iTRAQ‐coupled 2‐D LC‐MS/MS analysis of protein profile associated with HBV‐modulated DNA methylation

The development of hepatocellular carcinoma (HCC) is believed to be associated with multiple risk factors, including the infection of hepatitis B virus (HBV). Based on the analysis of individual genes, evidence has indicated the association between HCC and HBV and has also been expanded to epigenetic regulation, with an involvement of HBV in the DNA methylation of the promoter of cellular target genes leading to changes in their expression. Proteomic study has been widely used to map a comprehensive protein profile, which in turn could provide a better understanding of underlying mechanisms of disease onset. In the present study, we performed a proteomic profiling by using iTRAQ‐coupled 2‐D LC/MS‐MS analysis to identify cellular genes down‐regulated in HBV‐producing HepG2.2.15 cells compared with HepG2 cells. A total of 15 proteins including S100A6 and Annexin A2 were identified by our approach. The significance of these cellular proteins as target of HBV‐mediated epigenetic regulation was supported by our validation assays, including their reactivation in cells treated with 5‐aza‐2′‐deoxycytidine (a DNA methyltransferase inhibitor) by real‐time RT‐PCR and Western blot analysis, as well as the DNA methylation status analysis by bisulfite genome sequencing. Our approach provides a comprehensive analysis of cellular target proteins to HBV‐mediated epigenetic regulation and further analysis should facilitate a better understanding of its involvement in HCC development.     

Copper stress‐induced changes in leaf soluble proteome of Cu‐sensitive and tolerant Agrostis capillaris L. populations

Changes in leaf soluble proteome were explored in 3‐month‐old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1–50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO₄ spiked‐nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2‐DE (linear 4–7 pH gradient). Analysis of CCB‐stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC‐MS/MS. In both populations, Cu excess impacted both light‐dependent (OEE, cytochrome b6‐f complex, and chlorophyll a‐b binding protein), and ‐independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin‐NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S‐containing amino‐acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 ( http//proteomecentral.proteomexchange.org/dataset/PXD001930 ).     

Identification of lysosomal Npc1‐binding proteins: Cathepsin D activity is regulated by NPC1

Niemann–Pick type C (NPC) disease is an inherited lysosomal storage disorder, characterized by severe neurodegeneration. It is mostly produced by mutations in the NPC1 gene, encoding for a protein of the late endosomes/lysosomes membrane, involved in cholesterol metabolism. However, the specific role of this protein in NPC disease still remains unknown. We aimed to identify Npc1‐binding proteins in order to define new putative NPC1 lysosomal functions. By affinity chromatography using an Npc1 peptide (amino acids 1032–1066 of loop I), as bait, we fished 31 lysosomal proteins subsequently identified by LC‐MS/MS. Most of them were involved in proteolysis and lipid catabolism and included the protease cathepsin D. Cathepsin D and NPC1 interaction was validated by immunoprecipitation and the functional relevance of this interaction was studied. We found that fibroblasts from NPC patients with low levels of NPC1 protein have high amounts of procathepsin D but reduced quantities of the mature protein, thus showing a diminished cathepsin D activity. The increase of NPC1 protein levels in NPC cells by treatment with the proteasome inhibitor bortezomib, induced an elevation of cathepsin D activity. All these results suggest a new lysosomal function of NPC1 as a regulator of cathepsin D processing and activity.     

Accumulation of 24 nucleotide transgene‐derived siRNAs is associated with crinivirus immunity in transgenic plants

