International Journal of Molecular Sciences

265 KiB

Open AccessReview

Protease Inhibitors from Plants with Antimicrobial Activity

by Jin-Young Kim, Seong-Cheol Park, Indeok Hwang, Hyeonsook Cheong, Jae-Woon Nah, Kyung-Soo Hahm and Yoonkyung Park

Int. J. Mol. Sci. 2009, 10(6), 2860-2872; https://doi.org/10.3390/ijms10062860 - 23 Jun 2009

Cited by 183 | Viewed by 22931

Abstract

Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the [...] Read more.

Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C₁₈ reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. Full article

(This article belongs to the Special Issue Antimicrobial Agents)

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650 KiB

Open AccessArticle

Inactivation and Unfolding of the Hyperthermophilic Inorganic Pyrophosphatase from Thermus thermophilus by Sodium Dodecyl Sulfate

by Hang Mu, Sheng-Mei Zhou, Yong Xia, Hechang Zou, Fanguo Meng and Yong-Bin Yan

Int. J. Mol. Sci. 2009, 10(6), 2849-2859; https://doi.org/10.3390/ijms10062849 - 23 Jun 2009

Cited by 9 | Viewed by 10961

Abstract

Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential constitutive enzyme for energy metabolism and clearance of excess pyrophosphate. In this research, we investigated the sodium dodecyl sulfate (SDS)-induced inactivation and unfolding of PPase from Thermus thermophilus (T-PPase), a hyperthermophilic enzyme. The results indicated [...] Read more.

Inorganic pyrophosphatase (PPase, EC 3.6.1.1) is an essential constitutive enzyme for energy metabolism and clearance of excess pyrophosphate. In this research, we investigated the sodium dodecyl sulfate (SDS)-induced inactivation and unfolding of PPase from Thermus thermophilus (T-PPase), a hyperthermophilic enzyme. The results indicated that like many other mesophilic enzymes, T-PPase could be fully inactivated at a low SDS concentration of 2 mM. Using an enzyme activity assay, SDS was shown to act as a mixed type reversible inhibitor, suggesting T-PPase contained specific SDS binding sites. At high SDS concentrations, T-PPase was denatured via a two-state process without the accumulation of any intermediate, as revealed by far-UV CD and intrinsic fluorescence. A comparison of the inactivation and unfolding data suggested that the inhibition might be caused by the specific binding of the SDS molecules to the enzyme, while the unfolding might be caused by the cooperative non-specific binding of SDS to T-PPase. The possible molecular mechanisms underlying the mixed type inhibition by SDS was proposed to be caused by the local conformational changes or altered charge distributions. Full article

(This article belongs to the Section Biochemistry)

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Graphical abstract

Graphical abstract
Full article ">
Subunit structure of PPase from Thermus thermophilus (T-PPase, PDB ID 2PRD). (A) Subunit structure of T-PPase by ribbon representation. The center of the active site is indicated by the position of the sulfate molecule. N and C are the N- and C-terminus of the polypeptide. (B) The cavity of the active site. The hydrophobic side chains are in white, while red and blue represent the acidic and basic side chains. The cavity is surrounded by charged residues, while the bottom of the cavity is hydrophobic. The plots were generated using WebLab ViewerLite 3.7 from Molecular Simulations. Full article ">
Inactivation of T-PPase by SDS. The inactivation was performed by denaturing 11.5 μg/mL enzyme by various concentrations of SDS for 2 h, and the final concentration of the enzyme was 1.0 μg/mL in the activity assay. Full article ">
Dependence of T-PPase inactivation by SDS on enzyme concentration. Full article ">
Characterization of the type of PPase reversible inhibition by SDS. (A) Lineweaver-Burk plots. (B) Dixon plots. The lines are the best non-linear regression fit of the data to the parabolic function. (C) Slopes and intercepts on the Y-axis (Y-intercept) from the double reciprocal plot were plotted as a function of SDS concentration. The solid lines are the best non-linear regression fit of the data to the parabolic function. The dashed lines are the linear fit of the three lowest SDS concentrations, which yield the apparent inhibition constants using <a href="#FD1" class="html-disp-formula">Equations (1)</a> and <a href="#FD2" class="html-disp-formula">(2)</a>. Full article ">
Secondary and tertiary structural changes of T-PPase during SDS-induced denaturation. The protein was incubated in buffers with the addition of various concentrations of SDS for 2 h, and then the CD or intrinsic fluorescence spectra were measured. (A) CD spectra of T-PPase denatured by various concentrations of SDS. The final enzyme concentration was 0.2 mg/mL. The arrow indicates the CD spectra are recorded in the presence of 0, 0.25, 0.75, 1.25, 1.75, 2.0, 2.5, 3.0 and 4.0 mM SDS, respectively. (B) Intrinsic fluorescence spectra of T-PPase by SDS. The excitation wavelength was 295 nm. The arrow indicates the fluorescence emission spectra measured in the presence of 0, 0.5, 1.0, 1.5, 1.75, 2.0, 2.5, 3.0, 4.0 and 5.0 mM SDS, respectively. Full article ">
Transition curves of T-PPase unfolding by SDS monitored by the ellipticity at 222 nm, the emission maximum wavelength (Emax), intensity at Emax(Imax) and I320/I365 of intrinsic fluorescence. The inactivation data in <a href="#f2-ijms-10-02849" class="html-fig">Figure 2</a> is also presented. Full article ">
General Scheme for mixed type reversible inhibition. E, S and I denote enzyme, substrate and inhibitor respectively. Full article ">

