COMT polymorphism modulates the resting‐state EEG alpha oscillatory response to acute nicotine in male non‐smokers

Performance improvements in cognitive tasks requiring executive functions are evident with nicotinic acetylcholine receptor (nAChR) agonists, and activation of the underlying neural circuitry supporting these cognitive effects is thought to involve dopamine neurotransmission. As individual difference in response to nicotine may be related to a functional polymorphism in the gene encoding catechol‐O‐methyltransferase (COMT), an enzyme that strongly influences cortical dopamine metabolism, this study examined the modulatory effects of the COMT Val158Met polymorphism on the neural response to acute nicotine as measured with resting‐state electroencephalographic (EEG) oscillations. In a sample of 62 healthy non‐smoking adult males, a single dose (6 mg) of nicotine gum administered in a randomized, double‐blind, placebo‐controlled design was shown to affect α oscillatory activity, increasing power of upper α oscillations in frontocentral regions of Met/Met homozygotes and in parietal/occipital regions of Val/Met heterozygotes. Peak α frequency was also found to be faster with nicotine (vs. placebo) treatment in Val/Met heterozygotes, who exhibited a slower α frequency compared to Val/Val homozygotes. The data tentatively suggest that interindividual differences in brain α oscillations and their response to nicotinic agonist treatment are influenced by genetic mechanisms involving COMT.     

Proteomic Analysis of Urine to Identify Breast Cancer Biomarker Candidates Using a Label-Free LC-MS/MS Approach

Introduction

Breast cancer is a complex heterogeneous disease and is a leading cause of death in women. Early diagnosis and monitoring progression of breast cancer are important for improving prognosis. The aim of this study was to identify protein biomarkers in urine for early screening detection and monitoring invasive breast cancer progression.

Method

We performed a comparative proteomic analysis using ion count relative quantification label free LC-MS/MS analysis of urine from breast cancer patients (n = 20) and healthy control women (n = 20).

Results

Unbiased label free LC-MS/MS-based proteomics was used to provide a profile of abundant proteins in the biological system of breast cancer patients. Data analysis revealed 59 urinary proteins that were significantly different in breast cancer patients compared to the normal control subjects (p<0.05, fold change >3). Thirty-six urinary proteins were exclusively found in specific breast cancer stages, with 24 increasing and 12 decreasing in their abundance. Amongst the 59 significant urinary proteins identified, a list of 13 novel up-regulated proteins were revealed that may be used to detect breast cancer. These include stage specific markers associated with pre-invasive breast cancer in the ductal carcinoma in-situ (DCIS) samples (Leucine LRC36, MAST4 and Uncharacterized protein CI131), early invasive breast cancer (DYH8, HBA, PEPA, uncharacterized protein C4orf14 (CD014), filaggrin and MMRN2) and metastatic breast cancer (AGRIN, NEGR1, FIBA and Keratin KIC10). Preliminary validation of 3 potential markers (ECM1, MAST4 and filaggrin) identified was performed in breast cancer cell lines by Western blotting. One potential marker MAST4 was further validated in human breast cancer tissues as well as individual human breast cancer urine samples with immunohistochemistry and Western blotting, respectively.

Conclusions

Our results indicate that urine is a useful non-invasive source of biomarkers and the profile patterns (biomarkers) identified, have potential for clinical use in the detection of BC. Validation with a larger independent cohort of patients is required in the following study.     

Temporal Monitoring of Differentiated Human Airway Epithelial Cells Using Microfluidics

The airway epithelium is exposed to a variety of harmful agents during breathing and appropriate cellular responses are essential to maintain tissue homeostasis. Recent evidence has highlighted the contribution of epithelial barrier dysfunction in the development of many chronic respiratory diseases. Despite intense research efforts, the responses of the airway barrier to environmental agents are not fully understood, mainly due to lack of suitable in vitro models that recapitulate the complex in vivo situation accurately. Using an interdisciplinary approach, we describe a novel dynamic 3D in vitro model of the airway epithelium, incorporating fully differentiated primary human airway epithelial cells at the air-liquid interface and a basolateral microfluidic supply of nutrients simulating the interstitial flow observed in vivo. Through combination of the microfluidic culture system with an automated fraction collector the kinetics of cellular responses by the airway epithelium to environmental agents can be analysed at the early phases for the first time and with much higher sensitivity compared to common static in vitro models. Following exposure of primary differentiated epithelial cells to pollen we show that CXCL8/IL–8 release is detectable within the first 2h and peaks at 4–6h under microfluidic conditions, a response which was not observed in conventional static culture conditions. Such a microfluidic culture model is likely to have utility for high resolution temporal profiling of toxicological and pharmacological responses of the airway epithelial barrier, as well as for studies of disease mechanisms.     