RNA silencing is a conserved antiviral defence mechanism that has been used to develop robust resistance against plant virus infections. Previous efforts have been made to develop RNA silencing‐mediated resistance to criniviruses, yet none have given immunity. In this study, transgenic Nicotiana benthamiana plants harbouring a hairpin construct of the Lettuce infectious yellows virus (LIYV) RNA‐dependent RNA polymerase (RdRp) sequence exhibited immunity to systemic LIYV infection. Deep sequencing analysis was performed to characterize virus‐derived small interfering RNAs (vsiRNAs) generated on systemic LIYV infection in non‐transgenic N. benthamiana plants as well as transgene‐derived siRNAs (t‐siRNAs) derived from the immune‐transgenic plants before and after LIYV inoculation. Interestingly, a similar sequence distribution pattern was obtained with t‐siRNAs and vsiRNAs mapped to the transgene region in both immune and susceptible plants, except for a significant increase in t‐siRNAs of 24 nucleotides in length, which was consistent with small RNA northern blot results that showed the abundance of t‐siRNAs of 21, 22 and 24 nucleotides in length. The accumulated 24‐nucleotide sequences have not yet been reported in transgenic plants partially resistant to criniviruses, and thus may indicate their correlation with crinivirus immunity. To further test this hypothesis, we developed transgenic melon (Cucumis melo) plants immune to systemic infection of another crinivirus, Cucurbit yellow stunting disorder virus (CYSDV). As predicted, the accumulation of 24‐nucleotide t‐siRNAs was detected in transgenic melon plants by northern blot. Together with our findings and previous studies on crinivirus resistance, we propose that the accumulation of 24‐nucleotide t‐siRNAs is associated with crinivirus immunity in transgenic plants.     

A functional variant in the 5′‐flanking region of the chicken serotonin transporter gene is associated with increased body weight and locomotor activity

In this study, we identified a polymorphism in the 5′‐flanking region of the chicken serotonin transporter (5‐HTT) gene. Sequencing analysis revealed that in comparison with the wild‐type variant (W), a deleted variant (D) is generated by deletion of four nucleotides (5′‐AATT‐3′) and a single nucleotide change (A→T). Using a polyacrylamide gel electrophoresis system, we found that the 360‐bp DNA fragment containing the W variant with the wild‐type sequence 5′‐AATTAATT‐3′ shows intrinsic DNA curvature while the 356‐bp fragment containing the D variant lacking the four base pairs AATT is not curved. Quantitative real‐time RT‐PCR and ELISA demonstrated that the expression of 5‐HTT in D/D chickens was higher than that in W/W and W/D chickens. In addition, transient transfection experiments with chloramphenicol acetyltransferase reporter gene constructs revealed increased 5‐HTT promoter activity mediated by the D variant and a silencer activity of the W variant. Interestingly, females and males with D/D genotype showed significant greater increase in body weight from 6 weeks and 16 weeks of age, respectively, and higher body mass index. Moreover, we found that D/D chickens of both genders were physically more active than W/W and W/D chickens.

     

Stereoselective Determination of Metoprolol and its Metabolite α‐Hydroxymetoprolol in Plasma by LC‐MS/MS: Application to Pharmacokinetics during Pregnancy

Metoprolol is available for clinical use as a racemic mixture. The S‐(?)‐metoprolol enantiomer is the one expressing higher activity in the blockade of the β₁‐adrenergic receptor. The α‐hydroxymetoprolol metabolite also has activity in the blockade of the β₁‐adrenergic receptor. The present study describes the development and validation of a stereoselective method for sequential analysis of metoprolol and of α‐hydroxymetoprolol in plasma using high‐performance liquid chromatography with tandem mass spectrometry (LC‐MS/MS). 1‐ml aliquots of plasma were extracted with dichloromethane : diisopropyl ether (1:1, v/v). Metoprolol enantiomers and α‐hydroxymetoprolol isomers were separated on a Chiralpak AD column (Daicel Chemical Industries, New York, NY, USA) and quantitated by LC‐MS/MS. The limit of quantitation obtained was 0.2 ng of each metoprolol enantiomer/ml plasma and 0.1 ng/ml of each α‐hydroxymetoprolol isomer/ml plasma. The method was applied to the study of kinetic disposition of metoprolol in plasma samples collected up to 24 h after the administration of a single oral dose of 100‐mg metoprolol tartrate to a hypertensive parturient with a gestational age of 42 weeks. The clinical study showed that the metoprolol pharmakokinetics is enantioselective, with the observation of higher area under the curve (AUC)^0?∞ values for S‐(?)‐metoprolol (AUC_S‐(?)/AUC_R‐(+) = 1.81) and the favoring of the formation of the new chiral center 1′R of α‐hydroxymetoprolol (AUC^0?∞_1′R/1′S = 2.78). Chirality, 25:1–7, 2013. © 2012 Wiley Periodicals, Inc.     