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Open AccessReview

Folding Mechanism of Beta-Hairpin Trpzip2: Heterogeneity, Transition State and Folding Pathways

by Yi Xiao, Changjun Chen and Yi He

Int. J. Mol. Sci. 2009, 10(6), 2838-2848; https://doi.org/10.3390/ijms10062838 - 22 Jun 2009

Cited by 31 | Viewed by 12485

Abstract

We review the studies on the folding mechanism of the β-hairpin tryptophan zipper 2 (trpzip2) and present some additional computational results to refine the picture of folding heterogeneity and pathways. We show that trpzip2 can have a two-state or a multi-state folding pattern, [...] Read more.

We review the studies on the folding mechanism of the β-hairpin tryptophan zipper 2 (trpzip2) and present some additional computational results to refine the picture of folding heterogeneity and pathways. We show that trpzip2 can have a two-state or a multi-state folding pattern, depending on whether it folds within the native basin or through local state basins on the high-dimensional free energy surface; Trpzip2 can fold along different pathways according to the packing order of tryptophan pairs. We also point out some important problems related to the folding mechanism of trpzip2 that still need clarification, e.g., a wide distribution of the computed conformations for the transition state ensemble. Full article

(This article belongs to the Special Issue Protein Folding 2009)

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Open AccessArticle

Calculation of the Aqueous Thermodynamic Properties of Citric Acid Cycle Intermediates and Precursors and the Estimation of High Temperature and Pressure Equation of State Parameters

by Peter Dalla-Betta and Mitchell Schulte

Int. J. Mol. Sci. 2009, 10(6), 2809-2837; https://doi.org/10.3390/ijms10062809 - 22 Jun 2009

Cited by 13 | Viewed by 13427

Abstract

The citric acid cycle (CAC) is the central pathway of energy transfer for many organisms, and understanding the origin of this pathway may provide insight into the origins of metabolism. In order to assess the thermodynamics of this key pathway for microorganisms that [...] Read more.

The citric acid cycle (CAC) is the central pathway of energy transfer for many organisms, and understanding the origin of this pathway may provide insight into the origins of metabolism. In order to assess the thermodynamics of this key pathway for microorganisms that inhabit a wide variety of environments, especially those found in high temperature environments, we have calculated the properties and parameters for the revised Helgeson-Kirkham-Flowers equation of state for the major components of the CAC. While a significant amount of data is not available for many of the constituents of this fundamental pathway, methods exist that allow estimation of these missing data. Full article

(This article belongs to the Special Issue Origin of Life)

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254 KiB

Open AccessArticle

Lattice Strain Due to an Atomic Vacancy

by Shidong Li, Michael S. Sellers, Cemal Basaran, Andrew J. Schultz and David A. Kofke

Int. J. Mol. Sci. 2009, 10(6), 2798-2808; https://doi.org/10.3390/ijms10062798 - 19 Jun 2009

Cited by 43 | Viewed by 13875

Abstract

Volumetric strain can be divided into two parts: strain due to bond distance change and strain due to vacancy sources and sinks. In this paper, efforts are focused on studying the atomic lattice strain due to a vacancy in an FCC metal lattice [...] Read more.

Volumetric strain can be divided into two parts: strain due to bond distance change and strain due to vacancy sources and sinks. In this paper, efforts are focused on studying the atomic lattice strain due to a vacancy in an FCC metal lattice with molecular dynamics simulation (MDS). The result has been compared with that from a continuum mechanics method. It is shown that using a continuum mechanics approach yields constitutive results similar to the ones obtained based purely on molecular dynamics considerations. Full article

(This article belongs to the Special Issue Composite Materials)

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Graphical abstract

104 KiB

Open AccessArticle

Termite Resistance of MDF Panels Treated with Various Boron Compounds

by Mustafa Usta, Derya Ustaomer, Saip Nami Kartal and Sedat Ondaral

Int. J. Mol. Sci. 2009, 10(6), 2789-2797; https://doi.org/10.3390/ijms10062789 - 19 Jun 2009

Cited by 12 | Viewed by 11392

Abstract

In this study, the effects of various boron compounds on the termite resistance of MDF panels were evaluated. Either borax (BX), boric acid (BA), zinc borate (ZB), or sodium perborate tetrahydrate (SPT) were added to urea-formaldehyde (UF) resin at target contents of 1%, [...] Read more.

