Targeted eicosanoids lipidomics of exhaled breath condensate in healthy subjects

Background

Exhaled breath condensate collection is a non-invasive method of sampling the respiratory tract that can be repeated several times in a wide range of clinical settings. Quantitation of non-volatile compounds in the condensate requires highly sensitive analytical methods, e.g. mass spectrometry.

Objective

To validate cross-platform measurements of eicosanoids using high performance liquid chromatography or gas chromatography coupled with mass spectrometry in exhaled breath condensate sampled from 58 healthy individuals.

Methods

Twenty different eicosanoid compounds, representing major arachidonic acid lipoxygenation and cyclooxygenation pathways were measured using a stable isotope dilution method. We applied a free palmitic acid concentration as a surrogate marker for the condensate dilution factor.

Results

Eicosanoids concentrations in the condensates were consistent with their content in other biological fluids. Prostaglandin E₂ was the most abundant mediator, represented by its stable metabolite tetranor-PGEM. Prostaglandin D₂ products were at low concentration, while hydroxyacids derived from lipoxygenation were abundant. 5-HETE was elevated in current tobacco smokers. Leukotriene B₄ has the highest concentration of all 5-LO products. 15-LO analogues of cysteinyl leukotrienes–eoxins were detectable and metabolized to eoxin E₄. Two main vascular prostanoids: prostacyclin and thromboxane B₂ were present as metabolites. A marker for non-enzymatic lipid peroxidation, 8-iso-PGF_2α isoprostane was increased in smokers.

Conclusion

Presented targeted lipidomics analysis of exhaled breath condensate in healthy subjects justifies its application to investigation of inflammatory lung diseases. Measurements of non-volatile mediators of inflammation in the condensates might characterize disease-specific pathological mechanisms and responses to treatment.     

Manipulating the bioactivity of hydroxyapatite nano-rods structured networks: Effects on mineral coating morphology and growth kinetic

Background

Nano-hydroxyapatite particles have better bioactivity than the coarse crystals. So, they can be utilized for engineered tissue implants with improved efficiency over other materials. The development of materials with specific bioactive characteristics is still under investigation.

Methods

The surface properties of four hydroxyapatite materials templated by different micelle-polymer structured network are studied. The synergistic interaction of each block copolymer in contact with CTAB rod-like micelles results in crystalline HAp nano-rods of 25–50 nm length organized in hierarchical structures with different micro-rough characteristics.

Results

It was observed that the material in vitro bioactivity strongly depends on the surface structure while in a minor extent on their Ca/P ratio. So, MIII and MIV materials with Skewness parameter R_sk > 2.62 favored the formation on their surfaces of net-like phase with a high growth kinetic constant; while MI and MII (R_sk ≤ 2.62) induced the appearance of spherulitic-like structures and a growth rate 1.75 times inferior. Material biocompatibility was confirmed by interaction with rat calvarial osteoblasts.

Conclusions

The different structures growth is attributed to a dissimilar matching of crystal planes in the material and the apatite layer formed. In specific synthesis conditions, a biocompatible material with a Ca/P ratio close to that for the trabecular bone and a morphology that are considered essential for bone-bonding was obtained.

General significance

The creation of implantable devices with a specific bioactive characteristic may be useful to manipulate the attachment of cells on mineral coating directly affecting the stability and life of the implant.     

Evaluation of mutagenic and genotoxic activities of lobeline and its modulation on genomic instability induced by ethanol

Aim

Lobeline is a natural alkaloid derived from Lobelia inflata that has been investigated as a clinical candidate for the treatment of alcoholism. In a pre-clinical trial, lobeline decreased the preference for and consumption of ethanol, due to the modulation of the nicotinic acetylcholine receptor. However, the interaction between lobeline and ethanol is poorly known and thus there are safety concerns.The present study was conducted to evaluate the mutagenic and genotoxic effects of lobeline and assess its modulation of ethanol-induced toxicological effects.

Main methods

CF-1 male mice were divided into five groups. Groups received an intraperitoneal injection of saline solution, lobeline (5 or 10 mg/kg), ethanol (2.5 g/kg), or lobeline plus ethanol, once a day for three consecutive days. Genotoxicity was evaluated in peripheral blood using the alkaline comet assay. The mutagenicity was evaluated using both Salmonella/microsome assay in TA1535, TA97a, TA98, TA100, and TA102 Salmonella typhimurium strains and the micronucleus test in bone marrow. Possible liver and kidney injuries were evaluated using biochemical analysis.