In vitro oxidative footprinting provides insight into apolipoprotein B‐100 structure in low‐density lipoprotein

Low‐density lipoprotein (LDL) is a major cholesterol carrier in human blood. Oxidations of apolipoprotein B‐100 (apo B‐100, LDL protein) could be proatherogenic and play critical roles in early stages of plaque formation in the arterial wall. The structure of apo B‐100 is still poorly understood, partially due to its size (550 KDa, 4563 amino acids). To gain an insight into LDL structure, we mapped the regions of apo B‐100 in human LDL that were prone to oxidation using peroxynitrite and hypochlorite as probes. In this study, LDL was incubated with various concentrations of peroxynitrite and sodium hypochlorite in bicarbonate buffer. The LDL protein apo B‐100 was delipidated, denatured, alkylated, and subjected to tryptic digestion. Tryptic peptides were analyzed employing LC‐MS/MS. Database search was performed against the apo B‐100 database (SwissProt accession #P04114) using “SEQUEST” algorithm to identify peroxynitrite and hypochlorite‐mediated oxidations markers nitrotyrosine, nitrotryptophan, hydroxy‐tryptophan, and 3‐chlorotyrosine. Several site‐specific oxidations were identified in apo B‐100 after treatment of intact LDL particles with the oxidants. We hypothesize that these regions could be accessible to oxidant and critical for early events in atherosclerotic plaque deposition.     

Bower‐building behaviour is associated with increased sperm longevity in Tanganyikan cichlids

We investigated the evolutionary relationship between spawning behaviour and sperm motility traits among Tanganyikan mouth‐brooding cichlid species that have developed diverse mating behaviours and male sexual traits. Mouth‐brooding behaviour is common among these fish, but different species demonstrate a range of spawning behaviours, bower construction, male sexual traits and timing of gamete release. We observed spawning behaviours and compared sperm motility traits of 28 Tanganyikan mouth‐brooding cichlids to elucidate the evolutionary correlations between these traits. Sperm longevity was considerably longer in bower‐building species that construct crater‐shaped spawning sites compared with species that do not build bowers. Male bower builders released sperm in the pit of the bower prior to spawning, and the time from ejaculation to fertilization was longer. Conversely, most mouth‐brooding cichlids deposited semen directly into the female buccal cavity, and spawned eggs were immediately picked up to be placed inside the cavity; thus, the time from ejaculation to fertilization was short. These observations suggest that increased sperm longevity is favoured in bower builders. Comparative phylogenetic analyses suggested that bower‐building behaviour and greater time from ejaculation to fertilization are associated with the extension of sperm longevity, whereas sperm competition rank does not play a major role. In addition, bower‐building behaviour preceded the emergence of increased sperm longevity. These results indicate that the extension of sperm longevity as a result of the emergence of bower builders may have acted as an evolutionary attractor for sperm longevity.     

LC‐MS/MS as an alternative for SDS‐PAGE in blue native analysis of protein complexes

Two‐dimensional blue native/SDS‐PAGE is widely applied to investigate native protein–protein interactions, particularly those within membrane multi‐protein complexes. MS has enabled the application of this approach at the proteome scale, typically by analysis of picked protein spots. Here, we investigated the potential of using LC‐MS/MS as an alternative for SDS‐PAGE in blue native (BN) analysis of protein complexes. By subjecting equal slices from BN gel lanes to label‐free semi‐quantitative LC‐MS/MS, we determined an abundance profile for each protein across the BN gel, and used these profiles to identify potentially interacting proteins by protein correlation profiling. We demonstrate the feasibility of this approach by considering the oxidative phosphorylation complexes I–V in the native human embryonic kidney 293 mitochondrial fraction, showing that the method is capable of detecting both the fully assembled complexes as well as assembly/turnover intermediates of complex I (NADH:ubiquinone oxidoreductase). Using protein correlation profiling with a profile for subunits NDUFS2, 3, 7 and 8 we identified multiple proteins possibly involved in the biogenesis of complex I, including the recently implicated chaperone C6ORF66 and a novel candidate, C3ORF60.     