In this study, the effects of various boron compounds on the termite resistance of MDF panels were evaluated. Either borax (BX), boric acid (BA), zinc borate (ZB), or sodium perborate tetrahydrate (SPT) were added to urea-formaldehyde (UF) resin at target contents of 1%, 1.5%, 2% and 2.5% based on dry fiber weight. The panels were then manufactured using 12% urea-formaldehyde resin and 1% NH₄Cl. MDF samples from the panels were tested against the subterranean termites, Coptotermes formosanus Shiraki. Laboratory termite resistance tests showed that all samples containing boron compounds had greater resistance against termite attack compared to untreated MDF samples. At the second and third weeks of exposure, nearly 100% termite mortalities were recorded in all boron compound treated samples. The highest termite mortalities were determined in the samples with either BA or BX. Also, it was found that SPT showed notable performance on the termite mortality. As chemical loadings increased, termite mortalities increased, and at the same time the weight losses of the samples decreased. Full article

(This article belongs to the Section Biochemistry)

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317 KiB

Open AccessReview

Yeast Two-Hybrid, a Powerful Tool for Systems Biology

by Anna Brückner, Cécile Polge, Nicolas Lentze, Daniel Auerbach and Uwe Schlattner

Int. J. Mol. Sci. 2009, 10(6), 2763-2788; https://doi.org/10.3390/ijms10062763 - 18 Jun 2009

Cited by 381 | Viewed by 45110

Abstract

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very [...] Read more.

A key property of complex biological systems is the presence of interaction networks formed by its different components, primarily proteins. These are crucial for all levels of cellular function, including architecture, metabolism and signalling, as well as the availability of cellular energy. Very stable, but also rather transient and dynamic protein-protein interactions generate new system properties at the level of multiprotein complexes, cellular compartments or the entire cell. Thus, interactomics is expected to largely contribute to emerging fields like systems biology or systems bioenergetics. The more recent technological development of high-throughput methods for interactomics research will dramatically increase our knowledge of protein interaction networks. The two most frequently used methods are yeast two-hybrid (Y2H) screening, a well established genetic in vivo approach, and affinity purification of complexes followed by mass spectrometry analysis, an emerging biochemical in vitro technique. So far, a majority of published interactions have been detected using an Y2H screen. However, with the massive application of this method, also some limitations have become apparent. This review provides an overview on available yeast two-hybrid methods, in particular focusing on more recent approaches. These allow detection of protein interactions in their native environment, as e.g. in the cytosol or bound to a membrane, by using cytosolic signalling cascades or split protein constructs. Strengths and weaknesses of these genetic methods are discussed and some guidelines for verification of detected protein-protein interactions are provided. Full article

(This article belongs to the Special Issue Molecular System Bioenergetics)

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The classical yeast two-hybrid system. (A) The protein of interest X, is fused to the DNA binding domain (DBD), a construct called bait. The potential interacting protein Y is fused to the activation domain (AD) and is called prey. (B) The bait, i.e. the DBD-X fusion protein, binds the upstream activator sequence (UAS) of the promoter. The interaction of bait with prey, i.e. the AD-Y fusion protein, recruits the AD and thus reconstitutes a functional transcription factor, leading to further recruitment of RNA polymerase II and subsequent transcription of a reporter gene. Full article ">
Yeast two-hybrid systems, their subcellular location within a yeast cell, and their operating mode (represented at the moment of bait-prey interaction).Protein X (dark blue puzzle piece, part of bait construct) and protein Y (light blue puzzle piece, part of prey construct) directly interact (fitting puzzle pieces), thus inducing reconstitution of split-proteins (puzzle pieces of different colors in A, D, E), membrane recruitment (B, C) or protein dimerization (F). Protein fusions in bait or prey constructs are shown as solid black lines between puzzle pieces. Bait-prey interaction activates further downstream events (arrows) that directly (A) or indirectly (B, C, D, F) lead to transcriptional activation, or are independent of transcriptional activation (D, E), finally yielding screenable readouts like growth on specific media or color reactions. (A) Nuclear Y2H systems all require protein recruitment and bait-prey interaction at nuclear DNA. The classic Y2H and RTA Y2H both engage RNA polymerase II (RNA Pol II) transcription either by its activation or its inhibition. By contrast, the Pol III Y2H, involves RNA polymerase III (RNA Pol III) transcription. (B) Ras signalling based Y2H at the plasma membrane. The SRS Y2H, RRS Y2H, and rRRS Y2H are all based on protein recruitment to the plasma membrane via bait-prey interaction and subsequent activation of MAPK downstream signalling. While in the SRS and RRS Y2H the prey constructs harboring protein Y are anchored at the membrane via myristoylation to analyze interactions with cytosolic bait constructs harboring protein X, the rRRS is used to analyze interactions between soluble preys containing protein Y and partner X being a membrane protein. (C) G-protein signalling-based Y2H at the plasma membrane. In the G-protein fusion Y2H, bait X is a membrane or membrane-associated protein whose interaction with the prey construct disrupts protein G downstream signalling. (D) Split-ubiquitin based Y2H systems involve reconstitution of ubiquitin from two domains upon bait-prey interaction. Their subcellular localization depends on the nature of interacting proteins X or Y, and on the reporter proteins used. The Split ubiquitin Y2H uses non-transcriptional reporting of protein interactions in the cytosol, but can also be used for membrane proteins (not shown). The MbY2H is used for interaction analysis with membrane baits and thus occurs at the membrane location of protein X, e.g. the plasma membrane. The CytoY2H is used for membrane anchored cytosolic baits and occurs close to the ER membrane (E) Split-protein sensor Y2H. The Split-Trp Y2H is used to assay cytosolic bait-prey interactions based on reconstitution of an enzyme in tryptophan synthesis, allowing for non-transcriptional reporting. (F) ER Y2H system. The SCINEX-P Y2H allows bait-prey interaction analysis in the reducing environment of the ER, based on protein dimerization in unfolded protein signalling. ER, endoplasmic reticulum; for further abbreviations and details see chapter 3.2. Full article ">

