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Biomolecules
Volume 3
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Biomolecules, Volume 3, Issue 3 (September 2013) – 19 articles , Pages 351-732

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1349 KiB  
Article
Biophysical Characterization of α-Synuclein and Rotenone Interaction
by Blanca A. Silva, Ólöf Einarsdóttir, Anthony L. Fink and Vladimir N. Uversky
Biomolecules 2013, 3(3), 703-732; https://doi.org/10.3390/biom3030703 - 24 Sep 2013
Cited by 28 | Viewed by 9813
Abstract
Previous studies revealed that pesticides interact with α-synuclein and accelerate the rate of fibrillation. These results are consistent with the prevailing hypothesis that the direct interaction of α-synuclein with pesticides is one of many suspected factors leading to α-synuclein fibrillation and ultimately to [...] Read more.
Previous studies revealed that pesticides interact with α-synuclein and accelerate the rate of fibrillation. These results are consistent with the prevailing hypothesis that the direct interaction of α-synuclein with pesticides is one of many suspected factors leading to α-synuclein fibrillation and ultimately to Parkinson’s disease. In this study, the biophysical properties and fibrillation kinetics of α-synuclein in the presence of rotenone were investigated and, more specifically, the effects of rotenone on the early-stage misfolded forms of α-synuclein were considered. The thioflavine T (ThT) fluorescence assay studies provide evidence that early-phase misfolded α-synuclein forms are affected by rotenone and that the fibrillation process is accelerated. Further characterization by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) shows that rotenone increases the amount of ordered secondary structure in this intrinsically disordered protein. Morphological characterization by transmission electron microscopy (TEM) and atomic force microscopy (AFM) provide visualization of the differences in the aggregated α-synuclein species developing during the early kinetics of the fibrillation process in the absence and presence of rotenone. We believe that these data provide useful information for a better understanding of the molecular basis of rotenone-induced misfolding and aggregation of α-synuclein. Full article
(This article belongs to the Collection Intrinsically Disordered Proteins)
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Figure 1

Figure 1
<p>The effects of rotenone on α-synuclein fibrillation. T indicates the time during the fibrillation process at which rotenone was introduced. Chemical formula of rotenone is shown to the right.</p> Full article ">Figure 2
<p>Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) spectra of supernatant samples (left panels) and precipitate samples (right panels) taken at time of rotenone addition. Black curves are experimental signals; red curves are fits. Signals under curve represent deconvoluted experimental curve-fit signals bands used to determine structural content in a given sample and positioned at ~1635 cm<sup>−1</sup> (pink lines), ~1657 cm<sup>−1</sup> (blue lines), ~1678 cm<sup>−1</sup> (yellow lines), ~1695 cm<sup>−1</sup> (green lines).</p> Full article ">Figure 3
<p>Superimposed ATR-FTIR spectra of precipitate samples (top panels) and supernatant samples (bottom panels) taken at time of rotenone addition (black, red green, yellow and blue curves correspond to measurements taken at 0, 0.25, 0.5, 1, and 3 h, respectively).</p> Full article ">Figure 4
<p>ATR-FTIR spectra of supernatant samples (left panels) and precipitate samples (right panels) taken at the end of the fibrillation process.</p> Full article ">Figure 5
<p>Superimposed ATR-FTIR spectra of precipitate samples (top panels) and supernatant samples (bottom panels) taken at the end of the fibrillation process.</p> Full article ">Figure 6
<p>ATR-FTIR spectra of bovine serum albumin (BSA) in the presence of various rotenone equivalents after a 30-min incubation period.</p> Full article ">Figure 7
<p>Superimposed inverted BSA second derivative signal in the presence of various rotenone equivalents following a 30-min incubation period.</p> Full article ">Figure 8
<p>AFM micrographs obtained at time of rotenone addition: four images, upper left corner, 0 h; six images, upper right corner, 15 min, right; six images, middle left side, 30 min; six images, bottom left corner, 1 h; six images, bottom right corner, 3 h.</p> Full article ">Figure 9
<p>Multiple TEM micrographs, samples obtained at time of rotenone addition: 0 h; 15 min; 30 min; 1 h; 3 h; 5 h.</p> Full article ">
1451 KiB  
Review
Quantum Mechanical Modeling: A Tool for the Understanding of Enzyme Reactions
by Gábor Náray-Szabó, Julianna Oláh and Balázs Krámos
Biomolecules 2013, 3(3), 662-702; https://doi.org/10.3390/biom3030662 - 23 Sep 2013
Cited by 22 | Viewed by 12803
Abstract
Most enzyme reactions involve formation and cleavage of covalent bonds, while electrostatic effects, as well as dynamics of the active site and surrounding protein regions, may also be crucial. Accordingly, special computational methods are needed to provide an adequate description, which combine quantum [...] Read more.
Most enzyme reactions involve formation and cleavage of covalent bonds, while electrostatic effects, as well as dynamics of the active site and surrounding protein regions, may also be crucial. Accordingly, special computational methods are needed to provide an adequate description, which combine quantum mechanics for the reactive region with molecular mechanics and molecular dynamics describing the environment and dynamic effects, respectively. In this review we intend to give an overview to non-specialists on various enzyme models as well as established computational methods and describe applications to some specific cases. For the treatment of various enzyme mechanisms, special approaches are often needed to obtain results, which adequately refer to experimental data. As a result of the spectacular progress in the last two decades, most enzyme reactions can be quite precisely treated by various computational methods. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Hierarchical composition of a full enzyme system. Active site or quantum mechanical region (<b>A</b>); protein core or molecular mechanical region (<b>P</b>); bulk or dielectric continuum shell (<b>B</b>) (figure drawn on the basis of the crystal structure of human aromatase, 3EQM).</p> Full article ">Figure 2
<p>Reaction steps during serine protease catalyzed cleavage of the peptide bond (<b>left</b>). The acyl-enzyme intermediate hydrolyses via the reverse route (<b>right</b>).</p> Full article ">Figure 3
<p>The oxyanion hole stabilizing the tetrahedral intermediate in α-chymotrypsin. Backbone amide NH groups of Gly193 and Ser195 form hydrogen bonds with the amide oxygen atom of the substrate.</p> Full article ">Figure 4
<p>Reaction energy profile of the chymotrypsin-catalyzed cleavage of a model substrate (after Hudáky and Perczel [<a href="#B53-biomolecules-03-00662" class="html-bibr">53</a>]). Note that the tetrahedral intermediates as well as the acyl enzyme represent local minima.</p> Full article ">Figure 5
<p>Reaction paths for phosphoryl transfer reactions. (<b>a</b>) Dissociative (top), (<b>b</b>) associative (middle), (<b>c</b>) <span class="html-italic">S</span><sub>N</sub>2-type (bottom) mechanism. Square brackets represent the transition state.</p> Full article ">Figure 6
<p>Quantum mechanically calculated reaction profile for the phosphoryl transfer reaction catalyzed by HIV integrase (figure drawn on the basis of Ref. 56).</p> Full article ">Figure 7
<p>Molecular graphics model of the transition state in the reaction catalyzed by phoshoenol-pyruvate (PEP) mutase (on the basis of <a href="#biomolecules-03-00662-f003" class="html-fig">Figure 3</a> by Xu and Guo [<a href="#B70-biomolecules-03-00662" class="html-bibr">70</a>]). Note the planar metaphosphate intermediate stabilized by hydrogen bonds to amino-acid residues of the active site.</p> Full article ">Figure 8
<p>Two possible reaction routes for the hydrolysis of ribonuclease H. Top: schematic structure of the active site, bottom: blue line: attack by a water molecule, red line: attack by a hydroxyl group (figure drawn on the basis of <a href="#biomolecules-03-00662-f003" class="html-fig">Figure 3</a> of Ref. 72).</p> Full article ">Figure 9
<p>Schematic active-site models of cytochrome <span class="html-italic">c</span> peroxidase (CCP) (<b>left</b>) and ascorbate peroxidase (APX) (<b>right</b>). Top: distal position, bottom: proximal position, a separated red dot represents a water molecule (figure drawn on the basis of the crystal structures 1ZBZ and 2XI6).</p> Full article ">Figure 10
<p>Catalytic mechanism of the formation of Compound I. The distal His assists in removing a proton from the incoming peroxide and delivering it to the peroxide O<sub>2</sub> atom.</p> Full article ">Figure 11
<p>Singly occupied molecular orbitals of Compound I.</p> Full article ">Figure 12
<p>Thermodynamic cycle showing the relationship between ΔE<sub>1</sub>, ΔE<sub>2</sub>, (Fe-O bond enthalpy) and ΔE<sub>3</sub>.</p> Full article ">Figure 13
<p>Quantum mechanical/molecular mechanical (QM/MM) optimized snapshot of Compound I with residues hydrogen-bonded to the axial cysteinate in P450 2D6.</p> Full article ">Figure 14
<p>Metabolic routes of dextromethorphan in man as indicated by arrows.</p> Full article ">Figure 15
<p>(<b>a</b>) QM region used in the calculations (<b>b</b>) Barriers of O-demethylation and aromatic carbon oxidation obtained from quantum mechanical and QM/MM calculations.</p> Full article ">Figure 16
<p>Active site of P450 2D6 with dextromethorphan. The movement of dextromethorphan in the active site is hindered by its salt bridge to Glu216 and by the steric hindrance of the bulky amino acid side-chains.</p> Full article ">Figure 17
<p>The position of the NADH ligand in the docked structure (structure <b>A</b>) and in the crystal structure (structure <b>B</b>).</p> Full article ">Figure 18
<p>Energy profiles for closed-shell and open-shell singlet pathways for hydride transfer in the P450<sub>nor</sub> and the schematic structure of the transition state.</p> Full article ">Figure 19
<p>Catalytic mechanism of xylose isomerase<b>.</b> Top: ring opening, middle: substrate deprotonation, bottom: hydride shift.</p> Full article ">Figure 20
<p>Initial, transition and final structures in the proton transfer steps of the ring opening reaction (relative energies are given in kJ/mol).</p> Full article ">Figure 21
<p>Reaction mechanism of heme peroxidases. P is the porphyrin group of the enzyme whose heme iron is indicated, S is the substrate.</p> Full article ">Figure 22
<p>Conversion reaction catalyzed by P450<sub>nor</sub>.</p> Full article ">
398 KiB  
Article
Production of Fungal Glucoamylase for Glucose Production from Food Waste
by Wan Chi Lam, Daniel Pleissner and Carol Sze Ki Lin
Biomolecules 2013, 3(3), 651-661; https://doi.org/10.3390/biom3030651 - 19 Sep 2013
Cited by 46 | Viewed by 10199
Abstract
The feasibility of using pastry waste as resource for glucoamylase (GA) production via solid state fermentation (SSF) was studied. The crude GA extract obtained was used for glucose production from mixed food waste. Our results showed that pastry waste could be used as [...] Read more.
