Altitude acclimatization via hypoxia-mediated oxidative eustress involves interplay of protein nitrosylation and carbonylation: A redoxomics perspective.

AIM: Hypoxia is an important feature of multiple diseases like cancer and obesity and also an environmental stressor to high altitude travelers. Emerging research suggests the importance of redox signaling in physiological responses transforming the notion of oxidative stress into eustress and distress. However, the behavior of redox protein post-translational modifications (PTMs), and their correlation with stress acclimatization in humans remains sketchy. Scant information exists about modifications in redoxome during physiological exposure to environmental hypoxia. In this study, we investigated redox PTMs, nitrosylation and carbonylation, in context of extended environmental hypoxia exposure. METHODS: The volunteers were confirmed to be free of any medical conditions and matched for age and weight. The human global redoxome and the affected networks were investigated using TMT-labeled quantitative proteo-bioinformatics and biochemical assays. The percolator PSM algorithm was used for peptide-spectrum match (PSM) validation in database searches. The FDR for peptide matches was set to 0.01. 1-way ANOVA and Tukey's Multiple Comparison test were used for biochemical assays. p-value<0.05 was considered statistically significant. Three independent experiments (biological replicates) were performed. Results were presented as Mean ± standard error of mean (SEM). KEY FINDINGS: This investigation revealed direct and indirect interplay between nitrosylation and carbonylation especially within coagulation and inflammation networks; interlinked redox signaling (via nitrosylation­carbonylation); and novel nitrosylation and carbonylation sites in individual proteins. SIGNIFICANCE: This study elucidates the role of redox PTMs in hypoxia signaling favoring tolerance and survival. Also, we demonstrated direct and indirect interplay between nitrosylation and carbonylation is crucial to extended hypoxia tolerance.

Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of mitochondrial translation elongation.

The mammalian mitochondrial ribosome (mitoribosome) and its associated translational factors have evolved to accommodate greater participation of proteins in mitochondrial translation. Here we present the 2.68-3.96 Å cryo-EM structures of the human 55S mitoribosome in complex with the human mitochondrial elongation factor G1 (EF-G1mt) in three distinct conformational states, including an intermediate state and a post-translocational state. These structures reveal the role of several mitochondria-specific (mito-specific) mitoribosomal proteins (MRPs) and a mito-specific segment of EF-G1mt in mitochondrial tRNA (tRNAmt) translocation. In particular, the mito-specific C-terminal extension in EF-G1mt is directly involved in translocation of the acceptor arm of the A-site tRNAmt. In addition to the ratchet-like and independent head-swiveling motions exhibited by the small mitoribosomal subunit, we discover significant conformational changes in MRP mL45 at the nascent polypeptide-exit site within the large mitoribosomal subunit that could be critical for tethering of the elongating mitoribosome onto the inner-mitochondrial membrane.

Salivary proteome patterns of individuals exposed to High Altitude.

OBJECTIVE: Identification of molecular signatures having key roles in hypobaric hypoxia by analysing the salivary proteome. Saliva holds a promising future in the search for new clinical biomarkers that are easily accessible, less complex, accurate, and cost effective as well as being non-invasive. METHODOLOGY: We employed qualitative proteomics approach to develop discriminatory biomarker signatures from human saliva exposed to hypobaric hypoxia. Salivary proteins were analyzed and compared between age-matched healthy subjects exposed to high altitude (∼13700 ft) for seven days (HAD7) with control subjects at sea level (Normoxia) by using 2-Dimensional gel electrophoresis/Mass Spectrometry approach. RESULTS: Several proteins with significant differential expression were found. The up-regulated proteins were apoptosis inducing factor-2, cystatin S, cystatin SN and carbonic anhydrase 6. The down regulated proteins were polymeric immunoglobulin receptor, alpha-enolase and prolactin-inducible protein. Further confirmation of the altered proteins such as alpha enolase, carbonic anhydrase 6, prolactin-inducible protein, apoptosis inducing factor 2, cystatin S and cystatin SN were performed using immunoblotting. The expression patterns of the selected proteins observed by immunoblot were in concurrence with 2-Dimesional gel electrophoresis results, therefore affirming the authenticity of the proteomic investigation. CONCLUSION: This study provides the proof of concept of salivary biomarkers for the non-invasive detection of hypobaric hypoxia induced effects. It is highly feasible to turn these biomarkers into an applicable clinical test after large scale validation.