Key findings

Lobeline did not show genotoxic or mutagenic effects and did not increase the ethanol-induced genotoxic effects in blood. Lobeline also protected blood cells against oxidative damage induced by hydrogen peroxide. Biochemical parameters were not altered, indicating no liver or kidney injuries or alterations in lipid and carbohydrate metabolisms.

Significance

These findings suggest that lobeline does not induce gene or chromosomal mutations, and that this lack of genetic toxicity is maintained in the presence of ethanol, providing further evidence of the safety of this drug to treat alcohol dependence.     

Antibacterial performance of nanoscaled visible-light responsive platinum-containing titania photocatalyst in vitro and in vivo

Background

Traditional antibacterial photocatalysts are primarily induced by ultraviolet light to elicit antibacterial reactive oxygen species. New generation visible-light responsive photocatalysts were discovered, offering greater opportunity to use photocatalysts as disinfectants in our living environment. Recently, we found that visible-light responsive platinum-containing titania (TiO₂–Pt) exerted high performance antibacterial property against soil-borne pathogens even in soil highly contaminated water. However, its physical and photocatalytic properties, and the application in vivo have not been well-characterized.

Methods

Transmission electron microscopy, energy dispersive spectroscopy, X-ray photoelectron spectroscopy, X-ray diffraction, ultraviolet–visible absorption spectrum and the removal rate of nitrogen oxides were therefore analyzed. The antibacterial performance under in vitro and in vivo conditions was evaluated.

Results

The apparent quantum efficiency for visible light illuminated TiO₂–Pt is relatively higher than several other titania photocatalysts. The killing effect achieved approximately 2 log reductions of pathogenic bacteria in vitro. Illumination of injected TiO₂–Pt successfully ameliorated the subcutaneous infection in mice.

Conclusions

This is the first demonstration of in vivo antibacterial use of TiO₂–Pt nanoparticles. When compared to nanoparticles of some other visible-light responsive photocatalysts, TiO₂–Pt nanoparticles induced less adverse effects such as exacerbated platelet clearance and hepatic cytotoxicity in vivo.

General significance

These findings suggest that the TiO₂–Pt may have potential application on the development of an antibacterial material in both in vitro and in vivo settings.     

In vitro cross-linking of elastin peptides and molecular characterization of the resultant biomaterials

Background

Elastin is a vital protein and the major component of elastic fibers which provides resilience to many vertebrate tissues. Elastin's structure and function are influenced by extensive cross-linking, however, the cross-linking pattern is still unknown.

Methods

Small peptides containing reactive allysine residues based on sequences of cross-linking domains of human elastin were incubated in vitro to form cross-links characteristic of mature elastin. The resultant insoluble polymeric biomaterials were studied by scanning electron microscopy. Both, the supernatants of the samples and the insoluble polymers, after digestion with pancreatic elastase or trypsin, were furthermore comprehensively characterized on the molecular level using MALDI-TOF/TOF mass spectrometry.

Results

MS² data was used to develop the software PolyLinX, which is able to sequence not only linear and bifunctionally cross-linked peptides, but for the first time also tri- and tetrafunctionally cross-linked species. Thus, it was possible to identify intra- and intermolecular cross-links including allysine aldols, dehydrolysinonorleucines and dehydromerodesmosines. The formation of the tetrafunctional cross-link desmosine or isodesmosine was unexpected, however, could be confirmed by tandem mass spectrometry and molecular dynamics simulations.

Conclusions

The study demonstrated that it is possible to produce biopolymers containing polyfunctional cross-links characteristic of mature elastin from small elastin peptides. MALDI-TOF/TOF mass spectrometry and the newly developed software PolyLinX proved suitable for sequencing of native cross-links in proteolytic digests of elastin-like biomaterials.

General significance

The study provides important insight into the formation of native elastin cross-links and represents a considerable step towards the characterization of the complex cross-linking pattern of mature elastin.     

DNA double‐strand break response in stem cells: Mechanisms to maintain genomic integrity

Background

Embryonic stem cells (ESCs) represent the point of origin of all cells in a given organism and must protect their genomes from both endogenous and exogenous genotoxic stress. DNA double-strand breaks (DSBs) are one of the most lethal forms of damage, and failure to adequately repair DSBs would not only compromise the ability of SCs to self-renew and differentiate, but will also lead to genomic instability and disease.