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Open AccessArticle

Titration Calorimetry Standards and the Precision of Isothermal Titration Calorimetry Data

by Lina Baranauskienė, Vilma Petrikaitė, Jurgita Matulienė and Daumantas Matulis

Int. J. Mol. Sci. 2009, 10(6), 2752-2762; https://doi.org/10.3390/ijms10062752 - 18 Jun 2009

Cited by 77 | Viewed by 15986

Abstract

Current Isothermal Titration Calorimetry (ITC) data in the literature have relatively high errors in the measured enthalpies of protein-ligand binding reactions. There is a need for universal validation standards for titration calorimeters. Several inorganic salt co-precipitation and buffer protonation reactions have been suggested [...] Read more.

Current Isothermal Titration Calorimetry (ITC) data in the literature have relatively high errors in the measured enthalpies of protein-ligand binding reactions. There is a need for universal validation standards for titration calorimeters. Several inorganic salt co-precipitation and buffer protonation reactions have been suggested as possible enthalpy standards. The performances of several commercial calorimeters, including the VP-ITC, ITC200, and Nano ITC-III, were validated using these suggested standard reactions. Full article

(This article belongs to the Special Issue Isothermal Titration Calorimetry)

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Typical titration of 0.5 mM HNO3 with 5 mM Tris base using a VP-ITC (Microcal, Inc.) microcalorimeter at 37 °C. Both the cell and syringe solutions contained 100 mM NaCl. Full article ">
Tris-base – nitric acid ITC titration data obtained with a Microcal VP-ITC calorimeter at 25 °C. Filled symbols: 0.5 mM Tris base in the cell and 5 mM HNO3 in the syringe. Open symbols: 0.5 mM HNO3 in the cell and 5 mM Tris base in the syringe. All solutions contained 100 mM NaCl. When Tris base is in the cell (0.5 mM), the available concentration is reduced by dissolved CO2. Therefore, the stoichiometry was reduced to about 0.7. When Tris base is in the syringe (5 mM), the curve is practically unaffected by CO2. Using pre-boiled water in the preparation of Tris base solution solves the problem of reduced stoichiometry. Datapoints are the integrals of ITC raw data and the lines are fitted with Origin 5.0 using one- or two-binding site models. Full article ">
Titration of 0.1 mM NaI with 1.0 mM AgNO3 at 25 °C using VP-ITC (Microcal, Inc.) microcalorimeter. Full article ">
Comparison of measured ligand binding enthalpies to recombinant human carbonic anhydrase II using VP-ITC and Nano ITC-III microcalorimeters. Titrations were performed at 25 °C in 50 mM sodium phosphate buffer, pH 7.0, containing 50 mM NaCl and 1% DMSO. Note that there is a significant systematic overestimation of the enthalpies measured using the Nano ITC-III calorimeter or underestimation of the enthalpies using the VP-ITC calorimeter. All raw titration curves were good with a binding stoichiometry of 0.9±0.1. Full article ">

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Open AccessArticle

Variation in Dehydration Tolerance, ABA Sensitivity and Related Gene Expression Patterns in D-Genome Progenitor and Synthetic Hexaploid Wheat Lines

by Yumeto Kurahashi, Akihiro Terashima and Shigeo Takumi

Int. J. Mol. Sci. 2009, 10(6), 2733-2751; https://doi.org/10.3390/ijms10062733 - 18 Jun 2009

Cited by 39 | Viewed by 14014

Abstract

The wild wheat Aegilops tauschii Coss. has extensive natural variation available for breeding of common wheat. Drought stress tolerance is closely related to abscisic acid (ABA) sensitivity. In this study, 17 synthetic hexaploid wheat lines, produced by crossing the tetraploid wheat cultivar Langdon [...] Read more.

The wild wheat Aegilops tauschii Coss. has extensive natural variation available for breeding of common wheat. Drought stress tolerance is closely related to abscisic acid (ABA) sensitivity. In this study, 17 synthetic hexaploid wheat lines, produced by crossing the tetraploid wheat cultivar Langdon with 17 accessions of Ae. tauschii, were used for comparative analysis of natural variation in drought tolerance and ABA sensitivity. Ae. tauschii showed wide natural variation, with weak association between the traits. Drought-sensitive accessions of Ae. tauschii exhibited significantly less ABA sensitivity. D-genome variations observed at the diploid genome level were not necessarily reflected in synthetic wheats. However, synthetic wheats derived from the parental Ae. tauschii accessions with high drought tolerance were significantly more tolerant to drought stress than those from drought-sensitive accessions. Moreover, synthetic wheats with high drought tolerance showed significantly higher ABA sensitivity than drought-sensitive synthetic lines. In the hexaploid genetic background, therefore, weak association of ABA sensitivity with drought tolerance wasobserved. To study differences in gene expression patterns between stress-tolerant and -sensitive lines, levels of two Cor/Lea and three transcription factor gene transcripts were compared. The more tolerant accession of Ae. tauschii tended to accumulate more abundant transcripts of the examined genes than the sensitive accession under stress conditions. The expression patterns in the synthetic wheats seemed to be additive for parental lines exposed to drought and ABA treatments. However, the transcript levels of transcription factor genes in the synthetic wheats did not necessarily correspond to the postulated levels based on expression in parental lines. Allopolyploidization altered the expression levels of the stress-responsive genes in synthetic wheats. Full article

