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

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

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

Improved affinity selection using phage display technology and off-rate based selection

Flow systems such as a BIAcore biosensor can be very efficient tools to isolate high affinity antibody fragments from affinity matured phage display libraries. Here we show that using flow based selection, we can readily isolate a variant with a 35-fold higher affinity, especially with a 7 fold better off-rate, compared to the parent clone after only a single round of selection from a second generation affinity matured phage display library. The flow system represents a fast method to isolate affinity improved antibody fragments and can be particularly useful for isolating antibodies to antigens that have poor solubility, are toxic to the host cell, or prone to aggregation

Toxic Oligomeric Alpha-Synuclein Variants Present In Human Parkinson’S Disease Brains Are Differentially Generated In Mammalian Cell Models

Misfolding and aggregation of α-synuclein into toxic soluble oligomeric α-synuclein aggregates has been strongly correlated with the pathogenesis of Parkinson’s disease (PD). Here, we show that two different morphologically distinct oligomeric α-synuclein aggregates are present in human post-mortem PD brain tissue and are responsible for the bulk of α-synuclein induced toxicity in brain homogenates from PD samples. Two antibody fragments that selectively bind the different oligomeric α-synuclein variants block this α-synuclein induced toxicity and are useful tools to probe how various cell models replicate the α-synuclein aggregation pattern of human PD brain. Using these reagents, we show that mammalian cell type strongly influences α-synuclein aggregation, where neuronal cells best replicate the PD brain α-synuclein aggregation profile. Overexpression of α-synuclein in the different cell lines increased protein aggregation but did not alter the morphology of the oligomeric aggregates generated. Differentiation of the neuronal cells into a cholinergic-like or dopaminergic-like phenotype increased the levels of oligomeric α-synuclein where the aggregates were localized in cell neurites and cell bodies

The Main Belt Comets and ice in the Solar System

We review the evidence for buried ice in the asteroid belt; specifically the questions around the so-called Main Belt Comets (MBCs). We summarise the evidence for water throughout the Solar System, and describe the various methods for detecting it, including remote sensing from ultraviolet to radio wavelengths. We review progress in the first decade of study of MBCs, including observations, modelling of ice survival, and discussion on their origins. We then look at which methods will likely be most effective for further progress, including the key challenge of direct detection of (escaping) water in these bodies

Conformational Targeting of Fibrillar Polyglutamine Proteins in Live Cells Escalates Aggregation and Cytotoxicity

Misfolding- and aggregation-prone proteins underlying Parkinson's, Huntington's and Machado-Joseph diseases, namely alpha-synuclein, huntingtin, and ataxin-3 respectively, adopt numerous intracellular conformations during pathogenesis, including globular intermediates and insoluble amyloid-like fibrils. Such conformational diversity has complicated research into amyloid-associated intracellular dysfunction and neurodegeneration. To this end, recombinant single-chain Fv antibodies (scFvs) are compelling molecular tools that can be selected against specific protein conformations, and expressed inside cells as intrabodies, for investigative and therapeutic purposes.Using atomic force microscopy (AFM) and live-cell fluorescence microscopy, we report that a human scFv selected against the fibrillar form of alpha-synuclein targets isomorphic conformations of misfolded polyglutamine proteins. When expressed in the cytoplasm of striatal cells, this conformation-specific intrabody co-localizes with intracellular aggregates of misfolded ataxin-3 and a pathological fragment of huntingtin, and enhances the aggregation propensity of both disease-linked polyglutamine proteins. Using this intrabody as a tool for modulating the kinetics of amyloidogenesis, we show that escalating aggregate formation of a pathologic huntingtin fragment is not cytoprotective in striatal cells, but rather heightens oxidative stress and cell death as detected by flow cytometry. Instead, cellular protection is achieved by suppressing aggregation using a previously described intrabody that binds to the amyloidogenic N-terminus of huntingtin. Analogous cytotoxic results are observed following conformational targeting of normal or polyglutamine-expanded human ataxin-3, which partially aggregate through non-polyglutamine domains.These findings validate that the rate of aggregation modulates polyglutamine-mediated intracellular dysfunction, and caution that molecules designed to specifically hasten aggregation may be detrimental as therapies for polyglutamine disorders. Moreover, our findings introduce a novel antibody-based tool that, as a consequence of its general specificity for fibrillar conformations and its ability to function intracellularly, offers broad research potential for a variety of human amyloid diseases

OSIRIS – The scientific camera system onboard Rosetta

The Optical, Spectroscopic, and Infrared Remote Imaging System OSIRIS is the scientific camera system onboard the Rosetta spacecraft (Figure 1). The advanced high performance imaging system will be pivotal for the success of the Rosetta mission. OSIRIS will detect 67P/Churyumov-Gerasimenko from a distance of more than 106 km, characterise the comet shape and volume, its rotational state and find a suitable landing spot for Philae, the Rosetta lander. OSIRIS will observe the nucleus, its activity and surroundings down to a scale of ~2 cm px−1. The observations will begin well before the onset of cometary activity and will extend over months until the comet reaches perihelion. During the rendezvous episode of the Rosetta mission, OSIRIS will provide key information about the nature of cometary nuclei and reveal the physics of cometary activity that leads to the gas and dust coma. OSIRIS comprises a high resolution Narrow Angle Camera (NAC) unit and a Wide Angle Camera (WAC) unit accompanied by three electronics boxes. The NAC is designed to obtain high resolution images of the surface of comet 7P/Churyumov-Gerasimenko through 12 discrete filters over the wavelength range 250–1000 nm at an angular resolution of 18.6 μrad px−1. The WAC is optimised to provide images of the near-nucleus environment in 14 discrete filters at an angular resolution of 101 μrad px−1. The two units use identical shutter, filter wheel, front door, and detector systems. They are operated by a common Data Processing Unit. The OSIRIS instrument has a total mass of 35 kg and is provided by institutes from six European countrie