Scope of Review

Herein, we describe the mechanisms by which ESCs respond to DSB-inducing agents such as reactive oxygen species (ROS) and ionizing radiation, compared to somatic cells. We will also discuss whether the DSB response is fully reprogrammed in induced pluripotent stem cells (iPSCs) and the role of the DNA damage response (DDR) in the reprogramming of these cells.

Major Conclusions

ESCs have distinct mechanisms to protect themselves against DSBs and oxidative stress compared to somatic cells. The response to damage and stress is crucial for the maintenance of self-renewal and differentiation capacity in SCs. iPSCs appear to reprogram some of the responses to genotoxic stress. However, it remains to be determined if iPSCs also retain some DDR characteristics of the somatic cells of origin.

General Significance

The mechanisms regulating the genomic integrity in ESCs and iPSCs are critical for its safe use in regenerative medicine and may shed light on the pathways and factors that maintain genomic stability, preventing diseases such as cancer. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

The effects of selenium and the GPx-1 selenoprotein on the phosphorylation of H2AX

Background

Significant data supports the health benefits of selenium although supplementation trials have yielded mixed results. GPx-1, whose levels are responsive to selenium availability, is implicated in cancer etiology by human genetic data. Selenium's ability to alter the phosphorylation of the H2AX, a histone protein that functions in the reduction of DNA damage by recruiting repair proteins to the damage site, following exposure to ionizing radiation and bleomycin was investigated.

Methods

Human cell lines that were either exposed to selenium or were transfected with a GPx-1 expression construct were exposed to ionizing radiation or bleomycin. Phosphorylation of histone H2AX was quantified by flow cytometry and survival by the MTT assay. Phosphorylation of the Chk1 and Chk2 checkpoint proteins was quantified by western blotting.

Results

In colon-derived cells, selenium increases GPx-1 and attenuated H2AX phosphorylation following genotoxic exposures while the viability of these cells was unaffected. MCF-7 cells and transfectants that express high GPx-1 levels were exposed to ionizing radiation and bleomycin, and H2AX phosphorylation and cell viability were assessed. GPx-1 increased H2AX phosphorylation and viability following the induction of DNA damage while enhancing the levels of activated Chk1 and Chk2.

Conclusions

Exposure of mammalian cells to selenium can alter the DNA damage response and do so by mechanisms that are dependent and independent of its effect on GPx-1.

General significance

Selenium and GPx-1 may stimulate the repair of genotoxic DNA damage and this may account for some of the benefits attributed to selenium intake and elevated GPx-1 activity.     

Penetration of verapamil across blood brain barrier following cerebral ischemia depending on both paracellular pathway and P-glycoprotein transportation

Background

Blood brain barrier (BBB) dysfunction is a common facet of cerebral ischemia, and the alteration of drug transporter, P-glycoprotein (P-gp), has been documented.

Aims

This study explores influence of damaged BBB and elevated P-gp on cerebral verapamil penetration after ischemia both in vivo and in vitro.

Methods

Middle cerebral artery occlusion (MCAO) induced ischemia/reperfusion (I/R) of rats, and Na₂S₂O₄ induced hypoxia/reoxygenation (H/R) damage of rat brain mirovessel endothelial cells (RBMECs) respectively, served as BBB breakdown model in vivo and in vitro. Evans-Blue (EB) extravagation and ¹²⁵I-albumin were used to quantify BBB dysfunction; UPLC–MS/MS analytical method was performed to determine accurately the concentration of verapamil in brain tissue and cell. Flow cytometry, immunohistochemistry and western blotting were applied to evaluate transport function and protein expression of P-gp.

Results

Overexpressed ICAM-1 and MMP-9 mediated BBB dysfunction after ischemia, which induced EB leakage and ¹²⁵I-albumin uptake increase. Enhanced accumulation of verapamil in brain tissue, but intracellular concentration reduced evidently after H/R injury. Transcellular transportation of verapamil elevated when P-gp function or expression was inhibited after H/R injury.

Conclusion

These data indicated that BBB penetration of verapamil under ischemia condition was not only depending on BBB breakdown, but also regulated by P-gp.     

Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells

Background

The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake.

Methods

These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model membrane systems was evaluated by Langmuir balance and confocal microscopy followed by the study of the internalization of a lipid oligodeoxyribonucleotide conjugate bearing a double-tail lipid modification (C₂₈) into different cell lines by confocal microscopy and flow cytometry. This compound was also compared with other lipid containing conjugates and with the classical lipoplex formulation using Transfectin as transfection reagent.