(This article belongs to the Special Issue Biotic and Abiotic Stress)

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458 KiB

Open AccessArticle

Amino Acid Synthesis in a Supercritical Carbon Dioxide - Water System

by Kouki Fujioka, Yasuhiro Futamura, Tomoo Shiohara, Akiyoshi Hoshino, Fumihide Kanaya, Yoshinobu Manome and Kenji Yamamoto

Int. J. Mol. Sci. 2009, 10(6), 2722-2732; https://doi.org/10.3390/ijms10062722 - 15 Jun 2009

Cited by 6 | Viewed by 12986

Abstract

Mars is a CO₂-abundant planet, whereas early Earth is thought to be also CO₂-abundant. In addition, water was also discovered on Mars in 2008. From the facts and theory, we assumed that soda fountains were present on both planets, [...] Read more.

Mars is a CO₂-abundant planet, whereas early Earth is thought to be also CO₂-abundant. In addition, water was also discovered on Mars in 2008. From the facts and theory, we assumed that soda fountains were present on both planets, and this affected amino acid synthesis. Here, using a supercritical CO₂/liquid H₂O (10:1) system which mimicked crust soda fountains, we demonstrate production of amino acids from hydroxylamine (nitrogen source) and keto acids (oxylic acid sources). In this research, several amino acids were detected with an amino acid analyzer. Moreover, alanine polymers were detected with LC-MS. Our research lights up a new pathway in the study of life’s origin. Full article

(This article belongs to the Special Issue Origin of Life)

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Graphical abstract

2482 KiB

Open AccessReview

Precambrian Lunar Volcanic Protolife

by Jack Green

Int. J. Mol. Sci. 2009, 10(6), 2681-2721; https://doi.org/10.3390/ijms10062681 - 11 Jun 2009

Cited by 2 | Viewed by 13201

Abstract

Five representative terrestrial analogs of lunar craters are detailed relevant to Precambrian fumarolic activity. Fumarolic fluids contain the ingredients for protolife. Energy sources to derive formaldehyde, amino acids and related compounds could be by flow charging, charge separation and volcanic shock. With no [...] Read more.

Five representative terrestrial analogs of lunar craters are detailed relevant to Precambrian fumarolic activity. Fumarolic fluids contain the ingredients for protolife. Energy sources to derive formaldehyde, amino acids and related compounds could be by flow charging, charge separation and volcanic shock. With no photodecomposition in shadow, most fumarolic fluids at 40 K would persist over geologically long time periods. Relatively abundant tungsten would permit creation of critical enzymes, Fischer-Tropsch reactions could form polycyclic aromatic hydrocarbons and soluble volcanic polyphosphates would enable assembly of nucleic acids. Fumarolic stimuli factors are described. Orbital and lander sensors specific to protolife exploration including combined Raman/laser-induced breakdown spectrocsopy are evaluated. Full article

(This article belongs to the Special Issue Origin of Life)

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615 KiB

Open AccessArticle

Measurement of Nanomolar Dissociation Constants by Titration Calorimetry and Thermal Shift Assay – Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII

by Asta Zubrienė, Jurgita Matulienė, Lina Baranauskienė, Jelena Jachno, Jolanta Torresan, Vilma Michailovienė, Piotras Cimmperman and Daumantas Matulis

Int. J. Mol. Sci. 2009, 10(6), 2662-2680; https://doi.org/10.3390/ijms10062662 - 10 Jun 2009

Cited by 51 | Viewed by 14720

Abstract

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90aN) and the binding of ethoxzolamide to human carbonic [...] Read more.

The analysis of tight protein-ligand binding reactions by isothermal titration calorimetry (ITC) and thermal shift assay (TSA) is presented. The binding of radicicol to the N-terminal domain of human heat shock protein 90 (Hsp90aN) and the binding of ethoxzolamide to human carbonic anhydrase (hCAII) were too strong to be measured accurately by direct ITC titration and therefore were measured by displacement ITC and by observing the temperature-denaturation transitions of ligand-free and ligand-bound protein. Stabilization of both proteins by their ligands was profound, increasing the melting temperature by more than 10 ºC, depending on ligand concentration. Analysis of the melting temperature dependence on the protein and ligand concentrations yielded dissociation constants equal to 1 nM and 2 nM for Hsp90aN-radicicol and hCAII-ethoxzolamide, respectively. The ligand-free and ligand-bound protein fractions melt separately, and two melting transitions are observed. This phenomenon is especially pronounced when the ligand concentration is equal to about half the protein concentration. The analysis compares ITC and TSA data, accounts for two transitions and yields the ligand binding constant and the parameters of protein stability, including the Gibbs free energy and the enthalpy of unfolding. Full article

(This article belongs to the Special Issue Isothermal Titration Calorimetry)