The feasibility of using pastry waste as resource for glucoamylase (GA) production via solid state fermentation (SSF) was studied. The crude GA extract obtained was used for glucose production from mixed food waste. Our results showed that pastry waste could be used as a sole substrate for GA production. A maximal GA activity of 76.1 ± 6.1 U/mL was obtained at Day 10. The optimal pH and reaction temperature for the crude GA extract for hydrolysis were pH 5.5 and 55 °C, respectively. Under this condition, the half-life of the GA extract was 315.0 minutes with a deactivation constant (kd) 2.20 × 10−3minutes−1. The application of the crude GA extract for mixed food waste hydrolysis and glucose production was successfully demonstrated. Approximately 53 g glucose was recovered from 100 g of mixed food waste in 1 h under the optimal digestion conditions, highlighting the potential of this approach as an alternative strategy for waste management and sustainable production of glucose applicable as carbon source in many biotechnological processes. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Glucoamylase (GA) production from pastry waste with <span class="html-italic">Aspergillus awamori</span> during solid state fermentation(SSF) for 11 days at 30°C. Experiments were duplicated. The mean values are plotted and the standard errors are reported.</p> Full article ">Figure 2
<p>Effect of (<b>A</b>) pH at 55°C and (<b>B</b>) temperature at pH 5.5 on crude GA extract activity. Experiments were duplicated. The mean values are plotted and the standard errors are reported.</p> Full article ">Figure 3
<p>Thermal deactivation of crude GA extract at (◆) 55°C, (■) 60°C and (▲) 65°C over 90 min at pH 5.5. Experiments were duplicated. The mean values are plotted and the standard errors were reported.</p> Full article ">Figure 4
<p>Hydrolysis of mixed food waste for glucose production in the presence of crude GA extract with (●) no enzyme, (◆) 7.1 U/mL, (■) 14.2 U/mL and (▲) 28.4 U/mL at pH 5.5 and 55°C for 3 h.Experiments were duplicated. The mean values are plotted and the standard errors are reported.</p> Full article ">Figure 5
<p>Material balance of the process in this study for processing 1 kg mixed food waste to produce glucose using crude GA extract produced from pastry waste based on the laboratory-scale experimental data.</p> Full article ">
704 KiB  
Review
Arming Technology in Yeast—Novel Strategy for Whole-cell Biocatalyst and Protein Engineering
by Kouichi Kuroda and Mitsuyoshi Ueda
Biomolecules 2013, 3(3), 632-650; https://doi.org/10.3390/biom3030632 - 9 Sep 2013
Cited by 50 | Viewed by 11931
Abstract
Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to [...] Read more.
Cell surface display of proteins/peptides, in contrast to the conventional intracellular expression, has many attractive features. This arming technology is especially effective when yeasts are used as a host, because eukaryotic modifications that are often required for functional use can be added to the surface-displayed proteins/peptides. A part of various cell wall or plasma membrane proteins can be genetically fused to the proteins/peptides of interest to be displayed. This technology, leading to the generation of so-called “arming technology”, can be employed for basic and applied research purposes. In this article, we describe various strategies for the construction of arming yeasts, and outline the diverse applications of this technology to industrial processes such as biofuel and chemical productions, pollutant removal, and health-related processes, including oral vaccines. In addition, arming technology is suitable for protein engineering and directed evolution through high-throughput screening that is made possible by the feature that proteins/peptides displayed on cell surface can be directly analyzed using intact cells without concentration and purification. Actually, novel proteins/peptides with improved or developed functions have been created, and development of diagnostic/therapeutic antibodies are likely to benefit from this powerful approach. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Figure 1

Figure 1
<p>Cell surface display system in <span class="html-italic">S. cerevisiae</span> (<b>a</b>) α-agglutinin-based display system; (<b>b</b>) <b>a</b>-agglutinin-based display system; (<b>c</b>) Flo1p-based display system; (<b>d</b>) Membrane display system by anchoring domain of Yps1p.</p> Full article ">Figure 2
<p>Whole-cell biocatalyst constructed by arming technology and their applications.</p> Full article ">Figure 3
<p>Creation of novel proteins/peptides with improved or developed function by arming technology.</p> Full article ">
294 KiB  
Review
Fungal Beta-Glucosidases: A Bottleneck in Industrial Use of Lignocellulosic Materials
by Annette Sørensen, Mette Lübeck, Peter S. Lübeck and Birgitte K. Ahring
Biomolecules 2013, 3(3), 612-631; https://doi.org/10.3390/biom3030612 - 3 Sep 2013
Cited by 210 | Viewed by 13795
Abstract
Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, beta-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose [...] Read more.
Profitable biomass conversion processes are highly dependent on the use of efficient enzymes for lignocellulose degradation. Among the cellulose degrading enzymes, beta-glucosidases are essential for efficient hydrolysis of cellulosic biomass as they relieve the inhibition of the cellobiohydrolases and endoglucanases by reducing cellobiose accumulation. In this review, we discuss the important role beta-glucosidases play in complex biomass hydrolysis and how they create a bottleneck in industrial use of lignocellulosic materials. An efficient beta-glucosidase facilitates hydrolysis at specified process conditions, and key points to consider in this respect are hydrolysis rate, inhibitors, and stability. Product inhibition impairing yields, thermal inactivation of enzymes, and the high cost of enzyme production are the main obstacles to commercial cellulose hydrolysis. Therefore, this sets the stage in the search for better alternatives to the currently available enzyme preparations either by improving known or screening for new beta-glucosidases. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
173 KiB  
Review
Microbial Enzymes with Special Characteristics for Biotechnological Applications
by Poonam Singh Nigam
Biomolecules 2013, 3(3), 597-611; https://doi.org/10.3390/biom3030597 - 23 Aug 2013
Cited by 262 | Viewed by 20084
Abstract
This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes [...] Read more.
This article overviews the enzymes produced by microorganisms, which have been extensively studied worldwide for their isolation, purification and characterization of their specific properties. Researchers have isolated specific microorganisms from extreme sources under extreme culture conditions, with the objective that such isolated microbes would possess the capability to bio-synthesize special enzymes. Various Bio-industries require enzymes possessing special characteristics for their applications in processing of substrates and raw materials. The microbial enzymes act as bio-catalysts to perform reactions in bio-processes in an economical and environmentally-friendly way as opposed to the use of chemical catalysts. The special characteristics of enzymes are exploited for their commercial interest and industrial applications, which include: thermotolerance, thermophilic nature, tolerance to a varied range of pH, stability of enzyme activity over a range of temperature and pH, and other harsh reaction conditions. Such enzymes have proven their utility in bio-industries such as food, leather, textiles, animal feed, and in bio-conversions and bio-remediations. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
344 KiB  
Review
Decarboxylation of Pyruvate to Acetaldehyde for Ethanol Production by Hyperthermophiles
by Mohammad S. Eram and Kesen Ma
Biomolecules 2013, 3(3), 578-596; https://doi.org/10.3390/biom3030578 - 21 Aug 2013
Cited by 44 | Viewed by 18606
Abstract
Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The [...] Read more.