Results

This double-tail lipid modification showed better incorporation into both lipid model membranes and cell systems. Indeed, this lipid conjugation was capable of inserting the oligodeoxyribonucleotide into both liquid-disordered and liquid-ordered domains of model lipid bilayer systems and produced an enhancement of oligodeoxyribonucleotide uptake in cells, even better than the effect caused by lipoplexes. In addition, in β₂ integrin (CR3) expressing cells this receptor was directly involved in the enhanced internalization of this compound.

Conclusions

All these features confirm that the dual lipid modification (C₂₈) is an excellent modification for enhancing nucleic acid delivery without altering their binding properties.

General significance

Compared to the commercial lipoplex approach, oligodeoxyribonucleotide conjugation with C₂₈ dual lipid modification seems to be promising to improve oligonucleotide delivery in mammalian cells.     

EGF and angiotensin II modulate lysophosphatidic acid LPA1 receptor function and phosphorylation state

Background

Lysophosphatidic acid (LPA) is a local mediator that exerts its actions through G protein coupled receptors. Knowledge on the regulation of such receptors is scarce to date. Here we show that bidirectional cross-talk exits between LPA₁ and EGF receptors.

Methods

C9 cells expressing LPA₁ receptor fussed to the enhanced green fluorescent protein were used. We studied intracellular calcium concentration, Akt/PKB phosphorylation, LPA₁ and EGF receptor phosphorylation.

Results

EGF diminished LPA-mediated intracellular calcium response and induced LPA₁ receptor phosphorylation, which was sensitive to protein kinase C inhibitors. Angiotensin II and LPA induced EGF receptor transactivation as evidenced by Akt/PKB phosphorylation through metalloproteinase-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF receptors. This process was found to be of major importance in angiotensin II-induced LPA₁ receptor phosphorylation. Attempts to define a role for EGF receptor transactivation in homologous LPA₁ receptor desensitization and phosphorylation suggested that G protein-coupled receptor kinases are the major players in this process, overshadowing other events.

Conclusions

EGF receptors and LPA₁ receptors are engaged in an intense liaison, in that EGF receptors are capable of modulating LPA₁ receptor function through phosphorylation cascades. EGF transactivation plays a dual role: it mediates some LPA actions, and it modulates LPA₁ receptor function in inhibitory fashion.

General significance

EGF and LPA receptors coexist in many cell types and play key roles in maintaining the delicate equilibrium that we call health and in the pathogenesis of many diseases. The intense cross-talk described here has important physiological and pathophysiological implications.     

Facilitating joint chaos and fractal analysis of biosignals through nonlinear adaptive filtering

Background

Chaos and random fractal theories are among the most important for fully characterizing nonlinear dynamics of complicated multiscale biosignals. Chaos analysis requires that signals be relatively noise-free and stationary, while fractal analysis demands signals to be non-rhythmic and scale-free.

Methodology/Principal Findings

To facilitate joint chaos and fractal analysis of biosignals, we present an adaptive algorithm, which: (1) can readily remove nonstationarities from the signal, (2) can more effectively reduce noise in the signals than linear filters, wavelet denoising, and chaos-based noise reduction techniques; (3) can readily decompose a multiscale biosignal into a series of intrinsically bandlimited functions; and (4) offers a new formulation of fractal and multifractal analysis that is better than existing methods when a biosignal contains a strong oscillatory component.

Conclusions

The presented approach is a valuable, versatile tool for the analysis of various types of biological signals. Its effectiveness is demonstrated by offering new important insights into brainwave dynamics and the very high accuracy in automatically detecting epileptic seizures from EEG signals.     

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli

Background

F₁F_O ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli.

Methods

We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy.

Results

We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F₁F_O ATP synthases.

Conclusions

Our system now allows performing previously impossible structural and functional studies on A. aeolicus F₁F_O ATP synthase.

General significance

More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.     

Dynamics fingerprint and inherent asymmetric flexibility of a cold-adapted homodimeric enzyme. A case study of the Vibrio alkaline phosphatase

Background

Protein dynamics influence protein function and stability and modulate conformational changes. Such motions depend on the underlying networks of intramolecular interactions and communicating residues within the protein structure. Here, we provide the first characterization of the dynamic fingerprint of the dimeric alkaline phosphatase (AP) from the cold-adapted Vibrio strain G15-21 (VAP), which is among the APs with the highest known k_cat at low temperatures.