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Panel A. Isothermal titration calorimetry data from radicicol binding to Hsp90αN. Upper graph – raw ITC data, lower graph – integrated ITC data with the curve fit to the standard single binding site model. The cell contained 4 μM protein, while the syringe contained 40 μM radicicol in the same buffer - 50 mM sodium phosphate, pH 7.5, 0.5% DMSO, 100 mM NaCl, at 25 °C. Panel B. Isothermal titration calorimetry displacement assay. All conditions are the same as in Panel A, except there was 1 mM compound 1 added to the calorimeter cell. Panel C. Chemical structures of radicicol and compound 1. Panel D. Isothermal titration calorimetry data from ethoxzolamide binding to hCAII. The cell contained 7 μM protein, while the syringe contained 100 μM ethoxzolamide in the same buffer - 50 mM sodium phosphate, pH 7.0, 0.5% DMSO, 50 mM NaCl, at 37 °C. Full article ">
Denaturation profiles of Hsp90αN or hCAII in the presence of inhibitors. Panel A. Hsp90αN (14 μM) in 50 mM Tris buffer, pH 7.5, with added radicicol: ▪ - 0 μM, ▴ - 2 μM, × - 3 μM, Δ - 6 μM, ○ - 10 μM, + - 20 μM, and − - 50 μM. Panel B. hCAII (5 μM) in 50 mM phosphate buffer, pH 7.0, containing 50 mM NaCl, with added ethoxzolamide: ▪ - 0 μM, ▴ - 1.5 μM, × - 2 μM, Δ - 3 μM, and ○ - 50 μM. Increased inhibitor concentrations raise the melting temperature of both proteins by more than 10 ºC, depending on concentration. Two transitions are observed when both free and ligand-bound proteins are present. Heights of both transitions are additive and proportional to the fraction of saturation by the inhibitor. The data points represent experimental observations while the lines are fit to <a href="#FD10" class="html-disp-formula">Equation (10)</a>. Parameters are listed in <a href="#t1-ijms-10-02662" class="html-table">Table 1</a>. Panel C. DSC profiles of Hsp90αN (120 μM) with radicicol: dashed line – 0 μM, dot-dashed line – 60 μM, and solid line – 360 μM. Panel D. DSC profiles of hCAII (100 μM) with ethoxzolamide: dashed line – 0 μM, dotted line – 22 μM, dot-dashed line – 50 μM, and solid line – 1 mM. Full article ">
The protein melting temperature dependence on inhibitor concentration. Panel A. Hsp90αN with radicicol (from the melting curves in <a href="#f2-ijms-10-02662" class="html-fig">Figure 2A</a>). Panel B. hCAII with ethoxzolamide (from the melting curves in <a href="#f2-ijms-10-02662" class="html-fig">Figure 2B</a>). The data points show the melting temperatures of the ligand-bound (•) and ligand-free (▪) forms of the protein. The leftmost data point is the control where no inhibitor was added. Lines are the fit to the data according to <a href="#FD12" class="html-disp-formula">Equation (12)</a>. Full article ">
The dependence of the melting temperature of Hsp90αN on radicicol concentration. The data points show the melting temperatures of ligand-bound components at three protein concentrations: ▪ - 4.1 μM, Δ - 7.5 μM, and • - 14 μM. The Tm of the ligand-free Hsp90αN component is omitted for clarity. The leftmost data points are controls with no radicicol added. Lines are drawn according to <a href="#FD12" class="html-disp-formula">Equation (12)</a> and represent the best possible fit of the data. The solid line is for 4.1 μM, the dashed line for 7.5 μM, and the dotted line for 14 μM protein. Full article ">
The dependence of Hsp90αN melting temperature on radicicol concentration with various rates of heating. Data points show the melting temperatures of ligand-bound and free components at three heating rates: ▪ - 2 ºC/min, Δ - 1 ºC/min, and • - 0.5 ºC/min. Lines are drawn according to <a href="#FD12" class="html-disp-formula">Equation (12)</a>. Note that the Tm values obtained at different heating rates yield the same binding constant. Full article ">

1633 KiB

Open AccessReview

Periodic Density Functional Theory Investigation of the Uranyl Ion Sorption on Three Mineral Surfaces: A Comparative Study

by Jérôme Roques, Edouard Veilly and Eric Simoni

Int. J. Mol. Sci. 2009, 10(6), 2633-2661; https://doi.org/10.3390/ijms10062633 - 4 Jun 2009

Cited by 32 | Viewed by 21133

Abstract

Canister integrity and radionuclides retention is of prime importance for assessing the long term safety of nuclear waste stored in engineered geologic depositories. A comparative investigation of the interaction of uranyl ion with three different mineral surfaces has thus been undertaken in order [...] Read more.