Pyruvate decarboxylase (PDC encoded by pdc) is a thiamine pyrophosphate (TPP)-containing enzyme responsible for the conversion of pyruvate to acetaldehyde in many mesophilic organisms. However, no pdc/PDC homolog has yet been found in fully sequenced genomes and proteomes of hyper/thermophiles. The only PDC activity reported in hyperthermophiles was a bifunctional, TPP- and CoA-dependent pyruvate ferredoxin oxidoreductase (POR)/PDC enzyme from the hyperthermophilic archaeon Pyrococcus furiosus. Another enzyme known to be involved in catalysis of acetaldehyde production from pyruvate is CoA-acetylating acetaldehyde dehydrogenase (AcDH encoded by mhpF and adhE). Pyruvate is oxidized into acetyl-CoA by either POR or pyruvate formate lyase (PFL), and AcDH catalyzes the reduction of acetyl-CoA to acetaldehyde in mesophilic organisms. AcDH is present in some mesophilic (such as clostridia) and thermophilic bacteria (e.g., Geobacillus and Thermoanaerobacter). However, no AcDH gene or protein homologs could be found in the released genomes and proteomes of hyperthermophiles. Moreover, no such activity was detectable from the cell-free extracts of different hyperthermophiles under different assay conditions. In conclusion, no commonly-known PDCs was found in hyperthermophiles. Instead of the commonly-known PDC, it appears that at least one multifunctional enzyme is responsible for catalyzing the non-oxidative decarboxylation of pyruvate to acetaldehyde in hyperthermophiles. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Figure 1

Figure 1
<p>Two pathways of ethanol production from pyruvate. POR; Pyruvate ferredoxin oxidoreductase; PFL; Pyruvate formate lyase, AcDH; Acetaldehyde dehydrogenase, ADH; Alcohol dehydrogenase, PDC; pyruvate decarboxylase; CoASH; coenzyme A, Fd<sub>ox</sub>; oxidized ferredoxin, Fd<sub>red</sub>; reduced ferredoxin.</p> Full article ">
606 KiB  
Article
A Sensitive DNA Enzyme-Based Fluorescent Assay for Bacterial Detection
by Sergio D. Aguirre, M. Monsur Ali, Bruno J. Salena and Yingfu Li
Biomolecules 2013, 3(3), 563-577; https://doi.org/10.3390/biom3030563 - 20 Aug 2013
Cited by 60 | Viewed by 9673
Abstract
Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel “mix-and-read” assay where [...] Read more.
Bacterial detection plays an important role in protecting public health and safety, and thus, substantial research efforts have been directed at developing bacterial sensing methods that are sensitive, specific, inexpensive, and easy to use. We have recently reported a novel “mix-and-read” assay where a fluorogenic DNAzyme probe was used to detect model bacterium E. coli. In this work, we carried out a series of optimization experiments in order to improve the performance of this assay. The optimized assay can achieve a detection limit of 1000 colony-forming units (CFU) without a culturing step and is able to detect 1 CFU following as short as 4 h of bacterial culturing in a growth medium. Overall, our effort has led to the development of a highly sensitive and easy-to-use fluorescent bacterial detection assay that employs a catalytic DNA. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Design of <span class="html-italic">trans</span>-acting DNAzymes. (<b>A</b>) The sequences of EC1, EC1T, EC1TM and FS1. EC1 is the full length DNAzyme including two primer binding sites (nucleotides in italic) for polymerase chain reaction used in the original <span class="html-italic">in vitro</span> selection experiment. EC1T is the shortened version of EC1 with deleted primer binding sites. EC1TM is a mutant of EC1T wherein the nucleotides shown as lower-case letters are altered. The substrate FS1 contains an adenosine ribonucleotide (R) flanked by a fluorescein-dT (F) and a DABCYL-dT (Q). (<b>B</b>) dPAGE analysis of the cleavage reaction mixtures of FS1 with EC1, EC1T, or EC1TM in the absence (−) and presence (+) of CEM-EC. P1 represents the 5’-cleavage product, which can be observed by fluorescence scan as it contains the F unit. MK (marker) is a sample of FS1 fully cleaved by NaOH. Clv% for each sample was calculated following our previously reported method [<a href="#B20-biomolecules-03-00563" class="html-bibr">20</a>].</p> Full article ">Figure 2
<p>Cleavage reactions of EC1T/FS1 with (<b>A</b>) crude extracellular mixture (CEM)-EC and CIM-EC and (<b>B</b>) crude intracellular mixture (CIM)-EC collected from <span class="html-italic">E. coli</span> cells grown in various culture broths. NC is a negative control where the reaction was conducted in the absence of CEM-EC and CIM-EC. Each reaction mixture was analyzed by 10% dPAGE, followed by fluorimaging. NC: negative control where the reaction was conducted in RB without CEM-EC or CIM-EC.</p> Full article ">Figure 3
<p>(<b>A</b>) Cleavage activity of EC1T/FS1 in the presence of CEM-EC and various divalent metal ions. (<b>B</b>) Effect of the Ba<sup>2+</sup> concentration.</p> Full article ">Figure 4
<p>Cleavage activity of EC1T/FS1 with varying temperature (<b>A</b>), pH (<b>B</b>), and EC1T/FS1 ratio (<b>C</b>). The data are the average of two independent experiments.</p> Full article ">Figure 5
<p>Specificity of EC1T/FS1 for various gram-negative and gram-positive bacteria. PP: <span class="html-italic">Pseudomonas peli</span>, YR: <span class="html-italic">Yersinia rukeri</span>, HA: <span class="html-italic">Hafnea alvei</span>, AX: <span class="html-italic">Achromobacter xylosoxidans</span>, EC: <span class="html-italic">Escherichia coli</span>, BS: <span class="html-italic">Bacillus subtilis</span>, LM: <span class="html-italic">Leuconostoc mesenteroides</span>, LP: <span class="html-italic">Lactobacillus planturum</span>, PA: <span class="html-italic">Pediococcus acidilactici</span>.</p> Full article ">Figure 6
<p>Sensitivity test. (<b>A</b>) Real-time fluorescence monitoring and (<b>B</b>) dPAGE analysis of EC1T/FS1 in the presence of CIMs prepared from 10<sup>3</sup>–10<sup>7</sup> <span class="html-italic">E. coli</span> cells. (<b>C</b>) and (<b>D</b>) Similar experiments using RNA-cleaving fluorogenic DNAzyme (RFD-EC1) with CIMs prepared from 10<sup>2</sup>–10<sup>7</sup> <span class="html-italic">E. coli</span> cells. The data in (A) and (C) are the average of two independent experiments.</p> Full article ">Figure 7
<p>Culturing time required to detect a single <span class="html-italic">E. coli</span> cell (1 CFU). (<b>A</b>) Monitoring fluorescence of EC1T/FS1 with CIMs prepared from samples taken after a culturing time of 2, 4, 6, 8 and 10 h. (<b>B</b>) dPAGE analysis of the reaction mixtures in (A). (<b>C</b>) and (<b>D</b>) are equivalent experiments in which RFD-EC1 was used to replace EC1T/FS1. The data in (A) and (C) are the average of two independent experiments.</p> Full article ">
139 KiB  
Review
Carbonic Anhydrases and Their Biotechnological Applications
by Christopher D. Boone, Andrew Habibzadegan, Sonika Gill and Robert McKenna
Biomolecules 2013, 3(3), 553-562; https://doi.org/10.3390/biom3030553 - 19 Aug 2013
Cited by 38 | Viewed by 7663
Abstract
The carbonic anhydrases (CAs) are mostly zinc-containing metalloenzymes which catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. The CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. In particular, human CA [...] Read more.
The carbonic anhydrases (CAs) are mostly zinc-containing metalloenzymes which catalyze the reversible hydration/dehydration of carbon dioxide/bicarbonate. The CAs have been extensively studied because of their broad physiological importance in all kingdoms of life and clinical relevance as drug targets. In particular, human CA isoform II (HCA II) has a catalytic efficiency of 108 M−1 s−1, approaching the diffusion limit. The high catalytic rate, relatively simple procedure of expression and purification, relative stability and extensive biophysical studies of HCA II has made it an exciting candidate to be incorporated into various biomedical applications such as artificial lungs, biosensors and CO2 sequestration systems, among others. This review highlights the current state of these applications, lists their advantages and limitations, and discusses their future development. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
511 KiB  
Article
Pyranose Dehydrogenase from Agaricus campestris and Agaricus xanthoderma: Characterization and Applications in Carbohydrate Conversions
by Petra Staudigl, Iris Krondorfer, Dietmar Haltrich and Clemens K. Peterbauer
Biomolecules 2013, 3(3), 535-552; https://doi.org/10.3390/biom3030535 - 16 Aug 2013
Cited by 13 | Viewed by 7758
Abstract
Pyranose dehydrogenase (PDH) is a flavin-dependent sugar oxidoreductase that is limited to a rather small group of litter-degrading basidiomycetes. The enzyme is unable to utilize oxygen as an electron acceptor, using substituted benzoquinones and (organo) metal ions instead. PDH displays a broad substrate [...] Read more.
