Mutagenesis of the active site of glucoamylase from Aspergillus awamori

The Aspergillus awamori glucoamylase gene was modified by cassette mutagenesis, and eleven mutated enzymes were expressed by Saccharomyces cerevisiae;Four mutations were constructed to change the essential Trp 120 residue in subsite 4 to Tyr, Phe, His, and Leu. All the mutations bound maltose (G2) and isomaltose (iG2) more strongly than wild-type glucoamylase. A subsite map of the Tyr 120 mutation showed significantly higher affinities for D-glucosyl residues in subsites 1 and 2, suggesting an interaction of Trp 120 with residues located there;Three carboxylic acid residues in the active site, Asp 176, Glu 179, and Glu 180, were mutated to discover their role in catalysis. The Glu-Gln 179 mutation resulted in almost complete loss of activity with little change in binding, suggesting that Gln 179 is catalytically active. The Glu-Gln 180 mutation suggests that Glu 180 provides a strong bond with [alpha]-(1 → 4)-linked D-glucosyl residues in subsite 2, but is not involved in catalysis. The Asp-Asn 176 mutation resulted in a large decrease in catalytic activity and a moderate increase in binding of G2, iG2, and maltoheptaose (G7). A subsite map of this mutation suggests that Asp 176 may interact with Trp 120 and also be catalytically active;Four residues likely to be in the active site, based on homology with related enzymes, Tyr 116, Leu 177, Trp 178, and Asn 182, were changed to Ala, His, Arg, and Ala, respectively. The Tyr-Ala 116 mutation yielded similar though less dramatic results than the Trp 120 mutations, suggesting it has a similar role in catalysis. The Leu 177, Trp 178, and Asn 182 residues were suspected of affecting the selectivity of [alpha]-(1 → 4)- against [alpha]-(1 → 6)-D-glucosidic bond hydrolysis. The Leu-His 177 mutation showed moderate decreases in binding and catalytic rates. The Trp-Arg 178 mutation showed a substantial decrease in catalytic rate and a two-fold increase in selectivity of iG2 over G2 hydrolysis. The Asn-Ala 182 mutation gave slightly lower catalytic rates with an increased binding of G2 and decreased binging of iG2. This more than doubled the selectivity of G2 over iG2 hydrolysis with little effect on the catalytic rate

Process for enzymatic hydrolysis of starch to glucose

A process for converting starch or partially hydrolyzed starch into a syrup containing dextrose includes the steps of saccharifying starch hydrolyzate in the presence of a saccharifying starch hydrolyzate in the presence of a mutated glucoamylase or related enzyme and increasing the selectivity of the enzyme for α-(1→4)-glucosidic bonds by the glucoamylase or related enzyme by including at least one mutation, the mutation substituting an amino acid of the enzyme with at least one amino acid chosen by comparison with structurally related regions of other enzymes that selectively hydrolyze only α-(1→4) glucosidic bonds. Enzymes made in accordance with the present invention are also disclosed

Rainwater Harvesting: Diversifying the Irrigation Supply of the Coker Arboretum

The University of North Carolina, Chapel Hill (UNC) is considering a variety of measures to reduce flooding at the Coker Arboretum. As underground storage has been proposed as one recommended flood control measure by Rummel, Klepper, & Kahl LLP (RK&K) and Biohabitats Inc., this report explores the design of such storage to deliver external benefits of providing the arboretum with an auxiliary water supply for irrigation as well as reducing downstream nutrient loading. Historic rainfall data from 1947-2014 was used to analyze the performance of twenty four rainwater harvesting systems of varying capacity and location. Ultimately, the most cost effective system was determined to have 76 kgal capacity capable of replacing nearly 48% of annual arboretum irrigation demand.Master of Science in Environmental Engineerin

Curcumin reduces α-synuclein induced cytotoxicity in Parkinson's disease cell model

abstract: Background Overexpression and abnormal accumulation of aggregated α-synuclein (αS) have been linked to Parkinson's disease (PD) and other synucleinopathies. αS can misfold and adopt a variety of morphologies but recent studies implicate oligomeric forms as the most cytotoxic species. Both genetic mutations and chronic exposure to neurotoxins increase αS aggregation and intracellular reactive oxygen species (ROS), leading to mitochondrial dysfunction and oxidative damage in PD cell models. Results Here we show that curcumin can alleviate αS-induced toxicity, reduce ROS levels and protect cells against apoptosis. We also show that both intracellular overexpression of αS and extracellular addition of oligomeric αS increase ROS which induces apoptosis, suggesting that aggregated αS may induce similar toxic effects whether it is generated intra- or extracellulary. Conclusions Since curcumin is a natural food pigment that can cross the blood brain barrier and has widespread medicinal uses, it has potential therapeutic value for treating PD and other neurodegenerative disorders.The electronic version of this article is the complete one and can be found online at: http://bmcneurosci.biomedcentral.com/articles/10.1186/1471-2202-11-5

