Characterization of wheat germin (oxalate oxidase) expressed by Pichia pastoris

High-level secretory expression of wheat (Triticum aestivum) germin/oxalate oxidase was achieved in Pichia pastoris fermentation cultures as an alpha-mating factor signal peptide fusion, based on the native wheat cDNA coding sequence. The oxalate oxidase activity of the recombinant enzyme is substantially increased (7-fold) by treatment with sodium periodate, followed by ascorbate reduction. Using these methods, approximately 1 g (4x10(4) U) of purified, activated enzyme was obtained following eight days of induction of a high density Pichia fermentation culture, demonstrating suitability for large-scale production of oxalate oxidase for biotechnological applications. Characterization of the recombinant protein shows that it is glycosylated, with N-linked glycan attached at Asn47. For potential biomedical applications, a nonglycosylated (S49A) variant was also prepared which retains essentially full enzyme activity, but exhibits altered protein-protein interactions.     

Purification and characterization of thermostable H<Subscript>2</Subscript>O<Subscript>2</Subscript>-forming NADH oxidase from 2-phenylethanol-assimilating <Emphasis Type="Italic">Brevibacterium</Emphasis> sp. KU1309

A cytoplasmic NADH oxidase (NOX) was purified from a soil bacteria, Brevibacterium sp. KU1309, which is able to grow in the medium containing 2-phenylethanol as the sole source of carbon under an aerobic condition. The enzyme catalyzed the oxidation of NADH to NAD+ involving two-electron reduction of O2 to H2O2. The molecular weight of the enzyme was estimated to be 102 kDa by gel filtration and 57 kDa by SDS-PAGE, which indicates that the NOX was a homodimer consisting of a single subunit. The enzyme was stable up to 70 degrees C at a broad range of pH from 7 to 11. The enzyme activity increased about ten-fold with the addition of ammonium salt, while it was inhibited by Zn2+ (39%), Cu2+ (41%), Hg2+ (72%) and Ag+ (37%). The enzyme acts on NADH, but not on NADPH. The regeneration of NAD+ utilizing this enzyme made selective oxidation of mandelic acid or L: -phenylalanine possible. This thermostable enzyme is expected to be applicable as a useful biocatalyst for NAD+ recycling.     

Energetics of embryonic development: effects of temperature on egg and hatchling composition in a butterfly

Phenotypic plasticity may allow an organism to adjust its phenotype to environmental needs. However, little is known about environmental effects on offspring biochemical composition and turnover rates, including energy budgets and developmental costs. Using the tropical butterfly Bicyclus anynana and employing a full-factorial design with two oviposition and two developmental temperatures, we explore the consequences of temperature variation on egg and hatchling composition, and the associated use and turnover of energy and egg compounds. At the lower temperature, larger but fewer eggs were produced. Larger egg sizes were achieved by provisioning these eggs with larger quantities of all compounds investigated (and thus more energy), whilst relative egg composition was rather similar to that of smaller eggs laid at the higher temperature. Turnover rates during embryonic development differed across developmental temperatures, suggesting an emphasis on hatchling quality (i.e. protein content) at the more stressful lower temperature, but on storage reserves (i.e. lipids) at the higher temperature. These differences may represent adaptive maternal effects. Embryonic development was much more efficient at the lower temperature, providing a possible mechanism underlying the temperature-size rule.     

Multiphoton ANS fluorescence microscopy as an in vivo sensor for protein misfolding stress

The inability of cells to maintain protein folding homeostasis is implicated in the development of neurodegenerative diseases, malignant transformation, and aging. We find that multiphoton fluorescence imaging of 1-anilinonaphthalene-8-sulfonate (ANS) can be used to assess cellular responses to protein misfolding stresses. ANS is relatively nontoxic and enters live cells and cells or tissues fixed in formalin. In an animal model of Alzheimer’s disease, ANS fluorescence imaging of brain tissue sections reveals the binding of ANS to fibrillar deposits of amyloid peptide (Aβ) in amyloid plaques and in cerebrovascular amyloid. ANS imaging also highlights non-amyloid deposits of glial fibrillary acidic protein in brain tumors. Cultured cells under normal growth conditions possess a number of ANS-binding structures. High levels of ANS fluorescence are associated with the endoplasmic reticulum (ER), Golgi, and lysosomes—regions of protein folding and degradation. Nuclei are virtually devoid of ANS binding sites. Additional ANS binding is triggered by hyperthermia, thermal lesioning, proteasome inhibition, and induction of ER stress. We also use multiphoton imaging of ANS binding to follow the in vivo recovery of cells from protein-damaging insults over time. We find that ANS fluorescence tracks with the binding of the molecular chaperone Hsp70 in compartments where Hsp70 is present. ANS highlights the sensitivity of specific cellular targets, including the nucleus and particularly the nucleolus, to thermal stress and proteasome inhibition. Multiphoton imaging of ANS binding should be a useful probe for monitoring protein misfolding stress in cells.     