Canister integrity and radionuclides retention is of prime importance for assessing the long term safety of nuclear waste stored in engineered geologic depositories. A comparative investigation of the interaction of uranyl ion with three different mineral surfaces has thus been undertaken in order to point out the influence of surface composition on the adsorption mechanism(s). Periodic DFT calculations using plane waves basis sets with the GGA formalism were performed on the TiO₂(110), Al(OH)₃(001) and Ni(111) surfaces. This study has clearly shown that three parameters play an important role in the uranyl adsorption mechanism: the solvent (H₂O) distribution at the interface, the nature of the adsorption site and finally, the surface atoms’ protonation state. Full article

(This article belongs to the Special Issue Application of Density Functional Theory)

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129 KiB

Open AccessReview

The Eukaryotic Cell Originated in the Integration and Redistribution of Hyperstructures from Communities of Prokaryotic Cells Based on Molecular Complementarity

by Vic Norris and Robert Root-Bernstein

Int. J. Mol. Sci. 2009, 10(6), 2611-2632; https://doi.org/10.3390/ijms10062611 - 4 Jun 2009

Cited by 12 | Viewed by 12089

Abstract

In the “ecosystems-first” approach to the origins of life, networks of non-covalent assemblies of molecules (composomes), rather than individual protocells, evolved under the constraints of molecular complementarity. Composomes evolved into the hyperstructures of modern bacteria. We extend the ecosystems-first approach to explain the [...] Read more.

In the “ecosystems-first” approach to the origins of life, networks of non-covalent assemblies of molecules (composomes), rather than individual protocells, evolved under the constraints of molecular complementarity. Composomes evolved into the hyperstructures of modern bacteria. We extend the ecosystems-first approach to explain the origin of eukaryotic cells through the integration of mixed populations of bacteria. We suggest that mutualism and symbiosis resulted in cellular mergers entailing the loss of redundant hyperstructures, the uncoupling of transcription and translation, and the emergence of introns and multiple chromosomes. Molecular complementarity also facilitated integration of bacterial hyperstructures to perform cytoskeletal and movement functions. Full article

(This article belongs to the Special Issue Origin of Life)

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Open AccessReview

Application of Ionic Liquids in High Performance Reversed-Phase Chromatography

by Ye Wang, Minglei Tian, Wentao Bi and Kyung Ho Row

Int. J. Mol. Sci. 2009, 10(6), 2591-2610; https://doi.org/10.3390/ijms10062591 - 4 Jun 2009

Cited by 92 | Viewed by 14490

Abstract

Ionic liquids, considered “green” chemicals, are widely used in many areas of analytical chemistry due to their unique properties. Recently, ionic liquids have been used as a kind of novel additive in separation and combined with silica to synthesize new stationary phase as [...] Read more.

Ionic liquids, considered “green” chemicals, are widely used in many areas of analytical chemistry due to their unique properties. Recently, ionic liquids have been used as a kind of novel additive in separation and combined with silica to synthesize new stationary phase as separation media. This review will focus on the properties and mechanisms of ionic liquids and their potential applications as mobile phase modifier and surface-bonded stationary phase in reversed-phase high performance liquid chromatography (RP-HPLC). Ionic liquids demonstrate advantages and potential in chromatographic field. Full article

(This article belongs to the Section Green Chemistry)

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Scheme illustrating potential reorientation of bonded imidazolium ligands in response to deprotonation of residual silanols. Anion is not shown for clarity (adapted from [<a href="#b37-ijms-10-02591" class="html-bibr">37</a>]). Full article ">
Synthesis steps used in the preparation of zwitterionic stationary phase [<a href="#b43-ijms-10-02591" class="html-bibr">43</a>]. Full article ">
Scheme illustrating the modification of silica particles with the synthesized 1-allyl-3-(butyl-4-sulfonate)imidazolium ionic liquids [<a href="#b49-ijms-10-02591" class="html-bibr">49</a>]. Full article ">
Separation of test mixtures composed of cytosine (1), thymine (2), adenine (3), 2-aminopyrimidine (4), and 6-chloroguanine (5). Mobile phase: water, detection: UV at 254 nm [<a href="#b55-ijms-10-02591" class="html-bibr">55</a>]. Full article ">
Chromatograms of ephedrines with a mobile phase containing different concentrations of [BMIM][BF4] at pH 3.0. (a) 0, (b) 2.6, (c) 5.2, (d) 20.8, and (e) 62.4 mM. Chromatographic conditions: column: C18 (5 μm, 100×4.6 mm I.D.); rate-flow: 1.0 mL/min; detection: 252 nm. Peaks: (1) NE, (2) E, (3) PE, (4) ME, [<a href="#b57-ijms-10-02591" class="html-bibr">57</a>]. Full article ">
Effect of different ionic liquids on the separation of the seven antibiotics studied. Mobile phase: 10 mmol/L ammonium acetate at pH 3.0 with 13% (v/v) acetonitrile and (a) 6 mmol/L [Et4N][BF4]; (b) 6 mmol/L [EMIM][BF4]; (c) 6 mmol/L [BMIM][BF4]; (d) 6 mmol/L [HMIM][BF4]; (e) 6 mmol/L [MOIM][BF4]. Flow rate: 1 mL/min. Detection: λexc=280 nm and λem=450 nm. Peak identification: 1, FLERO; 2, CIPRO; 3, LOME; 4, DANO; 5, ENRO; 6, SARA and 7, DIFLO [<a href="#b74-ijms-10-02591" class="html-bibr">74</a>]. Full article ">

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Open AccessArticle

Tryptophanase-Catalyzed L-Tryptophan Synthesis from D-Serine in the Presence of Diammonium Hydrogen Phosphate

by Akihiko Shimada, Haruka Ozaki, Takeshi Saito and Fujii Noriko

Int. J. Mol. Sci. 2009, 10(6), 2578-2590; https://doi.org/10.3390/ijms10062578 - 3 Jun 2009

Cited by 12 | Viewed by 11950

Abstract

Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of L-tryptophan from L-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on D-serine. We previously reported that tryptophanase changed its stereospecificity to [...] Read more.