Post-translational modifications of eNOS augment nitric oxide availability and facilitates hypoxia adaptation in Ladakhi women.

The lower inhaled oxygen per volume at high altitude poses an intimidating challenge for humans to survive and reproduce. Indigenous populations of the Himalayas reportedly exhibit higher microcirculatory blood flow accompanied by higher orders of magnitude of nitric oxide (NO) products in lung, plasma and red blood cells as a vascular adaptation strategy for hypobaric hypoxia. The precise mechanism of such observed higher NO metabolites for hypoxia adaptation remains elusive. Studying high altitude native Ladakhi women, we observed significant higher eNOS mRNA and protein in blood/plasma as compared to lowland women. We also observed higher level of plasma l-citrulline and NOx (nitrates and nitrites) with concomitant lower levels of arginase mRNA and protein further suggesting higher eNOS activity and NO bioavailability. Interestingly, middle aged postmenopausal Ladakhi women exhibited significantly higher level of eNOS activity, NOx and cGMP as compared to age matched lowland women. Preferential phosphorylation of eNOS on stimulatory Ser1177 and Ser615 as well as dephosphorylation of inhibitory Thr495 site contributed to higher NO availability in Ladakhi women irrespective of age. We also observed higher levels of eNOS activating humoral factors like bradykinin and estrogen in both young and middle-aged Ladakhi women. These results suggest that an altered phosphorylation status, together with an enhanced expression of eNOS and potential humoral endothelial activators, are involved in enhanced activation of the eNOS-NO-cGMP pathway in Ladakhi women irrespective of age, reinforcing the hypothesis that NO metabolites play a major role in Himalayan pattern of hypoxia adaptation.

Competing trends of ROS and RNS-mediated protein modifications during hypoxia as an alternate mechanism of NO benefits.

Hypoxia, especially altitude associated hypoxia is known to cause severe physiological alterations and life-threatening conditions. Impaired redox balance along with oxidative stress, protein carbonylation and instigation of apoptotic events are common sub-cellular events that follow the hypoxic insult. The role of nitric oxide (NO) is very dynamic and versatile in preventing the ill effects of hypoxia vis-a-vis reacting with oxidative species and causing protein nitrosylation. Although several mechanisms of NO-mediated cytoprotection are known during hypoxic insult, limited pieces of evidence are available to support the relationship between two downstream events of oxidative stress, protein carbonylation (caused by carbonyl; CO radical) and protein nitrosylation/nitration (caused by NO/peroxynitrite; ONOO radical). In this study, we investigated an entirely new aspect of NO protection in hypoxia involving crosstalk between carbonylation and nitrosylation. Using standard NO inhibitor l-NAME and simulated hypoxic conditions in hypoxia-sensitive cell line H9c2, we evaluated the levels of radicals, cell death, mitochondrial membrane potential, levels of protein nitrosylation, protein nitration and carbonylation and glutathione content. The results were then carefully analyzed in light of NO bioavailability. Our study shows that reducing NO during hypoxia caused cell death via the increased degree of carbonylation in proteins. This provides a new aspect of NO benefits which furthers opens new possibilities to explore potential mechanisms and effects of cross-talk between nitrosylation, protein nitration and carbonylation, especially through some common antioxidant mediators such as glutathione and thioredoxin.

STAT3-RXR-Nrf2 activates systemic redox and energy homeostasis upon steep decline in pO2 gradient.