Amplification of a Cytochrome P450 Gene Is Associated with Resistance to Neonicotinoid Insecticides in the Aphid Myzus persicae

The aphid Myzus persicae is a globally significant crop pest that has evolved high levels of resistance to almost all classes of insecticide. To date, the neonicotinoids, an economically important class of insecticides that target nicotinic acetylcholine receptors (nAChRs), have remained an effective control measure; however, recent reports of resistance in M. persicae represent a threat to the long-term efficacy of this chemical class. In this study, the mechanisms underlying resistance to the neonicotinoid insecticides were investigated using biological, biochemical, and genomic approaches. Bioassays on a resistant M. persicae clone (5191A) suggested that P450-mediated detoxification plays a primary role in resistance, although additional mechanism(s) may also contribute. Microarray analysis, using an array populated with probes corresponding to all known detoxification genes in M. persicae, revealed constitutive over-expression (22-fold) of a single P450 gene (CYP6CY3); and quantitative PCR showed that the over-expression is due, at least in part, to gene amplification. This is the first report of a P450 gene amplification event associated with insecticide resistance in an agriculturally important insect pest. The microarray analysis also showed over-expression of several gene sequences that encode cuticular proteins (2–16-fold), and artificial feeding assays and in vivo penetration assays using radiolabeled insecticide provided direct evidence of a role for reduced cuticular penetration in neonicotinoid resistance. Conversely, receptor radioligand binding studies and nucleotide sequencing of nAChR subunit genes suggest that target-site changes are unlikely to contribute to resistance to neonicotinoid insecticides in M. persicae.     

Divalent Metal Ions in Plant Mitochondria and Their Role in Interactions with Proteins and Oxidative Stress-Induced Damage to Respiratory Function