Event-related Potential Study of the Effects of Emotional Facial Expressions on Task Performance in Euthymic Bipolar Patients

There appears to be a significant disconnect between symptomatic and functional recovery in bipolar disorder (BD). Some evidence points to interepisode cognitive dysfunction. We tested the hypothesis that some of this dysfunction was related to emotional reactivity in euthymic bipolar subjects may effect cognitive processing. A modification of emotional gender categorization oddball task was used. The target was gender (probability 25%) of faces with negative, positive, and neutral emotional expression. The experiment had 720 trials (3 blocks × 240 trials each). Each stimulus was presented for 150 ms, and the EEG/ERP responses were recorded for 1,000 ms. The inter-trial interval was varied in 1,100–1,500 ms range to avoid expectancy effects. Task took about 35 min to complete. There were 9 BD and 9 control subjects matched for age and gender. Reaction time (RT) was globally slower in BD subjects. The centro-parietal amplitudes at N170 and N200, and P200 and P300 were generally smaller in the BD group compared to controls. Latency was shorter to neutral and negative targets in BD. Frontal P200 amplitude was higher to emotional negative facial non-targets in BD subjects. The frontal N200 in response to positive facial emotion was less negative in BD subjects. The frontal P300 of BD subjects was lower to emotionally neutral targets. ERP responses to facial emotion in BD subjects varied significantly from normal controls. These variations are consistent with the common depressive symptomology seen in long term studies of bipolar subjects.     

Estimating the contribution of assembly activity to cortical dynamics from spike and population measures

The hypothesis that cortical networks employ the coordinated activity of groups of neurons, termed assemblies, to process information is debated. Results from multiple single-unit recordings are not conclusive because of the dramatic undersampling of the system. However, the local field potential (LFP) is a mesoscopic signal reflecting synchronized network activity. This raises the question whether the LFP can be employed to overcome the problem of undersampling. In a recent study in the motor cortex of the awake behaving monkey based on the locking of coincidences to the LFP we determined a lower bound for the fraction of spike coincidences originating from assembly activation. This quantity together with the locking of single spikes leads to a lower bound for the fraction of spikes originating from any assembly activity. Here we derive a statistical method to estimate the fraction of spike synchrony caused by assemblies—not its lower bound—from the spike data alone. A joint spike and LFP surrogate data model demonstrates consistency of results and the sensitivity of the method. Combining spike and LFP signals, we obtain an estimate of the fraction of spikes resulting from assemblies in the experimental data.     

Current knowledge of microbial community structures in landfills and its cover soils

Landfills are a vital component of our waste handling processes. Our lack of knowledge on the microbial processes in these systems, however, hampers our ability to design the next generation of landfills that: (1) enhance the rate and extent of waste decomposition, (2) produce byproducts of some value (e.g., methane that can be used for energy generation), and, (3) minimize their overall impact on driving climate change through the emission of greenhouse gases. In this review, the current state of knowledge the microbial community structure and activity in both the refuse and overlying cover soils is discussed, and suggestions provided for future research in this critical aspect of our infrastructure.     

An investigation on the genetic background of <Emphasis Type="Italic">Nostoc flagelliforme</Emphasis> by similarity analysis of its partial genomic DNA and phylogenetic comparison of deduced related species

Nostoc flagelliforme, which is distributed on arid and semi-arid steppes of northwestern parts of China, has attracted increasing interest for its stress tolerance. In order to gain more insight into the genetic background of N. flagelliforme, we sequenced its partial genomic DNA for similarity analyses against current public databases, followed by phylogenetic comparison of N. flagelliforme and the potentially related species deduced from the similarity analyses. Approximately 430 kb genomic sequence (~5% of genome as a rough estimate) was determined from 106 distinct genomic clones. Nucleotide BLAST showed that ~23.1% of the partial genomic sequence was similar to N. punctiforme genomic DNA and ~12.4% to its plasmid DNA. Similar protein search by online FASTA-protein program showed 46.2% of the similar proteins had their corresponding orthologs in N. punctiforme genome. Furthermore, phylogenetic comparison based on 16S rRNA sequences showed N. flagelliforme and N. punctiforme clustered closer among the deduced related species. These results indicated that N. punctiforme might also be potentially close neighbor species of N. flagelliforme, in addition to the formerly regarded close neighbor species N. commune and N. sphaeroids. In general, these data enriched our recognition of the evolutionary relationship between N. flagelliforme and other Nostoc species, especially N. punctiforme.