Hypobaric hypoxia elicits several patho-physiological manifestations, some of which are known to be lethal. Among various molecular mechanisms proposed so far, perturbation in redox state due to imbalance between radical generation and antioxidant defence is promising. These molecular events are also related to hypoxic status of cancer cells and therefore its understanding has extended clinical advantage beyond high altitude hypoxia. In present study, however, the focus was to understand and propose a model for rapid acclimatization of high altitude visitors to enhance their performance based on molecular changes. We considered using simulated hypobaric hypoxia at some established thresholds of high altitude stratification based on known physiological effects. Previous studies have focused on the temporal aspect while overlooking the effects of varying pO2 levels during exposure to hypobaric hypoxia. The pO2 levels, indicative of altitude, are crucial to redox homeostasis and can be the limiting factor during acclimatization to hypobaric hypoxia. In this study we present the effects of acute (24h) exposure to high (3049m; pO2: 71kPa), very high (4573m; pO2: 59kPa) and extreme altitude (7620m; pO2: 40kPa) zones on lung and plasma using semi-quantitative redox specific transcripts and quantitative proteo-bioinformatics workflow in conjunction with redox stress assays. It was observed that direct exposure to extreme altitude caused 100% mortality, which turned into high survival rate after pre-exposure to 59kPa, for which molecular explanation were also found. The pO2 of 59kPa (very high altitude zone) elicits systemic energy and redox homeostatic processes by modulating the STAT3-RXR-Nrf2 trio. Finally we posit the various processes downstream of STAT3-RXR-Nrf2 and the plasma proteins that can be used to ascertain the redox status of an individual.

ARM-microcontroller based portable nitrite electrochemical analyzer using cytochrome c reductase biofunctionalized onto screen printed carbon electrode.

Nitrite (NO2-) supplementation limits hypoxia-induced oxidative stress and activates the alternate NO pathway which may partially account for the nitrite-mediated cardioprotection. So, sensitive and selective biosensors with point-of-care devices need to be explored to detect the physiological nitrite level due to its important role in human pathophysiology. In this work, cytochrome c reductase (CcR) biofunctionalized self assembled monolayer (SAM) functionalized on gold nanoparticles (GNPs) in polypyrrole (PPy) nanocomposite onto the screen printed carbon electrode (SPCE) was investigated as a biosensor for the detection of nitrite based on its electrochemical and catalytic properties. CcR was covalently coupled with SAM layers on GNPs by using EDC and NHS. Direct electrochemical response of CcR biofunctionalized electrodes showed a couple of well-defined and nearly reversible cyclic voltammetric peaks at -0.34 and -0.45 vs. Ag/AgCl. Under optimal conditions, the biosensor could be used for the determination of NO2- with a linear range from 0.1-1600µm and a detection limit of 60nM with a sensitivity of 0.172µAµM-1cm-2. Further, we have designed and developed a novel and cost effective portable electrochemical analyzer for the measurement of NO2- in hypoxia induced H9c2 cardiac cells using ARM microcontroller. The results obtained here using the developed portable electrochemical nitrite analyzer were also compared with the standard cyclic voltammetry instrument and found in agreement with each other.

Cerium oxide nanoparticles promote neurogenesis and abrogate hypoxia-induced memory impairment through AMPK-PKC-CBP signaling cascade.

Structural and functional integrity of the brain is adversely affected by reduced oxygen saturation, especially during chronic hypoxia exposure and often encountered by altitude travelers or dwellers. Hypoxia-induced generation of reactive nitrogen and oxygen species reportedly affects the cortex and hippocampus regions of the brain, promoting memory impairment and cognitive dysfunction. Cerium oxide nanoparticles (CNPs), also known as nanoceria, switch between +3 and +4 oxidation states and reportedly scavenge superoxide anions, hydrogen peroxide, and peroxynitrite in vivo. In the present study, we evaluated the neuroprotective as well as the cognition-enhancing activities of nanoceria during hypobaric hypoxia. Using polyethylene glycol-coated 3 nm nanoceria (PEG-CNPs), we have demonstrated efficient localization of PEG-CNPs in rodent brain. This resulted in significant reduction of oxidative stress and associated damage during hypoxia exposure. Morris water maze-based memory function tests revealed that PEG-CNPs ameliorated hypoxia-induced memory impairment. Using microscopic, flow cytometric, and histological studies, we also provide evidences that PEG-CNPs augmented hippocampus neuronal survival and promoted neurogenesis. Molecular studies revealed that PEG-CNPs promoted neurogenesis through the 5'-adenine monophosphate-activated protein kinase-protein kinase C-cyclic adenosine monophosphate response element-binding protein binding (AMPK-PKC-CBP) protein pathway. Our present study results suggest that nanoceria can be translated as promising therapeutic molecules for neurodegenerative diseases.