Pyranose dehydrogenase (PDH) is a flavin-dependent sugar oxidoreductase that is limited to a rather small group of litter-degrading basidiomycetes. The enzyme is unable to utilize oxygen as an electron acceptor, using substituted benzoquinones and (organo) metal ions instead. PDH displays a broad substrate specificity and intriguing variations in regioselectivity, depending on substrate, enzyme source and reaction conditions. In contrast to the related enzyme pyranose 2-oxidase (POx), PDHs from several sources are capable of oxidizing α- or β-1→4-linked di- and oligosaccharides, including lactose. PDH from A. xanthoderma is able to perform C-1 and C-2 oxidation, producing, in addition to lactobionic acid, 2-dehydrolactose, an intermediate for the production of lactulose, whereas PDH from A. campestris oxidizes lactose nearly exclusively at the C-1 position. In this work, we present the isolation of PDH-encoding genes from A. campestris (Ac) and A. xanthoderma (Ax) and a comparison of other so far isolated PDH-sequences. Secretory overexpression of both enzymes in Pichia pastoris was successful when using their native signal sequences with yields of 371 U·L−1 for AxPDH and 35 U·L−1 for AcPDH. The pure enzymes were characterized biochemically and tested for applications in carbohydrate conversion reactions of industrial relevance. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Full article ">Figure 1
<p>Large scale production of pyranose dehydrogenases (PDHs) in <span class="html-italic">P. pastoris</span>. Black circles, wet biomass; grey triangles, extracellular protein concentration; black squares, volumetric activity.</p> Full article ">Figure 2
<p>SDS-PAGE of purified PDHs. M, molecular marker; 1, AcPDH; 2, AcPDH deglycosylated; 3, Ax PDH; 4, AxPDH deglycosylated.</p> Full article ">Figure 3
<p>Native PAGE of purified PDHs. M, molecular marker; 1, AcPDH; 2, AxPDH.</p> Full article ">Figure 4
<p>pH optima of AcPDH (black squares) and AxPDH (grey triangles) with the electron acceptors ferrocenium hexafluorophosphate and 1,4-benzoquinone; D-glucose as electron donor.</p> Full article ">Figure 5
<p>(<b>a</b>) HPLC analysis of lactose conversion by AcPDH; (<b>b</b>) and AxPDH at 0 h (A), 1 h (B), 3 h (C) and 7 h (D) incubation. Peaks: I, residual salt from enzyme preparation; II, lactobionic acid; III, lactose; IV, 2-dehydrolactose.</p> Full article ">
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Article
Lipases Immobilization for Effective Synthesis of Biodiesel Starting from Coffee Waste Oils
by Valerio Ferrario, Harumi Veny, Elisabetta De Angelis, Luciano Navarini, Cynthia Ebert and Lucia Gardossi
Biomolecules 2013, 3(3), 514-534; https://doi.org/10.3390/biom3030514 - 13 Aug 2013
Cited by 24 | Viewed by 8757
Abstract
Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior [...] Read more.
Immobilized lipases were applied to the enzymatic conversion of oils from spent coffee ground into biodiesel. Two lipases were selected for the study because of their conformational behavior analysed by Molecular Dynamics (MD) simulations taking into account that immobilization conditions affect conformational behavior of the lipases and ultimately, their efficiency upon immobilization. The enzymatic synthesis of biodiesel was initially carried out on a model substrate (triolein) in order to select the most promising immobilized biocatalysts. The results indicate that oils can be converted quantitatively within hours. The role of the nature of the immobilization support emerged as a key factor affecting reaction rate, most probably because of partition and mass transfer barriers occurring with hydrophilic solid supports. Finally, oil from spent coffee ground was transformed into biodiesel with yields ranging from 55% to 72%. The synthesis is of particular interest in the perspective of developing sustainable processes for the production of bio-fuels from food wastes and renewable materials. The enzymatic synthesis of biodiesel is carried out under mild conditions, with stoichiometric amounts of substrates (oil and methanol) and the removal of free fatty acids is not required. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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<p>Tridimensional models of CaLB (<b>a</b>) (PDB code 1TCA) and PcL (<b>b</b>) (PDB code 1YS1). The structures are colored according to their secondary structures; lids are highlighted in red. PcL (<b>b</b>) has the second “putative” lid highlighted in green and the Ca<sup>2+</sup> ion represented as orange sphere.</p> Full article ">Figure 2
<p>GRID analysis of hydrophobic (yellow) and hydrophilic (blue) surface of PcL. On the left: open conformation corresponding to the crystal structure obtained in the presence of an inhibitor (PDB code 1YS1). On the right: partially closed conformation computed after 20 ns MD simulation in explicit water.</p> Full article ">Figure 3
<p>Molecular dynamic (MD) simulation of PcL embedded in an explicit bi-phasic system octane-water. The hydrophobic side of the enzyme, corresponding to the opening of the active site, is highlighted in yellow beads. The octane phase is in grey points in the figure. At the starting point of the simulation (<b>a</b>) the water-octane interface is not completely defined; during the simulation the interface was formed (<b>b</b>) and the enzyme is oriented with its active site towards the octane part.</p> Full article ">Figure 4
<p>Effect of methanol concentration on CaL-S (star) and CaL-M (square) expressed as the conversion in methyl ester achieved after 25 h. The transesterification of triolein was carried out at 30 °C with molar ratios of 1:1, 2:1 and 3:1. Therefore, theoretical maximum conversions achievable were 33%, 66% and 100%, respectively.</p> Full article ">Figure 5
<p>Reaction profiles (24 h) of transesterification of triolein using a 1:1 molar ratio of oil and methanol (30 °C). CaL-S (star) and CaL-M (square). The maximum theoretical yield is 33%.</p> Full article ">Figure 6
<p>The appearance of the reaction system at the end of the methanolysis using the three preparations of CaLB considered in the study.</p> Full article ">Figure 7
<p>Effect of methanol concentration on CaL-S evaluated at 30 °C by monitoring the transesterification of triolein at different methanol/oil molar ratio.</p> Full article ">Figure 8
<p>Recyclability of CalB immobilized on styrenic support expressed as the percentage of methyl oleate formed after 4 h at 30 °C in the transesterification of triolein with methanol (1:1 molar ratio).</p> Full article ">Figure 9
<p>H<sup>1</sup>-NMR spectra of the oil from espresso spent coffee ground (<b>a</b>) and of the reaction mixture after 5 h (<b>b</b>) and 24 h (<b>c</b>).</p> Full article ">
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Review
The Impact of Sphingosine Kinase-1 in Head and Neck Cancer
by Paulette M. Tamashiro, Hideki Furuya, Yoshiko Shimizu, Kayoko Iino and Toshihiko Kawamori
Biomolecules 2013, 3(3), 481-513; https://doi.org/10.3390/biom3030481 - 12 Aug 2013
Cited by 20 | Viewed by 8369
Abstract
Head and neck squamous cell carcinoma (HNSCC) has a high reoccurrence rate and an extremely low survival rate. There is limited availability of effective therapies to reduce the rate of recurrence, resulting in high morbidity and mortality of advanced cases. Late presentation, delay [...] Read more.
Head and neck squamous cell carcinoma (HNSCC) has a high reoccurrence rate and an extremely low survival rate. There is limited availability of effective therapies to reduce the rate of recurrence, resulting in high morbidity and mortality of advanced cases. Late presentation, delay in detection of lesions, and a high rate of metastasis make HNSCC a devastating disease. This review offers insight into the role of sphingosine kinase-1 (SphK1), a key enzyme in sphingolipid metabolism, in HNSCC. Sphingolipids not only play a structural role in cellular membranes, but also modulate cell signal transduction pathways to influence biological outcomes such as senescence, differentiation, apoptosis, migration, proliferation, and angiogenesis. SphK1 is a critical regulator of the delicate balance between proliferation and apoptosis. The highest expression of SphK1 is found in the advanced stage of disease, and there is a positive correlation between SphK1 expression and recurrent tumors. On the other hand, silencing SphK1 reduces HNSCC tumor growth and sensitizes tumors to radiation-induced death. Thus, SphK1 plays an important and influential role in determining HNSCC proliferation and metastasis. We discuss roles of SphK1 and other sphingolipids in HNSCC development and therapeutic strategies against HNSCC. Full article
(This article belongs to the Special Issue Sphingolipids and Bioactive Lipids)
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<p>Suggested roles of SphK1 in HNSCC. EP: E-prostanoid receptor; TKR: tyrosine kinase receptor.</p> Full article ">Figure 2
<p>Overview of sphingolipid enzymes and metabolites and their influence on HNSCC. (<b>a</b>) <b>S</b>phK1 is positively associated with increased lymph node (LN) metastasis [<a href="#B32-biomolecules-03-00481" class="html-bibr">32</a>], proliferation [<a href="#B11-biomolecules-03-00481" class="html-bibr">11</a>] and resistance to radiation-induced cell death. (<b>b</b>) Perturbation of specific sphingolipid players affects invasion, proliferation and drug resistance in HNSCC. Abbreviations: SPPase (sphingosine phosphate phosphatase), GCS (glucosyl ceramidase), GCase (glucosyl ceramidase), CDase (ceramidase), CerS (ceramide synthase), C1P (ceramide 1-phosphate), SM (sphingomyelin), ER (endoplasmic reticulum).</p> Full article ">
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Article
Enzyme-Catalyzed Synthesis of Unsaturated Aliphatic Polyesters Based on Green Monomers from Renewable Resources
by Yi Jiang, Albert J.J. Woortman, Gert O.R. Alberda Van Ekenstein and Katja Loos
Biomolecules 2013, 3(3), 461-480; https://doi.org/10.3390/biom3030461 - 12 Aug 2013
Cited by 88 | Viewed by 15371
Abstract
Bio-based commercially available succinate, itaconate and 1,4-butanediol are enzymatically co-polymerized in solution via a two-stage method, using Candida antarctica Lipase B (CALB, in immobilized form as Novozyme® 435) as the biocatalyst. The chemical structures of the obtained products, poly(butylene succinate) (PBS) and poly(butylene [...] Read more.