The Opened Vein: Slaughterhouses as Sites of the Hidden Sacred

Focusing on hog slaughter in North Carolina, this ethnographic project uses scholarly models of sacrifice to interrogate the religious mechanisms that scaffold industrial slaughter in America today. Within the span of two hundred years, the killing of animals for human consumption has gone from being an accepted part of daily public life to an act shrouded in secrecy. Informed by this historical transformation, I argue that the ostensibly secular site of the contemporary slaughterhouse has taken on valences of the “negative sacred” that both respond to and fuel public sensibilities around animality, intimacy, and the act of killing. My project examines the ways in which the religious dynamics that structure this negative sacred space participate in the production and amplification of ostensibly non-religious social categories. By testing models of sacrifice in the context of hog slaughter, I open up a new set of theoretical questions around the relationships between prohibition, transgression, and social control.Doctor of Philosoph

Lithologic Mapping of HED Terrains on Vesta using Dawn Framing Camera Color Data

The surface composition of Vesta, the most massive intact basaltic object in the asteroid belt, is interesting because it provides us with an insight into magmatic differentiation of planetesimals that eventually coalesced to form the terrestrial planets. The distribution of lithologic and compositional units on the surface of Vesta provides important constraints on its petrologic evolution, impact history and its relationship with Vestoids and howardite-eucrite-diogenite (HED) meteorites. Using color parameters (band tilt and band curvature) originally developed for analyzing lunar data, we have identified and mapped HED terrains on Vesta in Dawn Framing Camera (FC) color data. The average color spectrum of Vesta is identical to that of howardite regions, suggesting an extensive mixing of surface regolith due to impact gardening over the course of solar system history. Our results confirm the hemispherical dichotomy (east-west and north-south) in albedo/color/composition that has been observed by earlier studies. The presence of diogenite-rich material in the southern hemisphere suggests that it was excavated during the formation of the Rheasilvia and Veneneia basins. Our lithologic mapping of HED regions provides direct evidence for magmatic evolution of Vesta with diogenite units in Rheasilvia forming the lower crust of a differentiated object.Comment: Accepted for Meteoritics and Planetary Science special issue for Composition of Vesta/Dawn Missio

The Electron Proton Helium INstrument as an Example for a Space Weather Radiation Instrument

The near-Earth energetic particle environment has been monitored since the 1970's. With the increasing importance of quantifying the radiation risk for, e.g. for the human exploration of the Moon and Mars, it is essential to continue and further improve these measurements. The Electron Proton Helium INstrument (EPHIN) on-board SOHO continually provides these data sets to the solar science and space weather communities since 1995. Here, we introduce the numerous data products developed over the years and present space weather related applications. Important design features that have led to EPHINs success as well as lessons learned and possible improvements to the instrument are also discussed with respect to the next generation of particle detectors

Human α4β2 Nicotinic Acetylcholine Receptor As A Novel Target Of Oligomeric α-Synuclein

Cigarette smoking is associated with a decreased incidence of Parkinson disease (PD) through unknown mechanisms. Interestingly, a decrease in the numbers of α4β2 nicotinic acetylcholine receptors (α4β2-nAChRs) in PD patients suggests an α4β2-nAChR-mediated cholinergic deficit in PD. Although oligomeric forms of α-synuclein have been recognized to be toxic and involved in the pathogenesis of PD, their direct effects on nAChR-mediated cholinergic signaling remains undefined. Here, we report for the first time that oligomeric α-synuclein selectively inhibits human α4β2-nAChR-mediated currents in a dose-dependent, non-competitive and use-independent manner. We show that pre-loading cells with guanyl-5′-yl thiophosphate fails to prevent this inhibition, suggesting that the α-synuclein-induced inhibition of α4β2-nAChR function is not mediated by nAChR internalization. By using a pharmacological approach and cultures expressing transfected human nAChRs, we have shown a clear effect of oligomeric α-synuclein on α4β2-nAChRs, but not on α4β4- or α7-nAChRs, suggesting nAChR subunit selectivity of oligomeric α-synuclein-induced inhibition. In addition, by combining the size exclusion chromatography and atomic force microscopy (AFM) analyses, we find that only large (\u3e4 nm) oligomeric α-synuclein aggregates (but not monomeric, small oligomeric or fibrillar α-synuclein aggregates) exhibit the inhibitory effect on human α4β2-nAChRs. Collectively, we have provided direct evidence that α4β2-nAChR is a sensitive target to mediate oligomeric α-synuclein-induced modulation of cholinergic signaling, and our data imply that therapeutic strategies targeted toward α4β2-nAChRs may have potential for developing new treatments for PD. © 2013 Liu et al