaques in group 3 were protected. Breakthrough infections had blunted acute viremias; drug resistance was seen in two of six animals. All six animals in group 4 that received intermittent PrEP were protected.

Conclusions

This model suggests that single drugs for daily PrEP can be protective but a combination of antiretroviral drugs may be required to increase the level of protection. Short but potent intermittent PrEP can provide protection comparable to that of daily PrEP in this SHIV/macaque model. These findings support PrEP trials for HIV prevention in humans and identify promising PrEP modalities.     

Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide‐dependent mechanism

Protein tyrosine (Tyr) nitration is a post‐translational modification yielding 3‐nitrotyrosine (NO₂–Tyr). Formation of NO₂–Tyr is generally considered as a marker of nitro‐oxidative stress and is involved in some human pathophysiological disorders, but has been poorly studied in plants. Leghemoglobin (Lb) is an abundant hemeprotein of legume nodules that plays an essential role as an O₂ transporter. Liquid chromatography coupled to tandem mass spectrometry was used for a targeted search and quantification of NO₂–Tyr in Lb. For all Lbs examined, Tyr30, located in the distal heme pocket, is the major target of nitration. Lower amounts were found for NO₂–Tyr25 and NO₂–Tyr133. Nitrated Lb and other as yet unidentified nitrated proteins were also detected in nodules of plants not receiving and were found to decrease during senescence. This demonstrates formation of nitric oxide (˙NO) and by alternative means to nitrate reductase, probably via a ˙NO synthase‐like enzyme, and strongly suggests that nitrated proteins perform biological functions and are not merely metabolic byproducts. In vitro assays with purified Lb revealed that Tyr nitration requires  + H₂O₂ and that peroxynitrite is not an efficient inducer of nitration, probably because Lb isomerizes it to . Nitrated Lb is formed via oxoferryl Lb, which generates nitrogen dioxide and tyrosyl radicals. This mechanism is distinctly different from that involved in heme nitration. Formation of NO₂–Tyr in Lb is a consequence of active metabolism in functional nodules, where Lb may act as a sink of toxic peroxynitrite and may play a protective role in the symbiosis.     

Isolation and molecular characterization of a mouse renal microvascular endothelial cell line

Murine endothelial cells (ECs) have proven difficult to obtain and maintain in culture. Long-term maintenance of normal ECs remains a difficult task. In this article we report the establishment of the first cellular line of renal microvascular endothelium obtained from normal tissue. Cells were isolated, cloned, and maintained by serial passages for longer than 24 mo, using endothelial cell growth supplement (ECGS) and gelatin-coated plates. Their morphology and ultrastructure, expression of von Willebrand factor, presence of smooth muscle alpha-actin, vimentin, cytokeratin filaments, capillary structures formed on Matrigel, and some typical ECs surface molecules were the criteria used to characterize cultured ECs. When examined for responsiveness to Shiga toxin-1, 13-20% of cytotoxicity was observed when coincubated with lipopolysaccharides. This cytotoxicity was not observed for normal lung ECs (1G11). Consequently, REC-A4 line retains characteristics of resting microvascular ECs and represents a useful in vitro model to study biological and physiopathological properties of renal endothelium.