Bio-based commercially available succinate, itaconate and 1,4-butanediol are enzymatically co-polymerized in solution via a two-stage method, using Candida antarctica Lipase B (CALB, in immobilized form as Novozyme® 435) as the biocatalyst. The chemical structures of the obtained products, poly(butylene succinate) (PBS) and poly(butylene succinate-co-itaconate) (PBSI), are confirmed by 1H- and 13C-NMR. The effects of the reaction conditions on the CALB-catalyzed synthesis of PBSI are fully investigated, and the optimal polymerization conditions are obtained. With the established method, PBSI with tunable compositions and satisfying reaction yields is produced. The 1H-NMR results confirm that carbon-carbon double bonds are well preserved in PBSI. The differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA) results indicate that the amount of itaconate in the co-polyesters has no obvious effects on the glass-transition temperature and the thermal stability of PBS and PBSI, but has significant effects on the melting temperature. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Full article ">Figure 1
<p><span class="html-italic">Candida antarctica</span> Lipase B (CALB)-catalyzed co-polymerization of succinate, itaconate and 1,4-butanediol.</p> Full article ">Figure 2
<p>The effect of diphenyl ether dosage on CALB-catalyzed synthesis of PBSI.</p> Full article ">Figure 3
<p><span class="html-italic">In situ</span> <sup>1</sup>H-NMR investigation of the oligomerization process of: (<b>a</b>) diethyl succinate (35%), dimethyl itaconate (15%) and 1,4-butanediol (50%); (<b>b</b>) diethyl succinate (25%), dimethyl itaconate (25%) and 1,4-butanediol (50%).</p> Full article ">Figure 4
<p>The effect of itaconate structure on CALB-catalyzed synthesis of PBSI: (<b>a</b>) the reaction yield of PBSI as a function of the feed ratio of itaconate; (<b>b</b>) the mole percentage of itaconate in PBSI (X<sub>I</sub>) as a function of the feed ratio of itaconate.</p> Full article ">Figure 5
<p>(<b>a</b>) The mole percentage of itaconate (X<sub>I</sub>) and the reaction yield of PBSI as a function of the feed ratio of dimethyl itaconate (F<sub>I</sub>); (<b>b</b>) Overlay of gel permeation chromatography (GPC) elution curves in chloroform; PBSI was synthesized <span class="html-italic">via</span> the optimal conditions.</p> Full article ">Figure 6
<p><sup>1</sup>H-NMR spectra of dimethyl itaconate, PB<sub>50</sub>S<sub>35</sub>I<sub>15</sub> and PB<sub>50</sub>S<sub>25</sub>I<sub>25</sub> in CDCl<sub>3</sub>.</p> Full article ">Figure 7
<p>Possible types of microstructures in PBSI, the formation sequence from easy to difficult: S-B-S &lt; S-B-I-1 &lt; S-B-I-2 &lt; I-B-I-1 &lt; I-B-I-2 &lt; I-B-I-3.</p> Full article ">Figure 8
<p>(<b>a</b>) the T<sub>g</sub> and T<sub>d</sub> of PBSI as a function of the mole percentage of itaconate (X<sub>I</sub>); (<b>b</b>) the T<sub>m</sub> of PBSI as a function of the mole percentage of itaconate (X<sub>I</sub>); PBSI was synthesized <span class="html-italic">via</span> the optimal conditions.</p> Full article ">Figure 9
<p>(<b>a</b>) <sup>1</sup>H-; (<b>b</b>) <sup>13</sup>C-NMR spectra of PBS and PBSI.</p> Full article ">Figure 10
<p>Calculation of the molar composition of PBSI from <sup>1</sup>H-NMR spectra.</p> Full article ">Figure 11
<p>Calculation of the M<sub>n</sub> from <sup>1</sup>H-NMR spectra.</p> Full article ">
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Article
Enantiocomplementary Yarrowia lipolytica Oxidoreductases: Alcohol Dehydrogenase 2 and Short Chain Dehydrogenase/Reductase
by Kamila Napora-Wijata, Gernot A. Strohmeier, Manoj N. Sonavane, Manuela Avi, Karen Robins and Margit Winkler
Biomolecules 2013, 3(3), 449-460; https://doi.org/10.3390/biom3030449 - 12 Aug 2013
Cited by 7 | Viewed by 8830
Abstract
Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The [...] Read more.
Enzymes of the non-conventional yeast Yarrowia lipolytica seem to be tailor-made for the conversion of lipophilic substrates. Herein, we cloned and overexpressed the Zn-dependent alcohol dehydrogenase ADH2 from Yarrowia lipolytica in Escherichia coli. The purified enzyme was characterized in vitro. The substrate scope for YlADH2 mediated oxidation and reduction was investigated spectrophotometrically and the enzyme showed a broader substrate range than its homolog from Saccharomyces cerevisiae. A preference for secondary compared to primary alcohols in oxidation direction was observed for YlADH2. 2-Octanone was investigated in reduction mode in detail. Remarkably, YlADH2 displays perfect (S)-selectivity and together with a highly (R)-selective short chain dehydrogenase/ reductase from Yarrowia lipolytica it is possible to access both enantiomers of 2-octanol in >99% ee with Yarrowia lipolytica oxidoreductases. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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<p>pH optimum of <span class="html-italic">Yl</span>ADH2 catalyzed oxidation of (<span class="html-italic">S</span>)-2-octanol. ♦: citrate; ■: potassium phosphate; ▲: Tris-HCl; ▬: borate; X: glycine; ●: carbonate.</p> Full article ">Figure 2
<p>pH optimum of <span class="html-italic">Yl</span>ADH2 catalyzed reduction of 2-octanone. ♦: citrate; ■: potassium phosphate; ▲: Tris-HCl.</p> Full article ">Figure 3
<p>Time dependent formation of ♦ (<span class="html-italic">S</span>)-2-octanol (&lt;99% <span class="html-italic">ee</span>) catalyzed by <span class="html-italic">Yl</span>ADH2 and ■ (<span class="html-italic">R</span>)-2-octanol (&lt;99% <span class="html-italic">ee</span>) catalyzed by <span class="html-italic">Yl</span>SDR.</p> Full article ">Figure 4
<p>Routes to enantiomerically pure (<span class="html-italic">S</span>)- and (<span class="html-italic">R</span>)-2-octanol via <span class="html-italic">Yarrowia lipolytica</span> oxidoreductases.</p> Full article ">
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Article
Altered Sphingolipid Metabolism in Patients with Metastatic Pancreatic Cancer
by Yixing Jiang, Nicole A. DiVittore, Megan M Young, Zhiliang Jia, Keping Xie, Timothy M. Ritty, Mark Kester and Todd E. Fox
Biomolecules 2013, 3(3), 435-448; https://doi.org/10.3390/biom3030435 - 25 Jul 2013
Cited by 42 | Viewed by 7803
Abstract
Although numerous genetic mutations and amplifications have been identified in pancreatic cancer, much of the molecular pathogenesis of the disease remains undefined. While proteomic and transcriptomic analyses have been utilized to probe and characterize pancreatic tumors, lipidomic analyses have not been applied to [...] Read more.