Reduced glucose‐induced insulin secretion in low‐protein‐fed rats is associated with altered pancreatic islets redox status

In the present study, we investigated the relationship between early life protein malnutrition‐induced redox imbalance, and reduced glucose‐stimulated insulin secretion. After weaning, male Wistar rats were submitted to a normal‐protein‐diet (17%‐protein, NP) or to a low‐protein‐diet (6%‐protein, LP) for 60 days. Pancreatic islets were isolated and hydrogen peroxide (H₂O₂), oxidized (GSSG) and reduced (GSH) glutathione content, CuZn‐superoxide dismutase (SOD1), glutathione peroxidase (GPx1) and catalase (CAT) gene expression, as well as enzymatic antioxidant activities were quantified. Islets that were pre‐incubated with H₂O₂ and/or N‐acetylcysteine, were subsequently incubated with glucose for insulin secretion measurement. Protein malnutrition increased CAT mRNA content by 100%. LP group SOD1 and CAT activities were 50% increased and reduced, respectively. H₂O₂ production was more than 50% increased whereas GSH/GSSG ratio was near 60% lower in LP group. Insulin secretion was, in most conditions, approximately 50% lower in LP rat islets. When islets were pre‐incubated with H₂O₂ (100 μM), and incubated with glucose (33 mM), LP rats showed significant decrease of insulin secretion. This effect was attenuated when LP islets were exposed to N‐acetylcysteine.     

Proteomic characterization of EL4 lymphoma‐derived tumors upon chemotherapy treatment reveals potential roles for lysosomes and caspase‐6 during tumor cell death in vivo

The murine mouse lymphoblastic lymphoma cell line (EL4) tumor model is an established in vivo apoptosis model for the investigation of novel cancer imaging agents and immunological treatments due to the rapid and significant response of the EL4 tumors to cyclophosphamide and etoposide combination chemotherapy. Despite the utility of this model system in cancer research, little is known regarding the molecular details of in vivo tumor cell death. Here, we report the first in‐depth quantitative proteomic analysis of the changes that occur in these tumors upon cyclophosphamide and etoposide treatment in vivo. Using a label‐free quantitative proteomic approach a total of 5838 proteins were identified in the treated and untreated tumors, of which 875 were determined to change in abundance with statistical significance. Initial analysis of the data reveals changes that may have been predicted, such as the downregulation of ribosomes, but demonstrates the robustness of the dataset. Analysis of the dataset also reveals the unexpected downregulation of caspase‐3 and an upregulation of caspase‐6 in addition to a global upregulation of lysosomal proteins in the bulk of the tumor.     

Biospeckle activity in coffee seeds is associated non‐destructively with seedling quality

Seeds age during storage, resulting in a decline in germination and seedling quality. Seed quality tests are important to monitor this decline. However, such tests are usually destructive and require large seed numbers and long time. For coffee seeds the standard germination test and assessment of seedling quality takes 30 days. Biospeckle has been used previously as a non‐destructive optical tool to monitor biological activity in a range of tissues. Biospeckle was applied 3–6 days after imbibition (DAI) to investigate an association with coffee seedling quality after 30 days. Two distinct areas of biospeckle activity were demonstrated, concurring with the locations of the embryonic axis and the cotyledons in the apical and central seed parts, respectively. Moisture content analysis revealed that embryos of imbibed seeds contained more water than endosperm. Different areas within the endosperm did not differ in moisture content, while the moisture content of the axis was higher than that of the cotyledons, and this did not change from 4 DAI. Therefore, it was concluded that high biospeckle activity was not the result of increased water content in any seed part, but more likely of growth and metabolism in the axis and cotyledons, which had been described previously. A threshold biospeckle ratio apical : central of 1.02 after 6 days distinguished between seeds that produced dead and viable seedlings after 30 days and provided similar results as a tetrazolium test, a widely acknowledged but destructive test for seed quality. Thus, biospeckle data provided a non‐destructive early parameter for seedling quality, based on embryo growth during germination.     

Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation‐sensitive amplified polymorphism

Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal‐contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation‐sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal‐contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal‐contaminated site compared to uncontaminated populations. Other genotypes from a different metal‐contaminated site within the same region appear to be recalcitrant to metal‐induced DNA alterations even ≥30 years of tree life exposure to nickel and copper . MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal‐contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.     

S‐nitrosoglutathione prevents blood–brain barrier disruption associated with increased matrix metalloproteinase‐9 activity in experimental diabetes

Hyperglycemia is known to induce microvascular complications, thereby altering blood–brain barrier (BBB) permeability. This study investigated the role of matrix metalloproteinases (MMPs) and their endogenous inhibitors in increased BBB permeability and evaluated the protective effect of S‐nitrosoglutathione (GSNO) in diabetes. Diabetes was induced in mice by intraperitoneal injection of streptozotocin (40 mg/kg body weight) for 5 days and GSNO was administered orally (100 μg/kg body weight) daily for 8 weeks after the induction of diabetes. A significant decline in cognitive functions was observed in diabetic mice assessed by Morris water maze test. Increased permeability to different molecular size tracers accompanied by edema and ion imbalance was observed in cortex and hippocampus of diabetic mice. Furthermore, activity of both pro and active MMP‐9 was found to be significantly elevated in diabetic animals. Increased in situ gelatinase activity was observed in tissue sections and isolated microvessels from diabetic mice brain. The increase in activity of MMP‐9 was attributed to increased mRNA and protein expression in diabetic mice. In addition, a significant decrease in mRNA and protein expression of tissue inhibitor of matrix metalloproteinase‐1 was also observed in diabetic animals. However, GSNO supplementation to diabetic animals was able to abridge MMP‐9 activation as well as tissue inhibitor of matrix metalloproteinase‐1 levels, restoring BBB integrity and also improving learning and memory. Our findings clearly suggest that GSNO could prevent hyperglycemia‐induced disruption of BBB by suppressing MMP‐9 activity.

     

Increased resistance to hydrogen peroxide‐induced cardiac contracture is associated with decreased myocardial oxidative stress in hypothyroid rats

The purpose of this study was to determine whether decreased oxidative stress would increase the resistance to cardiac contracture induced by H₂O₂ in hypothyroid rats. Male Wistar rats were divided into two groups: control and hypothyroid. Hypothyroidism was induced via thyroidectomy. Four weeks post surgery, blood samples were collected to perform thyroid hormone assessments, and excised hearts were perfused at a constant flow with or without H₂O₂ (1 mmol/L), being divided into two sub‐groups: control, hypothyroid, control + H₂O₂, hypothyroid + H₂O₂. Lipid peroxidation (LPO) was evaluated by chemiluminescence (CL) and thiobarbituric acid reactive substances (TBARS) methods, and protein oxidation by carbonyls assay in heart homogenates. Cardiac tissue was also screened for superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) activities, and for total radical‐trapping antioxidant potential (TRAP). Analyses of SOD and glutathione‐S‐transferase (GST) protein expression were also performed in heart homogenates. Hypothyroid hearts were found to be more resistant to H₂O₂‐induced contracture (60% elevation in LVEDP) as compared to control. CL, TBARS, carbonyl, as well as SOD, CAT, GPx activities and TRAP levels were reduced (35, 30, 40, 30, 16, 25, and 33%, respectively) in the cardiac homogenates of the hypothyroid group as compared to controls. A decrease in SOD and GST protein levels by 20 and 16%, respectively, was also observed in the hypothyroid group. These results suggest that a hypometabolic state caused by thyroid hormone deficiency can lead to an improved response to H₂O₂ challenge and is associated with decreased oxidative myocardial damage. Copyright © 2009 John Wiley & Sons, Ltd.