Nanoceria based electrochemical sensor for hydrogen peroxide detection.

Oxidative stress is a condition when the concentration of free radicals and reactive molecular species rise above certain level in living systems. This condition not only perturbs the normal physiology of the system but also has been implicated in many diseases in humans and other animals. Hydrogen peroxide (H2O2) is known to be involved in induction of oxidative stress and has also been linked to a variety of ailments such as inflammation, rheumatoid arthritis, diabetes, and cancer in humans. It is one of the more stable reactive molecular species present in living systems. Because of its stability and links with various diseases, sensing the level of H2O2 can be of great help in diagnosing these diseases, thereby easing disease management and amelioration. Nanoceria is a potent candidate in free radical scavenging as well as sensing because of its unique redox properties. These properties have been exploited, in the reported work, to sense and quantify peroxide levels. Nanoceria has been synthesized using different capping agents: Hexamethylene-tetra-amine (HMTA) and fructose. CeO2-HMTA show rhombohedral and cubic 6.4 nm particles whereas CeO2-fructose are found to be spherical with average particle diameter size 5.8 nm. CeO2-HMTA, due to the better exposure of the active (200) and (220) planes relative to (111) plane, exhibits superior electrocatalytic activity toward H2O2 reduction. Amperometric responses were measured by increasing H2O2 concentration. The authors observed a sensitivity of 21.13 and 9.6 µA cm(-2) mM(-1) for CeO2-HMTA and CeO2-fructose, respectively. The response time of 4.8 and 6.5 s was observed for CeO2-HMTA and CeO2-fructose, respectively. The limit of detection is as low as 0.6 and 2.0 µM at S/N ratio 3 for CeO2-HMTA and CeO2-fructose, respectively. Ceria-HMTA was further tested for its antioxidant activity in an animal cell line in vitro and the results confirmed its activity.

Designing label-free electrochemical immunosensors for cytochrome c using nanocomposites functionalized screen printed electrodes.

We have designed here a label-free direct electrochemical immunosensor for the detection of cytochrome c (cyt c), a heme containing metalloprotein using its specific monoclonal antibody. Two nanocomposite-based electrochemical immunosensor platforms were evaluated for the detection of cyt c; (i) self-assembled monolayer (SAM) on gold nanoparticles (GNP) in polypyrrole (PPy) grafted screen printed electrodes (SPE) and (ii) carbon nanotubes (CNT) integrated PPy/SPE. The nanotopologies of the modified electrodes were confirmed by scanning electron microscopy. Electrochemical impedance spectroscopy and cyclic voltammetry were employed to monitor the stepwise fabrication of the nanocomposite immunosensor platforms. In the present method, the label-free quantification of cyt c is based on the direct electron transfer between Fe (III)/Fe (II)-heme redox active site of cyt c selectively bound to anti-cyt c nanocomposite modified SPE. GNP/PPy and CNT/PPy nanocomposites promoted the electron transportation through the conductive pore channels. The overall analytical performance of GNP/PPy based immunosensor (detection limit 2 nM; linear range: 2 nM to 150 µM) was better than the anti-cyt c/CNT/PPy (detection limit 10 nM; linear range: 10 nM to 50 µM). Further, the measurement of cyt c release in cell lysates of cardiomyocytes using the GNP/PPy based immunosensor gave an excellent correlation with standard ELISA.