Although numerous genetic mutations and amplifications have been identified in pancreatic cancer, much of the molecular pathogenesis of the disease remains undefined. While proteomic and transcriptomic analyses have been utilized to probe and characterize pancreatic tumors, lipidomic analyses have not been applied to identify perturbations in pancreatic cancer patient samples. Thus, we utilized a mass spectrometry-based lipidomic approach, focused towards the sphingolipid class of lipids, to quantify changes in human pancreatic cancer tumor and plasma specimens. Subgroup analysis revealed that patients with positive lymph node metastasis have a markedly higher level of ceramide species (C16:0 and C24:1) in their tumor specimens compared to pancreatic cancer patients without nodal disease or to patients with pancreatitis. Also of interest, ceramide metabolites, including phosphorylated (sphingosine- and sphinganine-1-phosphate) and glycosylated (cerebroside) species were elevated in the plasma, but not the pancreas, of pancreatic cancer patients with nodal disease. Analysis of plasma level of cytokine and growth factors revealed that IL-6, IL-8, CCL11 (eotaxin), EGF and IP10 (interferon inducible protein 10, CXCL10) were elevated in patients with positive lymph nodes metastasis, but that only IP10 and EGF directly correlated with several sphingolipid changes. Taken together, these data indicate that sphingolipid metabolism is altered in human pancreatic cancer and associated with advanced disease. Assessing plasma and/or tissue sphingolipids could potentially risk stratify patients in the clinical setting. Full article
(This article belongs to the Special Issue Sphingolipids and Bioactive Lipids)
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<p>Altered ceramide levels in patient specimens with pancreatic cancer. LC-MS/MS was utilized to quantify ceramides from patient specimens. Ceramides with a d18:1 backbone with fatty acids from 14 to 26 carbons were assessed. Molecular species of ceramides were quantified from pancreas tissues (<b>A</b>); and corresponding plasma samples (<b>B</b>) from patients with pancreatitis, non-metastatic (nodal negative) pancreatic cancer, or metastatic (nodal positive) pancreatic cancer. <b>*</b> = <span class="html-italic">p &lt;</span> 0.05 assessed by <span class="html-italic">t</span>-test analysis.</p> Full article ">Figure 2
<p>Altered cerebroside levels in patient specimens with pancreatic cancer. LC-MS/MS was utilized to quantify cerebrosides from patient specimens. Cerobrosides with a d18:1 backbone with fatty acids from 14 to 26 carbons were assessed. Molecular species of cerebrosides were quantified from pancreas tissues (<b>A</b>); and corresponding plasma samples (<b>B</b>) from patients with pancreatitis, non-metastatic (nodal negative) pancreatic cancer, or metastatic (nodal positive) pancreatic cancer. <b>*</b> <span class="html-italic">p &lt;</span> 0.05 assessed by <span class="html-italic">t</span>-test analysis.</p> Full article ">Figure 3
<p>Sphingomyelin levels in patient specimens with pancreatic cancer. LC-MS/MS was utilized to quantify sphingomyelin levels from patient specimens. Sphingomyelins with a d18:1 backbone with fatty acids from 14 to 26 carbons were assessed. Molecular species of sphingomyelins were quantified from pancreas tissues (<b>A</b>) and corresponding plasma samples (<b>B</b>) from patients with pancreatitis, non-metastatic (nodal negative) pancreatic cancer, or metastatic (nodal positive) pancreatic cancer. <b>*</b> <span class="html-italic">p &lt;</span> 0.05 assessed by <span class="html-italic">t</span>-test analysis.</p> Full article ">Figure 4
<p>Altered plasma phosphorylated long chain sphingoid bases in patient samples with pancreatic cancer. LC-MS/MS was utilized to quantify perturbation in the long chain bases from plasma. Graphical representations of the data for (<b>A</b>) sphingosine; (<b>B</b>) sphinganine; (<b>C</b>) sphingosine-1-phosphate; and (<b>D</b>) sphinganine-1-phosphate are shown from the plasma from patients with pancreatitis, nodal negative pancreatic cancer or nodal positive pancreatic cancer. <b>*</b> <span class="html-italic">p &lt;</span> 0.05 assessed by t-test analysis.</p> Full article ">Figure 5
<p>Correlation between cytokines and sphingolipid alterations in patient specimens with pancreatic cancer. To determine if a statistical relationship exists between cytokines and sphingolipids in patient specimens, Pearson correlation analysis was performed. (<b>A</b>) Tissue C16-ceramide and plasma IP-10 levels (<span class="html-italic">r =</span> 0.583, <span class="html-italic">p =</span> 0.0035); and (<b>B</b>) tissue C24:1-ceramide and plasma IP-10 levels from tissue demonstrate a positive correlation (<span class="html-italic">r =</span> 0.6012, <span class="html-italic">p =</span> 0.0024); (<b>C</b>) as did plasma C16:0 and C24:1 cerebroside mass (<span class="html-italic">r =</span> 0.6174, <span class="html-italic">p =</span> 0.0013), with plasma IP10; and (<b>D</b>) plasma S1P with IP10 (<span class="html-italic">r</span> = 0.4655, <span class="html-italic">p</span> = 0.019). Plasma EGF levels demonstrated positive correlated with both plasma; (<b>E</b>) S1P (<span class="html-italic">r =</span> 0.6928, <span class="html-italic">p =</span> 0.0001) and (<b>F</b>) dhS1P (<span class="html-italic">r =</span> 0.6787, <span class="html-italic">p =</span> 0.0002) levels.</p> Full article ">Figure 6
<p>IP10 expression in pancreatic tumor specimens. IP 10 immunohistochemistry staining shows IP10 expression was down-regulated in tumor tissue (<b>B</b>) compared with non-cancerous, tissue (<b>A</b>)<b>.</b> (<b>C</b>) Fisher’s exact test shows the quantitative staining intensity difference between tumor and normal tissue is significant (<span class="html-italic">p</span> &lt; 0.01).</p> Full article ">
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Review
Emerging Role of Sphingosine-1-phosphate in Inflammation, Cancer, and Lymphangiogenesis
by Wei-Ching Huang, Masayuki Nagahashi, Krista P. Terracina and Kazuaki Takabe
Biomolecules 2013, 3(3), 408-434; https://doi.org/10.3390/biom3030408 - 23 Jul 2013
Cited by 61 | Viewed by 10817
Abstract
The main function of the lymphatic system is to control and maintain fluid homeostasis, lipid transport, and immune cell trafficking. In recent years, the pathological roles of lymphangiogenesis, the generation of new lymphatic vessels from preexisting ones, in inflammatory diseases and cancer progression [...] Read more.
The main function of the lymphatic system is to control and maintain fluid homeostasis, lipid transport, and immune cell trafficking. In recent years, the pathological roles of lymphangiogenesis, the generation of new lymphatic vessels from preexisting ones, in inflammatory diseases and cancer progression are beginning to be elucidated. Sphingosine-1-phosphate (S1P), a bioactive lipid, mediates multiple cellular events, such as cell proliferation, differentiation, and trafficking, and is now known as an important mediator of inflammation and cancer. In this review, we will discuss recent findings showing the emerging role of S1P in lymphangiogenesis, in inflammation, and in cancer. Full article
(This article belongs to the Special Issue Sphingolipids and Bioactive Lipids)
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<p>Sphingosine-1-phosphate (S1P) in cancer-induced lymphangiogenesis.</p> Full article ">Figure 2
<p>S1P and sprouting angiogenesis in normal development and in tumor-induced angiogenesis.</p> Full article ">
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Article
Regulation of Cytoskeleton Organization by Sphingosine in a Mouse Cell Model of Progressive Ovarian Cancer
by Amy L. Creekmore, C. Lynn Heffron, Bradley P. Brayfield, Paul C. Roberts and Eva M. Schmelz
Biomolecules 2013, 3(3), 386-407; https://doi.org/10.3390/biom3030386 - 16 Jul 2013
Cited by 14 | Viewed by 6936
Abstract
Ovarian cancer is a multigenic disease and molecular events driving ovarian cancer progression are not well established. We have previously reported the dysregulation of the cytoskeleton during ovarian cancer progression in a syngeneic mouse cell model for progressive ovarian cancer. In the present [...] Read more.