Understanding the metal ion content of plant mitochondria and metal ion interactions with the proteome are vital for insights into both normal respiratory function and the process of protein damage during oxidative stress. We have analyzed the metal content of isolated Arabidopsis (Arabidopsis thaliana) mitochondria, revealing a 26:8:6:1 molar ratio for iron:zinc:copper:manganese and trace amounts of cobalt and molybdenum. We show that selective changes occur in mitochondrial copper and iron content following in vivo and in vitro oxidative stresses. Immobilized metal affinity chromatography charged with Cu²⁺, Zn²⁺, and Co²⁺ was used to identify over 100 mitochondrial proteins with metal-binding properties. There were strong correlations between the sets of immobilized metal affinity chromatography-interacting proteins, proteins predicted to contain metal-binding motifs, and protein sets known to be oxidized or degraded during abiotic stress. Mitochondrial respiratory chain pathways and matrix enzymes varied widely in their susceptibility to metal-induced loss of function, showing the selectivity of the process. A detailed study of oxidized residues and predicted metal interaction sites in the tricarboxylic acid cycle enzyme aconitase identified selective oxidation of residues in the active site and showed an approach for broader screening of functionally significant oxidation events in the mitochondrial proteome.Transition metal ions are essential in myriad biochemical functions by being incorporated into or associating with proteins to elicit functions in living cells. In plant mitochondria, key functions of metal cofactors include metabolism, electron transport, ATP synthesis, and the detoxification of reactive oxygen species (ROS). For example, copper (Cu) and iron (Fe) ions facilitate the transfer of electrons in the electron transport chain (ETC; Bligny and Douce, 1977; Pascal and Douce, 1993), proteins of the tricarboxylic acid (TCA) cycle utilize metal ion cofactors to catalyze primary metabolic reactions (Miernyk and Randall, 1987; Jordanov et al., 1992), manganese (Mn) and Fe are required for antioxidant defense enzymes (Alscher et al., 2002), and zinc (Zn) is required for the protein import apparatus in both carrier protein transport to the inner membrane (Lister et al., 2002) and presequence degradation (Moberg et al., 2003). Cobalt (Co) is known to substitute for other metal ions in the activation of NAD-malic enzyme and succinyl-CoA ligase from plant mitochondrial extracts (Palmer and Wedding, 1966; Macrae, 1971), but it is not known whether there is an in vivo requirement for trace amounts of Co for plant respiratory metabolism.Metal ions, however, can also be highly toxic to cells and cell organelle functions. The redox-inactive heavy metal cadmium exhibits strong affinity for oxygen, nitrogen, and sulfur atoms (Nieboer and Richardson, 1980) and can inhibit enzyme activity by direct blocking of protein function or displacement of natural metal centers. There are numerous reports of heavy metals depleting cellular glutathione pools, leading to diminished antioxidant protection in the cell and resulting in ROS accumulation (Schutzendubel and Polle, 2002). Cadmium has been reported to both directly and indirectly inhibit plant mitochondrial function (Kesseler and Brand, 1994; Smiri et al., 2009), as have Co complexes (Guzhova et al., 1979). Redox-active metal catalysts such as Cu and Fe cations can also be cytotoxic, as they react with ROS via the Haber-Weiss reaction or Fenton-type reactions to produce the hydroxyl anion (Stohs and Bagchi, 1995). Inhibitory effects of exogenously added Cu and Fe on plant respiratory function have been reported (Kampfenkel et al., 1995; Padua et al., 1996, 1999). Therefore, the presence of free metal cations, redox active or inactive, in mitochondria may significantly contribute to the initiation and perpetuation of oxidative stress.One of the best described mechanisms for metal-linked damage is metal-catalyzed oxidation (MCO) of proteins, which involves the oxidation of susceptible amino acids such as Arg, Lys, Pro, and His, among a plethora of other poorly characterized consequences (Stadtman, 1990). It has been proposed that MCO of proteins can be a highly specific event where proteins are more susceptible to damage if they bind metal ions and when the site of protein oxidation can be defined on the protein surface that binds to the metal ions (Stadtman, 1990). One of the major consequences of MCO is the irreversible formation of reactive carbonyls on amino acid side chains (Stadtman, 1990). Such carbonyls are known to accumulate in the wheat (Triticum aestivum) mitochondrial proteome during environmental stress, even more so than in other ROS-producing subcellular organelles of plants (Bartoli et al., 2004). The selectivity of protein susceptibility to MCO was also demonstrated in rice (Oryza sativa), where distinct subpopulations of the mitochondrial matrix proteome were carbonylated following Cu²⁺ and hydrogen peroxide (H₂O₂) treatment (Kristensen et al., 2004). The targeted damage of select sets of plant mitochondrial proteins has also been observed in other studies, but without clear linkage to the role of metal ions. For example, altered protein abundance has been observed in Arabidopsis (Arabidopsis thaliana; Sweetlove et al., 2002) and pea (Pisum sativum; Taylor et al., 2005) mitochondria after the initiation of oxidative or environmental stress. Additionally, inhibition of respiratory metabolism by the lipid peroxidation by-product 4-hydroxy-2-nonenal has been shown to operate through modification of a specific subset of proteins (Taylor et al., 2002; Winger et al., 2005, 2007). However, the mechanisms of targeted oxidative modification, the role of metals, and the consequences for mitochondrial metabolic function are not very well understood. Furthermore, whether or not selectivity of protein damage in mitochondria is based on relative metal ion affinity and if the sites of damage can be predicted by the sites of metal ion binding are not known.In this study, we investigated metal homeostasis in the Arabidopsis mitochondrion during oxidative stress. The interactions between metal ions and proteins were also investigated using immobilized metal affinity chromatography (IMAC). Functional assays were used to determine the targets and consequences of metal ion interaction in the mitochondrion and to explore the linkages to the redox nature of the metal and the loss of mitochondrial functions. Finally, a detailed study of the oxidized peptides of aconitase was undertaken to probe the linkage between metal-binding sites, the oxidation of amino acids, and the inactivation of this critical TCA cycle enzyme.     