Copper, zinc superoxide dismutase and nitrate reductase coimmobilized bienzymatic biosensor for the simultaneous determination of nitrite and nitrate.

This work presents a novel bienzymatic biosensor for the simultaneous determination of nitrite (NO2(-)) and nitrate (NO3(-)) ions using copper, zinc superoxide dismutase (SOD1) and nitrate reductase (NaR) coimmobilized on carbon nanotubes (CNT)-polypyrrole (PPy) nanocomposite modified platinum electrode. Morphological changes of the PPy and CNT modified electrodes were investigated using scanning electron microscopy. The electrochemical behavior of the bienzymatic electrode (NaR-SOD1-CNT-PPy-Pt) was characterized by cyclic voltammetry exhibiting quasi-reversible redox peak at +0.06 V and reversible redox peaks at -0.76 and -0.62V vs. Ag/AgCl, for the immobilized SOD1 and NaR respectively. The electrocatalytic activity of SOD1 towards NO2(-) oxidation observed at +0.8 V was linear from 100 nM to 1mM with a detection limit of 50 nM and sensitivity of 98.5 ± 1.7 nA µM(-1)cm(-2). Similarly, the coimmobilized NaR showed its electrocatalytic activity towards NO3(-) reduction at -0.76 V exhibiting linear response from 500 nM to 10mM NO3(-) with a detection limit of 200 nM and sensitivity of 84.5 ± 1.56 nA µM(-1)cm(-2). Further, the present bienzymatic biosensor coated with cellulose acetate membrane for the removal of non-specific proteins was used for the sensitive and selective determinations of NO2(-) and NO3(-) present in human plasma, whole blood and saliva samples.

Nanomaterial-based electrochemical biosensors for cytochrome c using cytochrome c reductase.

Emerging evidences have pointed out that the release of cytochrome c (cyt c) from mitochondria into cytosol is a critical step in the activation of apoptosis. This article presents a novel approach for the detection of mitochondrial cyt c release for the first time using cytochrome c reductase (CcR) immobilized on nanoparticles decorated electrodes. Two kinds of nanomaterial-based biosensor platforms were used: (a) carbon nanotubes (CNT) incorporated polypyrrole (PPy) matrix on Pt electrode and (b) self-assembled monolayer (SAM) functionalized gold nanoparticles (GNP) in PPy-Pt. Scanning electron microscope was used to characterize the surface morphologies of the nanomaterial modified electrodes. Cyclic voltammograms of both the biosensors showed reversible redox peaks at -0.45 and -0.34 V vs Ag/AgCl, characteristic of CcR. In comparison, the CcR-CNT biosensor gave a detection limit of 0.5±0.03 µM cyt c, which was 4-fold better than the CcR-GNP biosensor (2±0.03 µM). Moreover, the CcR-CNT biosensor achieved a much larger linear range (1-1000 µM) over the CcR-GNP biosensor (5-600 µM) with 2-fold better sensitivity. The CcR-CNT-PPy-Pt biosensor was further applied to quantify the mitochondrial cyt c released in cytosol of A549 cells upon induction of apoptosis with doxorubicin, the results agreed well with standard western blot analysis.

Dietary nitrite attenuates oxidative stress and activates antioxidant genes in rat heart during hypobaric hypoxia.

The nitrite anion represents the circulatory and tissue storage form of nitric oxide (NO) and a signaling molecule, capable of conferring cardioprotection and many other health benefits. However, molecular mechanisms for observed cardioprotective properties of nitrite remain largely unknown. We have evaluated the NO-like bioactivity and cardioprotective efficacies of sodium nitrite supplemented in drinking water in rats exposed to short-term chronic hypobaric hypoxia. We observed that, nitrite significantly attenuates hypoxia-induced oxidative stress, modulates HIF-1α stability and promotes NO-cGMP signaling in hypoxic heart. To elucidate potential downstream targets of nitrite during hypoxia, we performed a microarray analysis of nitrite supplemented hypoxic hearts and compared with both hypoxic and nitrite supplemented normoxic hearts respectively. The analysis revealed a significant increase in the expression of many antioxidant genes, transcription factors and cardioprotective signaling pathways which was subsequently confirmed by qRT-PCR and Western blotting. Conversely, hypoxia exposure increased oxidative stress, activated inflammatory cytokines, downregulated ion channels and altered expression of both pro- and anti-oxidant genes. Our results illustrate the physiological function of nitrite as an eNOS-independent source of NO in heart profoundly modulating the oxidative status and cardiac transcriptome during hypoxia.