Ovarian cancer is a multigenic disease and molecular events driving ovarian cancer progression are not well established. We have previously reported the dysregulation of the cytoskeleton during ovarian cancer progression in a syngeneic mouse cell model for progressive ovarian cancer. In the present studies, we investigated if the cytoskeleton organization is a potential target for chemopreventive treatment with the bioactive sphingolipid metabolite sphingosine. Long-term treatment with non-toxic concentrations of sphingosine but not other sphingolipid metabolites led to a partial reversal of a cytoskeleton architecture commonly associated with aggressive cancer phenotypes towards an organization reminiscent of non-malignant cell phenotypes. This was evident by increased F-actin polymerization and organization, a reduced focal adhesion kinase expression, increased a-actinin and vinculin levels which together led to the assembly of more mature focal adhesions. Downstream focal adhesion signaling, the suppression of myosin light chain kinase expression and hypophosphorylation of its targets were observed after treatment with sphingosine. These results suggest that sphingosine modulate the assembly of actin stress fibers via regulation of focal adhesions and myosin light chain kinase. The impact of these events on suppression of ovarian cancer by exogenous sphingosine and their potential as molecular markers for treatment efficacy warrants further investigation. Full article
(This article belongs to the Special Issue Sphingolipids and Bioactive Lipids)
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<p>Sph treatment increases cytoskeleton organization. Mouse Ovarian Surface Epithelium (MOSE)-E and MOSE–L cells were grown in the absence (Ctrl) or presence of 1.5 µM sphingosine (So), fixed with paraformaldehyde and stained with AlexaFluor<sup>488</sup>-conjugated phalloidin to visualize filamentous actin (<b>A</b>) or with antibodies against ß-tubulin; (<b>B</b>) to visualize the microtubule network; (<b>C</b>) Quantitation of F-actin after 4–24 h treatment with 1.5 µM So, or continuous passaging. *significantly different from MOSE-E, <span class="html-italic">p &lt;</span> 0.05, + different from untreated corresponding control, <span class="html-italic"> p &lt;</span> 0.05; (<b>D</b>) MOSE-E and L cells were treated with 1.0 µM enigmol for 3 passages and stained with AlexaFluor<sup>488</sup>-conjugated phalloidin; (<b>E</b>) MOSE-E and –L cells were treated with 500nM S1P for 8 h and stained with AlexaFluor<sup>488</sup>-conjugated phalloidin; (Original magnification X600).</p> Full article ">Figure 2
<p>Sph treatment affects protein but not mRNA levels of cytoskeleton genes and regulators in MOSE cells. MOSE-E and MOSE–L cells were passaged three times in the absence or presence of 1.5 µM So. (<b>A</b>) Real-time PCR analyses of changes in mRNA levels of select cytoskeleton genes. *significantly different from MOSE-E, <span class="html-italic">p &lt;</span> 0.05; (<b>B</b>) Representative Western blot of MOSE cells treated with So; and (<b>C</b>) quantitated using γ-tubulin as as housekeeping protein; expressed as percent of MOSE-E levels ± SD. *significantly different from untreated MOSE-E, p &lt; 0.05; + significantly different from corresponding untreated controls, p &lt; 0.05.</p> Full article ">Figure 3
<p>Effects of Sph treatment on FAK phosphorylation. MOSE-E and MOSE-L cells were treated with sphingosine (So) or vehicle, and immunostained for FAK phosphorylated on (<b>A</b>) Tyr-397; or (<b>B</b>) Tyr-861; (<b>C</b>) Co-localization of non-phosphorylated FAK and FAK; (<b>D</b>) co-localization of FAK phosphorylated on Tyr-861 and Tyr-397</p> Full article ">Figure 4
<p>Increased focal adhesion assembly and maturation by treatment with non-toxic concentrations of Sph. MOSE-E and –L cells were treated with 1.5 µM sphingosine (So) or vehicle (Ctrl); FA were visualized by staining for FAK and foci were counted and measured in cells that were completely in the visual field using the NIS Elements software (Nikon). Data represent mean ± SEM. *significantly different from untreated MOSE-E, p &lt; 0.001; <sup>+</sup>significantly different from corresponding untreated control, p &lt; 0.001.</p> Full article ">Figure 5
<p>Changes in MLCK expression and its target phosphorylation after Sph treatment. (A) Immunofluorescent identification of myosin light chain kinase (MLCK); arrow indicates co-localization with F-actin (left panel); Western blot analysis and quantitation of MLCK in whole cell extracts, normalized to to γ tubulin. A representative blot is shown in the right panel. Data are expressed as percent of MOSE-E ± SEM; *significantly different from MOSE-E, p &lt; 0.05; +significantly different from corresponding untreated control, p &lt; 0.05. (B) Myosin light chain expression (left panels) or myosin light chain phosphorylated at Ser-19 (pMyosin light chain).</p> Full article ">Figure 6
<p>Suppression of invasion by Sph treatment. Number of cells able to invade matrigel afte r sphingosine (So) treatment. Date are presented as mean ± SEM. * significantly different from MOSE-E, <span class="html-italic">p</span> &lt; 0.01.</p> Full article ">
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Review
Architecture of Amylose Supramolecules in Form of Inclusion Complexes by Phosphorylase-Catalyzed Enzymatic Polymerization
by Jun-ichi Kadokawa
Biomolecules 2013, 3(3), 369-385; https://doi.org/10.3390/biom3030369 - 11 Jul 2013
Cited by 39 | Viewed by 7998
Abstract
This paper reviews the architecture of amylose supramolecules in form of inclusion complexes with synthetic polymers by phosphorylase-catalyzed enzymatic polymerization. Amylose is known to be synthesized by enzymatic polymerization using α-d-glucose 1-phosphate as a monomer, by phosphorylase catalysis. When the phosphorylase-catalyzed enzymatic polymerization [...] Read more.
This paper reviews the architecture of amylose supramolecules in form of inclusion complexes with synthetic polymers by phosphorylase-catalyzed enzymatic polymerization. Amylose is known to be synthesized by enzymatic polymerization using α-d-glucose 1-phosphate as a monomer, by phosphorylase catalysis. When the phosphorylase-catalyzed enzymatic polymerization was conducted in the presence of various hydrophobic polymers, such as polyethers, polyesters, poly(ester-ether), and polycarbonates as a guest polymer, such inclusion supramolecules were formed by the hydrophobic interaction in the progress of polymerization. Because the representation of propagation in the polymerization is similar to the way that a vine of a plant grows, twining around a rod, this polymerization method for the formation of amylose-polymer inclusion complexes was proposed to be named “vine-twining polymerization”. To yield an inclusion complex from a strongly hydrophobic polyester, the parallel enzymatic polymerization system was extensively developed. The author found that amylose selectively included one side of the guest polymer from a mixture of two resemblant guest polymers, as well as a specific range in molecular weights of the guest polymers poly(tetrahydrofuran) (PTHF) in the vine-twining polymerization. Selective inclusion behavior of amylose toward stereoisomers of chiral polyesters, poly(lactide)s, also appeared in the vine-twining polymerization. Full article
(This article belongs to the Special Issue Enzymes and Their Biotechnological Applications)
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Figure 1

Figure 1
<p>Structure of amylase.</p> Full article ">Figure 2
<p>Amylose forms inclusion complex with relatively low molecular weight hydrophobic molecule (<b>a</b>); but, mostly, does not form it with polymeric molecule (<b>b</b>).</p> Full article ">Figure 3
<p>Phosphorylase-catalyzed enzymatic polymerization of G-1-P to form amylase.</p> Full article ">Figure 4
<p>Image of “vine-twining polymerization”.</p> Full article ">Figure 5
<p>Architecture of inclusion complexes by vine-twining polymerization using hydrophobic guest polyethers.</p> Full article ">Figure 6
<p>XRD profiles of amylose (<b>a</b>); amylose-PTHF inclusion complex (<b>b</b>); amylose-P(GA-<span class="html-italic">co</span>-CL) inclusion complex (<b>c</b>); the product obtained by vine-twining polymerization using P(GA-<span class="html-italic">b</span>-CL) (<b>d)</b>; and amylose-PLLA inclusion complex (<b>e</b>).</p> Full article ">Figure 7
<p>Architecture of inclusion complexes by vine-twining polymerization using carbonyl-containing hydrophobic polymers.</p> Full article ">Figure 8
<p>IR spectra of PVL (<b>a</b>); and amylose-PVL inclusion complex (<b>b</b>).</p> Full article ">Figure 9
<p>Architecture of inclusion complexes composed of amylose and strongly hydrophobic polyester in parallel enzymatic polymerization system.</p> Full article ">Figure 10
<p>Amylose selectively includes one of two resemblant polyethers or polyesters.</p> Full article ">Figure 11
<p>Stereoselective inclusion complexation by amylose in vine-twining polymerization using poly(<span class="html-small-caps">l</span>-lactide) (PLLA).</p> Full article ">
503 KiB  
Article
Sphingosine Phosphate Lyase Regulates Murine Embryonic Stem Cell Proliferation and Pluripotency through an S1P2/STAT3 Signaling Pathway
by Gaelen S. Smith, Ashok Kumar and Julie D. Saba
Biomolecules 2013, 3(3), 351-368; https://doi.org/10.3390/biom3030351 - 24 Jun 2013
Cited by 19 | Viewed by 7109
Abstract
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) [...] Read more.