Methods

Multiple all-atom explicit solvent molecular dynamics simulations were employed in conjunction with different metrics to analyze the dynamical patterns and the paths of intra- and intermolecular communication.

Results

Interactions and coupled motions at the interface between the two VAP subunits have been characterized, along with the networks of intramolecular interactions. It turns out a low number of intermolecular interactions and coupled motions, which result differently distributed in the two monomers. The paths of long-range communication mediated from the catalytic residues to distal sites were also characterized, pointing out a different information flow in the two subunits.

Conclusions

A pattern of asymmetric flexibility is evident in the two identical subunits of the VAP dimer that is intimately linked to a different distribution of intra- and intermolecular interactions. The asymmetry was also evident in pairs of cross-correlated residues during the dynamics.

General significance

The results here discussed provide a structural rationale to the half-of-site mechanism previously proposed for VAP and other APs, as well as a general framework to characterize asymmetric dynamics in homomeric enzymes.     

Inhibition of peroxisomal hydroxypyruvate reductase (HPR1) by tyrosine nitration

Background

Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported.

Methods

We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches.

Results

Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H₂O₂, NO, GSH and peroxynitrite showed that the ONOO⁻ molecule caused the highest inhibition of activity (51% at 5 mM SIN-1), with 5 mM H₂O₂ having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite.

Conclusion

These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function.

General significance

This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions.     

A rubber transfer gasket to improve the throughput of liquid–liquid extraction in 96-well plates: Application to vitamin D testing

Background

The unmitigated rise in demand for the assessment of vitamin D status has taxed the ability of clinical mass spectrometry laboratories to preserve turn-around times. We aimed to improve the throughput of liquid–liquid extraction of plasma/serum for the assay of 25-hydroxy vitamin D.

Methods

We designed and fabricated a flexible rubber gasket that seals two 96-well plates together to quantitatively transfer the contents of one plate to another. Using the transfer gasket and a dry-ice acetone bath to freeze the aqueous infranatant, we developed a novel liquid–liquid extraction workflow in a 96-well plate format. We applied the technology to the mass spectrometric quantification of 25-hydroxy vitamin D.

Results

Cross-contamination between wells was ≤0.13%. The interassay imprecision over 132 days of clinical implementation was less than 10%. The method compared favorably to a standard liquid–liquid extraction in glass tubes (Deming slope = 1.018, S_x|y = 0.022). The accuracy of the assay was 102–105% as assessed with the recently released control materials from NIST.

Conclusions

The development of a plate-sealing gasket permits the liquid–liquid extraction of clinical specimens in a moderate-throughput workflow and the reliable assay of vitamin D status. In the future, the gasket may also prove useful in other sample preparation techniques for HPLC or mass spectrometry.     

Biochemical and pharmacological properties of a new thrombin-like serine protease (Russelobin) from the venom of Russell's Viper (Daboia russelii russelii) and assessment of its therapeutic potential

Background

Snake venoms are rich sources of bioactive molecules, and several venom-derived proteins have entered clinical trials for use in ischemic disorders; however, late-stage failure of a recent drug candidate due to low in vivo efficacy demonstrated the need for new sources of fibrinogenolytic drug candidates.

Methods

A 51.3 kDa thrombin-like serine protease (Russelobin) purified from the venom of Russell's Viper (Daboia russelii russelii) was subjected to extensive biochemical characterization, including N-terminal sequencing, substrate specificity, kinetic and inhibitor assays, glycosylation analysis and stability assays. Toxicity and pathology analyses were conducted in NSA mice.

Results

Russelobin has extensive N-terminus identity with a beta-fibrinogenase-like serine proteinase precursor from Daboia russelii siamensis venom, a mass of 51.3 kDa and contains extensive N-linked oligosaccharides. Serine protease inhibitors and heparin significantly decreased activity, with much lower inhibition by DTT, antithrombin-III and α₂-macroglobulin. Russelobin preferentially released FPA and slowly released FPB from human fibrinogen, forming a labile fibrin clot readily hydrolyzed by plasmin. The partially deglycosylated enzyme showed significantly lower activity toward fibrinogen and less resistance against neutralization by plasma α₂MG and antithrombin-III. Russelobin was non-cytotoxic, non-lethal and produced no histopathologies in mice, and it demonstrated in vivo dose-dependent defibrinogenating activity.