Copper nanoparticles entrapped in SWCNT-PPy nanocomposite on Pt electrode as NOx electrochemical sensor.

A highly sensitive NO(x) sensor was designed and developed by electrochemical incorporation of copper nanoparticles (CuNP) on single-walled carbon nanotubes (SWCNT)-polypyrrole (PPy) nanocomposite modified Pt electrode. The modified electrodes were characterized by scanning electron microscopy and energy dispersive X-ray analysis. Further, the electrochemical behavior of the CuNP-SWCNT-PPy-Pt electrode was investigated by cyclic voltammetry. It exhibited the characteristic CuNP reversible redox peaks at -0.15 V and -0.3 V vs. Ag/AgCl respectively. The electrocatalytic activity of the CuNP-SWCNT-PPy-Pt electrode towards NO(x) is four-fold than the CuNP-PPy-Pt electrode. These results clearly revealed that the SWCNT-PPy nanocomposite facilitated the electron transfer from CuNP to Pt electrode and provided an electrochemical approach for the determination of NO(x). A linear dependence (r(2)=0.9946) on the NO(x) concentrations ranging from 0.7 to 2000 µM, with a sensitivity of 0.22 ± 0.002 µA µM(-1)cm(-2) and detection limit of 0.7 µM was observed for the CuNP-SWCNT-PPy-Pt electrode. In addition, the sensor exhibited good reproducibility and retained stability over a period of one month.

Cordyceps sinensis promotes exercise endurance capacity of rats by activating skeletal muscle metabolic regulators.

ETHNOPHARMACOLOGICAL RELEVANCE: Cordyceps sinensis is a traditional Chinese medicine used for promotion of health, longevity and athletic power. However, the molecular mechanism for anti-fatigue activity and physical fitness has not yet been reported. AIM OF THE STUDY: The present study was conducted to evaluate the exercise endurance promoting activities of fungal traditional Chinese medicine (FTCM) Cordyceps sinensis cultured whole mycelium (CS) and the underlying mechanisms. MATERIALS AND METHODS: CS was orally supplemented (200mg/kg body weight/day) to rats for 15days with or without swimming exercise along with exercise and placebo groups. RESULTS: Both CS supplementation and supplementation concurrent with exercise improved exercise endurance by 1.79- (P<0.05) and 2.9-fold (P<0.01) respectively as compared to placebo rats. CS supplementation concurrent with exercise also increased the swimming endurance by 1.32-fold (P<0.05) over the exercise group. To study the molecular mechanism of the observed effect, we measured the expression levels of endurance responsive skeletal muscle metabolic regulators AMPK, PGC-1α and PPAR-δ as well as endurance promoting and antioxidant genes like MCT1, MCT4, GLUT4, VEGF, NRF-2, SOD1 and TRX in red gastrocnemius muscle. Our results indicate that CS supplementation significantly upregulates the skeletal muscle metabolic regulators, angiogenesis, better glucose and lactate uptake both in exercised and non-exercised rats. We have also observed increased expression of oxidative stress responsive transcription factor NRF-2 and its downstream targets SOD1 and TRX by CS supplementation. CONCLUSION: CS supplementation with or without exercise improves exercise endurance capacity by activating the skeletal muscle metabolic regulators and a coordinated antioxidant response. Consequently, CS can be used as a potent natural exercise mimetic.

Activity-dependent neuroprotective protein (ADNP)-derived peptide (NAP) ameliorates hypobaric hypoxia induced oxidative stress in rat brain.