Increasing cell biomass in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> increases recombinant protein yield: the use of a respiratory strain as a microbial cell factory

Background  

Recombinant protein production is universally employed as a solution to obtain the milligram to gram quantities of a given protein required for applications as diverse as structural genomics and biopharmaceutical manufacture. Yeast is a well-established recombinant host cell for these purposes. In this study we wanted to investigate whether our respiratory Saccharomyces cerevisiae strain, TM6*, could be used to enhance the productivity of recombinant proteins over that obtained from corresponding wild type, respiro-fermentative strains when cultured under the same laboratory conditions.     

recA genotyping of Salmonella enteritidis phage type 4 isolates by restriction fragment length polymorphism analysis

AIMS: To subtype Salmonella enteritidis phage type 4 isolates by using recA genotyping. METHODS AND RESULTS: Random amplified polymorphic DNA analysis using a primer ERIC2 of 76 isolates of Salmonella enteritidis phage type 4 obtained in Northern Ireland in 1998 and in 1999 demonstrated the presence of five genotypes. Restriction fragment length polymorphism analysis, using a degenerate primer pair designed to amplify a segment (about 640 bp in length) of the recA gene from several members of the Enterobacteriaceae with restriction enzymes, HhaI and Sau3AI, showed that the resulting fragments could differentiate the isolates into three groups, respectively. CONCLUSION: recA gene amplification and HhaI and Sau3AI restriction digestion was demonstrated to increase the differentiating power between isolates of Salmonella enteritidis phage type 4 by combining the patterns of the random amplified polymorphic DNA analysis procedure using a primer ERIC2. Significance and Impact of the Study: A novel restriction fragment length polymorphism assay for isolates of Salmonella enteritidis phage type 4, based on the amplification of the recA gene was attained and its comparison and its combination with random amplified polymorphic DNA analysis was provided.     

Mitochondrial acyl carrier proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> are predominantly soluble matrix proteins and none can be confirmed as subunits of respiratory Complex I

Arabidopsis mitochondria are predicted to contain three acyl carrier proteins (ACPs). These small proteins are involved in fatty acid and lipoic acid synthesis in other organisms and have been previously reported to be subunits of respiratory Complex I in mitochondria in mammals, fungi and plants. Recently, the mammalian mitochondrial ACP (mtACP) has been shown to be largely a soluble matrix protein but also to be minimally associated with Complex I (Cronan et al. 2005), consistent with its involvement in synthesis of lipoic acid for TCA cycle decarboxylating dehydrogenases in the matrix but contrary to earlier claims it was primarily a Complex I subunit. We have investigated the localization of the ACPs in Arabidopsis mitochondria. Evidence is presented that mtACP1 and mtACP2 dominate the ACP composition in Arabidopsis mitochondria, and both are present in the mitochondrial matrix rather than in the membrane. No significant amounts of mtACPs were detected in Complex I isolated by blue native gel electrophoresis, rather mtACPs were detected at low molecular mass in the soluble fraction, showing that in A. thaliana mtACPs are predominately free soluble matrix proteins.     

Detection and resolution of genetic loci affecting circadian period in <Emphasis Type="Italic">Brassica oleracea</Emphasis>

Circadian rhythms regulate many aspects of plant growth, fitness and vigour. The components and detailed mechanism of circadian regulation to date have been dissected in the reference species Arabidopsis thaliana. To determine the genetic basis and range of natural allelic variation for intrinsic circadian period in the closest crop relatives, we used an accurate and high throughput data capture system to record rhythmic cotyledon movement in two immortal segregating populations of Brassica oleracea, derived from parent lines representing different crop types. Periods varied between 24.4 and 26.1 h between the parent lines, with transgressive segregation between extreme recombinant lines in both populations of ∼3.5 h. The additive effect of individual QTL identified in each population varied from 0.17 to 0.36 h. QTL detected in one doubled haploid population were verified and the mapping intervals further resolved by determining circadian period in genomic substitution lines derived from the parental lines. Comparative genomic analysis based on collinearity between Brassica and Arabidopsis also allowed identification of candidate orthologous genes known to regulate period in Arabidopsis, that may account for the additive circadian effects of specific QTL. The distinct QTL positions detected in the two populations, and the extent of transgressive segregation suggest that there is likely to be considerable scope for modulating the range of available circadian periods in natural populations and crop species of Brassica. This may provide adaptive advantage for optimising growth and development in different latitudes, seasons or climate conditions.