Conclusions

Russelobin is an A/B fibrinogenase with high specificity toward fibrinogen, both in vitro and in vivo. Extensive glycosylation appears to protect the molecule against endogenous protease inhibitors, prolonging its in vivo efficacy.

General significance

Due to its low toxicity, stability and activity as a defibrinogenating agent, Russelobin shows high potential for cardiovascular drug development.     

Plasmodium falciparum antioxidant protein as a model enzyme for a special class of glutaredoxin/glutathione-dependent peroxiredoxins

Background

Peroxiredoxins are important heterogeneous thiol-dependent hydroperoxidases with a variety of isoforms and enzymatic mechanisms. A special subclass of glutaredoxin/glutathione-dependent peroxiredoxins has been discovered in bacteria and eukaryotes during the last decade, but the exact enzymatic mechanisms of these enzymes remain to be unraveled.

Methods

We performed a comprehensive analysis of the enzyme kinetics and redox states of one of these glutaredoxin/glutathione-dependent peroxiredoxins, the antioxidant protein from the malaria parasite Plasmodium falciparum, using steady-state kinetic measurements, site-directed mutagenesis, redox mobility shift assays, gel filtration, and mass spectrometry.

Results

P. falciparum antioxidant protein requires not only glutaredoxin but also glutathione as a true substrate for the reduction of hydroperoxides. One peroxiredoxin cysteine residue and one glutaredoxin cysteine residue are sufficient for catalysis, however, additional cysteine residues of both proteins result in alternative redox states and conformations in vitro with implications for redox regulation. Our data furthermore point to a glutathione-dependent peroxiredoxin activation and a negative subunit cooperativity.

Conclusions

The investigated glutaredoxin/glutathione/peroxiredoxin system provides numerous new insights into the mechanism and redox regulation of peroxiredoxins.

General significance

As a member of the special subclass of glutaredoxin/glutathione-dependent peroxiredoxins, the P. falciparum antioxidant protein could become a reference protein for peroxiredoxin catalysis and regulation.     

Bauhinia forficata lectin (BfL) induces cell death and inhibits integrin-mediated adhesion on MCF7 human breast cancer cells

Background

Plant lectins have attracted great interest in cancer studies due to their antitumor activities. These proteins or glycoproteins specifically and reversibly bind to different types of carbohydrates or glycoproteins. Breast cancer, which presents altered glycosylation of cell surface glycoproteins, is one of the most frequent malignant diseases in women. In this work, we describe the effect of the lectin Bauhinia forficata lectin (BfL), which was purified from B. forficata Link subsp. forficata seeds, on the MCF7 human breast cancer cellular line, investigating the mechanisms involved in its antiproliferative activity.

Methods

MCF7 cells were treated with BfL. Viability and adhesion alterations were evaluated using flow cytometry and western blotting.

Results

BfL inhibited the viability of the MCF7 cell line but was ineffective on MDA-MB-231 and MCF 10A cells. It inhibits MCF7 adhesion on laminin, collagen I and fibronectin, decreases α₁, α₆ and β₁ integrin subunit expression, and increases α₅ subunit expression. BfL triggers necrosis and secondary necrosis, with caspase-9 inhibition. It also causes deoxyribonucleic acid (DNA) fragmentation, which leads to cell cycle arrest in the G2/M phase and a decrease in the expression of the regulatory proteins pRb and p21.

Conclusion

BfL shows selective cytotoxic effect and adhesion inhibition on MCF7 breast cancer cells.

General significance

Cell death induction and inhibition of cell adhesion may contribute to understanding the action of lectins in breast cancer.     

Proteomics screening of molecular targets of curcumin in mouse brain

Aims

Curcumin is one of the most important constituent of Curcuma longa L. with antioxidant, anti-inflammatory and anticancer effects. In this study, we investigated potential intracellular targets of curcumin by affinity chromatography based on target deconvolution. Identification of curcumin interacting proteins may help in evaluating biological and side effects of this natural compound.

Main methods

Curcumin was immobilized through a linker to sepharose beads as solid matrix. Pull down assay was performed by passing tissue lysate of mouse brain through the column to enrich and purify curcumin interacting proteins. Then proteins were separated using two-dimensional gel electrophoresis and identified using MALDI/TOF/TOF mass spectrometry.

Key findings

Our results show that curcumin physically binds to a wide range of cellular proteins including structural proteins, metabolic enzymes and proteins involved in apoptosis pathway.

Significance

Finding curcumin interacting proteins may help in understanding a part of curcumin pharmacological effects.