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.     

In vivo release of dopamine during perfusion of neurotensin in substantia nigra of the unrestrained rat

The functional effect of neurotensin on the kinetics of dopamine (DA) release in the substantia nigra of the freely moving rat was investigated. After guide tubes for push-pull perfusion were implanted stereotaxically just above the substantia nigra, endogenous stores of DA in this structure were labelled by micro-injection of 0.02–0.05 μCi of [¹⁴C]-DA. Then an artificial cerebrospinal fluid (CSF) was perfused within the site at a rate of 20 μl/min at successive 5 min intervals. Neurotensin added to the CSF perfusate in concentrations of 0.05–0.1 μg/μl evoked an immediate, Ca⁺⁺ dependent release of DA from sites directly within the substantia nigra or a delayed efflux when the peptide was perfused at the edge of this structure. Neurotensin failed to affect the pattern of release of this monoamine at sites which were not within the substantia nigra. Further, the body temperature of the rat also was not altered by neurotensin at any of the sites of perfusions. A relatively inactive analogue of the peptide, [D-Arg]⁹ neurotensin, was essentially without effect on DA activity. In double isotope experiments in which the substantia nigra of the rat was labelled with both [³H]-5-HT and [¹⁴C]-DA, the perfusion with neurotensin failed to affect 5-HT efflux while the release of DA was enhanced. Chromatographic analysis of the metabolites of DA in samples of push-pull perfusates revealed that neurotensin enhanced significantly the level of DOPAC and HVA. Overall, these results demonstrate that in the unrestrained rat neurotensin acts selectively within the substantia nigra to alter the presynaptic, Ca⁺⁺ dependent release of DA. It is suggested that the mechanism by which the tri-decapeptide functions within this brainstem structure is through its modulation of nigral dopaminergic neurons.     

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.     

Functional conservation of an insect odorant receptor gene across 250 million years of evolution

Pest insects have a profound negative impact on agriculture and human health. Significant global losses of crops, stored agricultural products, timber and livestock can be attributed to damage and destruction by insects . Blood-feeding insects such as mosquitoes, flies and ticks transmit many of humanity's most devastating infectious diseases. Insect-borne diseases account for more than one million annual fatalities, and insect-associated illnesses surpass 300 million annual reported cases . The medical and economic impact of these animals can be ascribed in part to the sensitivity and selectivity of their olfactory systems, essential for location of their preferred plant and animal hosts.     

Multiple strategies to improve sensitivity,speed and robustness of isothermal nucleic acid amplification for rapid pathogen detection

Background  

In the past decades the rapid growth of molecular diagnostics (based on either traditional PCR or isothermal amplification technologies) meet the demand for fast and accurate testing. Although isothermal amplification technologies have the advantages of low cost requirements for instruments, the further improvement on sensitivity, speed and robustness is a prerequisite for the applications in rapid pathogen detection, especially at point-of-care diagnostics. Here, we describe and explore several strategies to improve one of the isothermal technologies, helicase-dependent amplification (HDA).     

Biological role of MicroRNA-103 based on expression profile and target genes analysis in pigs

MicroRNAs (miRNAs) are endogenously expressed RNAs consisting of 20–24 nucleotides. These molecules are thought to repress protein translation by binding to target mRNAs. However, biological functions have not been assigned to most of the 175 porcine miRNAs registered in miRBase (release 15.0). In an effort to uncover miR-103 important in pigs, we examined the integrative tissue expression profile and gene ontology (GO) term enrichment of predicted target genes to determine the global biological functions of miR-103. Our results demonstrated that miR-103 is involved in various biological processes including brain development, lipid metabolism, adipocyte differentiation, hematopoiesis, and immunity. Moreover, we also experimentally verified effects of miR-103 in porcine preadipocytes. miR-103 levels increased in differentiating adipocytes, and inhibition of miR-103 effectively inhibited preadipocyte differentiation. In addition, mRNA levels of the putative miR-103 target RAI14 were higher in miR-103 inhibitor-treated adipocytes. These results demonstrate that miR-103 is involved in porcine preadipocyte differentiation and may act through the putative target gene RAI14. In a word, our data provide new insights into the global biological role of miR-103.