Hypobaric hypoxia is a socio-economic problem affecting cognitive, memory and behavior functions. Severe oxidative stress caused by hypobaric hypoxia adversely affects brain areas like cortex, hippocampus, basal ganglia, and cerebellum. In the present study, we have investigated the antioxidant and memory protection efficacy of the synthetic NAP peptide (NAPVSIPQ) during long-term chronic hypobaric hypoxia (7, 14, 21 and 28 days, 25,000ft) in rats. Intranasal supplementation of NAP peptide (2µg/Kg body weight) improved antioxidant status of brain evaluated by biochemical assays for free radical estimation, lipid peroxidation, GSH and GSSG level. Analysis of expression levels of SOD revealed that NAP significantly activated antioxidant genes as compared to hypoxia exposed rats. We have also observed a significant increased expression of Nrf2, the master regulator of antioxidant defense system and its downstream targets such as HO-1, GST and SOD1 by NAP supplementation, suggesting activation of Nrf2-mediated antioxidant defense response. In corroboration, our results also demonstrate that NAP supplementation improved the memory function assessed with radial arm maze. These cumulative results suggest the therapeutic potential of NAP peptide for ameliorating hypobaric hypoxia-induced oxidative stress.

Simultaneous electrochemical determination of superoxide anion radical and nitrite using Cu,ZnSOD immobilized on carbon nanotube in polypyrrole matrix.

A novel highly sensitive biosensor for the direct and simultaneous determination of superoxide anion radical (O2-) and nitrite (NO2-) was developed by incorporation of carbon nanotube (CNT) solubilized in nafion in polypyrrole (PPy) matrix on Pt electrode followed by immobilization of Cu,ZnSOD (SOD1) on it. The CNT/PPy nanocomposite electrode enhanced the immobilization of SOD1 and promoted the electron transfer of SOD1 minimizing its fouling effect. The surface morphological images of PPy and CNT-PPy nanocomposite on Pt electrode were obtained by scanning electron microscopy exhibiting highly microporous structures. The electrochemical behavior of the biosensor investigated by cyclic voltammetry revealed that the SOD1 immobilized electrode showed characteristic of SOD1 quasi-reversible redox peaks with a formal potential of +0.065 V vs. Ag/AgCl. The biosensor exhibited a linear response over the concentration range from 0.1 to 750 µM, with a detection limit of 0.1±0.03 µM for O2- and a corresponding linear range of 0.5-2000 µM, with a detection limit of 0.5±0.025 µM for NO2-. In addition, the biosensor exhibited high sensitivity, good reproducibility and retained stability over 30 days. This modified electrode was quite effective not only in detecting O2- and NO2- independently but also determining the concentration of O2- and NO2- simultaneously in vitro and from cancer cells.

Proteomics and electron microscopic characterization of the unusual mitochondrial ribosome-related 45S complex in Leishmania tarentolae.

A novel type of ribonucleoprotein (RNP) complex has been described from the kinetoplast-mitochondria of Leishmania tarentolae. The complex, termed the 45S SSU*, contains the 9S small subunit rRNA but does not contain the 12S large subunit rRNA. This complex is the most stable and abundant mitochondrial RNP complex present in Leishmania. As shown by tandem mass spectrometry, the complex contains at least 39 polypeptides with a combined molecular mass of almost 2.1 MDa. These components include several homologs of small subunit ribosomal proteins (S5, S6, S8, S9, S11, S15, S16, S17, S18, MRPS29); however, most of the polypeptides present are unique. Only a few of them show recognizable motifs, such as protein-protein (coiled-coil, Rhodanese) or protein-RNA (pentatricopeptide repeat) interaction domains. A cryo-electron microscopy examination of the 45S SSU* fraction reveals that 27% of particles represent SSU homodimers arranged in a head-to-tail orientation, while the majority of particles are clearly different and show an asymmetric bilobed morphology. Multiple classes of two-dimensional averages were derived for the asymmetrical particles, probably reflecting random orientations of the particles and difficulties in correlating these views with the known projections of ribosomal complexes. One class of the two-dimensional averages shows a SSU moiety attached to a protein mass or masses in a monosome-like appearance. The combined mass spectrometry and electron microscopy data thus indicate that the majority 45S SSU* particles represents a heterodimeric complex in which the SSU of the Leishmania mitochondrial ribosome is associated with an additional protein mass. The biological role of these particles is not known.