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that activates a family of G protein coupled-receptors (GPCRs) implicated in mammalian development, angiogenesis, immunity and tissue regeneration. S1P functions as a trophic factor for many cell types, including embryonic stem cells (ESCs). Sphingosine phosphate lyase (SPL) is an intracellular enzyme that catalyzes the irreversible degradation of S1P. We found SPL to be highly expressed in murine ESCs (mESCs). To investigate the role of SPL in mESC biology, we silenced SPL in mESCs via stable transfection with a lentiviral SPL-specific short hairpin RNA (shRNA) construct. SPL-knockdown (SPL-KD) mESCs showed a 5-fold increase in cellular S1P levels, increased proliferation rates and high expression of cell surface pluripotency markers SSEA1 and OCT4 compared to vector control cells. Compared to control mESCs, SPL-KD cells showed robust activation of STAT3 and a 10-fold increase in S1P2 expression. Inhibition of S1P2 or STAT3 reversed the proliferation and pluripotency phenotypes of SPL-KD mESCs. Further, inhibition of S1P2 attenuated, in a dose-dependent fashion, the high levels of OCT4 and STAT3 activation observed in SPL-KD mESCs. Finally, we showed that SPL-KD cells are capable of generating embryoid bodies from which muscle stem cells, called satellite cells, can be isolated. These findings demonstrate an important role for SPL in ESC homeostasis and suggest that SPL inhibition could facilitate ex vivo ESC expansion for therapeutic purposes. Full article
(This article belongs to the Special Issue Sphingolipids and Bioactive Lipids)
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Graphical abstract

Graphical abstract
Full article ">Figure 1
<p>Generation of sphingosine phosphate lyase (SPL) murine embryonic stem cells (mESCs) knockdown and vector control cell lines. (<b>A</b>) Wild type (WT) E14 mES cells containing empty pLKO.1 vector show robust SPL expression by immunoblotting. SPL was undetectable by immunoblotting in mESCs in which SPL was knocked down (KD) using <span class="html-italic">Sgpl1</span> short hairpin RNA (shRNA) expressing construct in lentiviral vector pLKO.1. These results represent three separate experiments; (<b>B</b>) SPL enzyme activity is undetectable in whole cell extracts from SPL-KD mESCs. * For WT <span class="html-italic">vs.</span> KD, <span class="html-italic">p &lt;</span> 0.05; (<b>C</b>) S1P levels quantified by mass spectrometry in WT and SPL-KD mESCs.</p> Full article ">Figure 2
<p>Effects of SPL silencing on mESC proliferation and pluripotency marker expression. (<b>A</b>) Proliferation was determined by serial cell counts of exponentially growing cultures of SPL-KD (closed triangle) and vector control (closed square) mESCs. * For WT <span class="html-italic">vs.</span> KD at 72 h, <span class="html-italic">p &lt;</span> 0.05; (<b>B</b>) Quantification of pluripotency markers SSEA1, OCT4, SOX2 and NANOG protein expression relative to Actin loading control as determined by ImageJ software analysis. * For WT <span class="html-italic">vs.</span> KD expression of OCT4 and SSEA1, <span class="html-italic">p &lt;</span> 0.05; (<b>C</b>) Protein levels of pluripotency markers SSEA1, OCT4, SOX2 and NANOG and Actin control were measured by immunoblotting whole cell extracts of SPL-KD and vector control mESCs. Shown is representative immunoblot used for quantification of results depicted in (<b>B</b>). These results are representative of at least three separate experiments.</p> Full article ">Figure 3
<p>SPL silencing promotes proliferation and pluripotency marker expression through STAT3 activation in mESCs. (<b>A</b>) SPL-KD and WT mESCs were grown to confluence, trypsinized, counted, and seeded at 75,000 cells/mL. Exponentially growing cultures of SPL-KD and WT mESCs were then treated with 10 µM PD98059 for 72 h. Cell proliferation was determined at the indicated time points by cell counting of vehicle-treated SPL-KD (black open diamond), vehicle-treated WT cells (black open square), inhibitor-treated SPL-KD (red open diamond), and inhibitor-treated WT (red open square).* For inhibitor treated cells, difference between SPL-KD and WT cells remains significant, <span class="html-italic">p &lt;</span> 0.05; (<b>B</b>) SPL-KD and WT mESC cultures were prepared as in (<b>A</b>) but were treated instead with 1 µM LY294002 for 72 h, followed by cell counting at indicated time points. * For inhibitor treated cells, difference between SPL-KD and WT cells is no longer significant; (<b>C</b>) SPL-KD and WT mESC cultures were prepared as in (<b>A</b>) but were treated instead with 500 nM Stattic for 72 h, followed by cell counting at indicated time points. * For inhibitor treated cells, difference between SPL-KD and WT cells is no longer significant; (<b>D</b>) SPL-KD and WT mESC cultures were prepared as in (<b>A–C</b>), and cells were harvested at 72 h. Whole cell extracts were prepared and evaluated by immunoblotting for the proliferation marker SSEA1 with actin serving as a loading control. Note that LY284002 treatment resulted in cell death of both SPL-KD and vector control cells, shown by absence of actin; (<b>E</b>) Phosphorylated (activated) STAT3 (STAT3-P) and total STAT3 (STAT3-T) levels were measured by immunoblotting whole cell extracts of SPL-KD and WT mESCs. Actin was used as a loading control. These results are representative of at least three experiments.</p> Full article ">Figure 4
<p>S1P<sub>2</sub> is required to promote proliferation and pluripotency marker expression mediated by SPL silencing in mESCs. (<b>A</b>) Expression levels of S1P<sub>1–5</sub> were determined by qRT-PCR in total RNA isolated from SPL-KD and WT mESCs grown under proliferation conditions; (<b>B</b>) SPL-KD and WT mESCs were grown to confluence, trypsinized, counted, and seeded at 75,000 cells/mL. Cells were grown in the presence of 20 µM of the S1P<sub>1</sub> antagonist W123, 5 µM of the S1P<sub>2</sub> antagonist JTE013, or 10 µM of the S1P<sub>3</sub> antagonist BML241 (Cayman Chemical). Fresh media and inhibitors were added at 24-h time points. Cells were harvested at 72 h, and whole cell extracts were prepared and used for immunoblotting to measure proliferation antigen SSEA1, OCT4 and actin loading control; (<b>C</b>) SPL-KD and WT mESC cell cultures were prepared as in (<b>B</b>), treated with 20 µM W123. Cell proliferation was determined at the indicated time points by cell counting of vehicle-treated SPL-KD (black open diamond), vehicle-treated WT cells (black open square), antagonist-treated SPL-KD (red open diamond), and antagonist-treated WT (red open square). S1P<sub>1</sub> antagonist W123; (<b>D</b>) SPL-KD and WT mESCs were prepared as in (<b>A</b>) but were treated instead with S1P<sub>3</sub> antagonist BML241; (<b>E</b>) SPL-KD and WT mESCs were prepared as in (<b>A</b>) but were treated instead with S1P<sub>2</sub> antagonist JTE013. These results are representative of three separate experiments. * For 72 h, <span class="html-italic">p &lt;</span> 0.05 for SPL-KD cells with vehicle <span class="html-italic">vs.</span> JTE013.</p> Full article ">Figure 5
<p>SPL silencing in mESCs activates STAT3 via S1P<sub>2</sub> signaling. (<b>A</b>) SPL-KD and WT mESCs were grown to confluence, trypsinized, counted, and seeded at 75,000 cells/mL. Cells were grown in the presence of vehicle or varying concentrations from 2.5 to 20 µM of the S1P<sub>2</sub> antagonist JTE013. Cells were trypsinized and harvested for cell counting in duplicate at the indicated time points; (<b>B</b>) SPL-KD and WT mESCs were treated as described in (<b>A</b>) but were harvested at 72 h incubation, and evaluated by immunoblotting of whole cell extracts to detect the pluripotency marker SSEA1, and phosphorylated STAT3. Actin was used as a loading control; (<b>C</b>) SPL-KD and WT mESCs were grown to confluence, trypsinized, counted, and seeded at 75,000 cells/mL. Cells were grown in the presence of vehicle or the S1P receptor antagonists W123 (S1P<sub>1</sub>), JTE013 (S1P<sub>2</sub>) and BML241 (S1P<sub>3</sub>). Cells were trypsinized, and whole cell extracts were evaluated for total and phosphorylated STAT3 proteins. * in (<b>A</b>) For KD plus JTE013 at 5, 10 and 20 µM <span class="html-italic">vs.</span> vehicle, <span class="html-italic">p &lt;</span> 0.05.</p> Full article ">Figure 6
<p>SPL-KD cells are capable of generating satellite cells. WT and SPL-KD mESC cell lines were induced to form embryoid bodies using the hanging drop method. Cells were dissociated, and satellite cells were isolated from total cells using a FACSAria cell sorter to separate cells positive or negative for the monoclonal antibody SM/C-2.6, which recognizes an antigen on the cell surface of satellite cells. Total number of viable cells counted is shown in black bars, and SM/C-2.6+ cells are shown in white bars for each line.</p> Full article ">Figure 7
<p>S1P<sub>2</sub> antagonist JTE013 and STAT3 inhibitor Stattic do not induce necrotic cell death. Trypan blue staining was performed on cell cultures of SPL-KD and vector control mESCs grown in the presence of S1P<sub>2</sub> antagonist JTE013 and STAT3 inhibitor Stattic under similar conditions as described for proliferation assays shown in <a href="#biomolecules-03-00351-f002" class="html-fig">Figure 2</a>, <a href="#biomolecules-03-00351-f003" class="html-fig">Figure 3</a> and <a href="#biomolecules-03-00351-f004" class="html-fig">Figure 4</a>. Minimal Trypan Blue Dye uptake was observed.</p> Full article ">
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