Membrane binding, structure, and localization of cecropin-mellitin hybrid peptides: a site-directed spin-labeling study.

The interaction of antimicrobial peptides with membranes is a key factor in determining their biological activity. In this study we have synthesized a series of minimized cecropin-mellitin hybrid peptides each containing a single cysteine residue, modified the cysteine with the sulfhydryl-specific methanethiosulfonate spin-label, and used electron paramagnetic resonance spectroscopy to measure membrane-binding affinities and determine the orientation and localization of peptides bound to membranes that mimic the bacterial cytoplasmic membrane. All of the peptides were unstructured in aqueous solution but underwent a significant conformational change upon membrane binding that diminished the rotational mobility of the attached spin-label. Apparent partition coefficients were similar for five of the six constructs examined, indicating that location of the spin-label had little effect on peptide binding as long as the attachment site was in the relatively hydrophobic C-terminal domain. Depth measurements based on accessibility of the spin-labeled sites to oxygen and nickel ethylenediaminediacetate indicated that at high lipid/peptide ratios these peptides form a single alpha-helix, with the helical axis aligned parallel to the bilayer surface and immersed approximately 5 A below the membrane-aqueous interface. Such a localization would provide exposure of charged/polar residues on the hydrophilic face of the amphipathic helix to the aqueous phase, and allow the nonpolar residues along the opposite face of the helix to remain immersed in the hydrophobic phase of the bilayer. These results are discussed with respect to the mechanism of membrane disruption by antimicrobial peptides.

Transmembrane nitration of hydrophobic tyrosyl peptides. Localization, characterization, mechanism of nitration, and biological implications.

We have shown previously that peroxynitrite-induced nitration of a hydrophobic tyrosyl probe is greater than that of tyrosine in the aqueous phase (Zhang, H., Joseph, J., Feix, J., Hogg, N., and Kalyanaraman, B. (2001) Biochemistry 40, 7675-7686). In this study, we have tested the hypothesis that the extent of tyrosine nitration depends on the intramembrane location of tyrosyl probes and on the nitrating species. To this end, we have synthesized membrane spanning 23-mer containing a single tyrosyl residue at positions 4, 8, and 12. The location of the tyrosine residues in the phospholipid membrane was determined by fluorescence and electron spin resonance techniques. Nitration was initiated by slow infusion of peroxynitrite, co-generated superoxide and nitric oxide ((.)NO), or a myeloperoxidase/hydrogen peroxide/nitrite anion (MPO/H(2)O(2)/NO(2)(-)) system. Results indicate that with slow infusion of peroxynitrite, nitration of transmembrane tyrosyl peptides was much higher (10-fold or more) than tyrosine nitration in aqueous phase. Peroxynitrite-dependent nitration of tyrosyl-containing peptides increased with increasing depth of the tyrosyl residue in the bilayer. In contrast, MPO/H(2)O(2)/ NO(2)(-)-induced tyrosyl nitration decreased with increasing depth of tyrosyl residues in the membrane. Transmembrane nitrations of tyrosyl-containing peptides induced by both peroxynitrite and MPO/H(2)O(2)/NO(2)(-) were totally inhibited by (.)NO that was slowly released from spermine NONOate. Nitration of peptides in both systems was concentration-dependently inhibited by unsaturated fatty acid. Concomitantly, an increase in lipid oxidation was detected. A mechanism involving (.)NO(2) radical is proposed for peroxynitrite and MPO/H(2)O(2)/NO(2)(-)-dependent transmembrane nitration reactions.