Tryptophanase, an enzyme with extreme absolute stereospecificity for optically active stereoisomers, catalyzes the synthesis of L-tryptophan from L-serine and indole through a β-substitution mechanism of the ping-pong type, and has no activity on D-serine. We previously reported that tryptophanase changed its stereospecificity to degrade D-tryptophan in highly concentrated diammonium hydrogen phosphate, (NH₄)₂HPO₄ solution. The present study provided the same stereospecific change seen in the D-tryptophan degradation reaction also occurs in tryptophan synthesis from D-serine. Tryptophanase became active to D-serine to synthesize L-tryptophan in the presence of diammonium hydrogen phosphate. This reaction has never been reported before. D-serine seems to undergo β-replacement via an enzyme-bonded α-aminoacylate intermediate to yield L-tryptophan. Full article

(This article belongs to the Special Issue Origin of Life)

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Thin layer chromatogram of the tryptophan synthesized from l- or d-serine by tryptophanase. For comparison, d-tryptophan, l-tryptophan, d-serine and l-serine were developed in the left half. Reaction products were developed in the right half. Lane1: l-serine + indole + tryptophanase in a potassium phosphate buffer; lane 2: d-serine + indole + 20 % saturation diammoniumhydrogen phosphate in a potassium phosphate buffer; lane 3: d-serine + indole + tryptophanase in a potassium phosphate buffer; lane 4: d-serine + indole + tryptophanase + 20 % saturation diammoniumhydrogen phosphate in a potassium phosphate buffer. Full article ">
Tryptophan synthesis from l- or d-serine against diammonium hydrogen phosphate saturation concentration. ○: l-serine, •: d-serine. Full article ">
Resolution chromatograms of the reactant products (monitored by UV detection at λ = 280 nm). (a) Advanced resolution chromatography was carried out to determine a retention time of l-tryptophan. (b) There is no tryptophan peak in the absence of tryptophanase. (c) l-tryptophan (a peak at 14.6 min) was synthesized from l-serine and indole by tryptophanase in a potassium phosphate buffer solution. (d) Tryptophan was synthesized from d-serine and indole by tryptophanase in the presence of diammonium hydrogen phosphate. Full article ">
Detection of the reactant product with CD detector. (a) d, l-tryptophan as a standard substance was eluted onto a resolution column Crownpack CR (+). (b) The tryptophan synthesized was eluted onto the same column. Optical isomeric form of the tryptophan was established to be l type. Full article ">

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Open AccessReview

QSPR Studies on Aqueous Solubilities of Drug-Like Compounds

by Pablo R. Duchowicz and Eduardo A. Castro

Int. J. Mol. Sci. 2009, 10(6), 2558-2577; https://doi.org/10.3390/ijms10062558 - 3 Jun 2009

Cited by 53 | Viewed by 14764

Abstract

A rapidly growing area of modern pharmaceutical research is the prediction of aqueous solubility of drug-sized compounds from their molecular structures. There exist many different reasons for considering this physico-chemical property as a key parameter: the design of novel entities with adequate aqueous [...] Read more.

A rapidly growing area of modern pharmaceutical research is the prediction of aqueous solubility of drug-sized compounds from their molecular structures. There exist many different reasons for considering this physico-chemical property as a key parameter: the design of novel entities with adequate aqueous solubility brings many advantages to preclinical and clinical research and development, allowing improvement of the Absorption, Distribution, Metabolization, and Elimination/Toxicity profile and “screenability” of drug candidates in High Throughput Screening techniques. This work compiles recent QSPR linear models established by our research group devoted to the quantification of aqueous solubilities and their comparison to previous research on the topic. Full article

(This article belongs to the Special Issue Recent Advances in QSAR/QSPR Theory)

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1267 KiB

Open AccessReview

Trends in the Molecular Pathogenesis and Clinical Therapeutics of Common Neurodegenerative Disorders

by Yahya E. Choonara, Viness Pillay, Lisa C. Du Toit, Girish Modi, Dinesh Naidoo, Valence M.K. Ndesendo and Sibongile R. Sibambo

Int. J. Mol. Sci. 2009, 10(6), 2510-2557; https://doi.org/10.3390/ijms10062510 - 3 Jun 2009

Cited by 66 | Viewed by 20023

Abstract

The term neurodegenerative disorders, encompasses a variety of underlying conditions, sporadic and/or familial and are characterized by the persistent loss of neuronal subtypes. These disorders can disrupt molecular pathways, synapses, neuronal subpopulations and local circuits in specific brain regions, as well as higher-order [...] Read more.

The term neurodegenerative disorders, encompasses a variety of underlying conditions, sporadic and/or familial and are characterized by the persistent loss of neuronal subtypes. These disorders can disrupt molecular pathways, synapses, neuronal subpopulations and local circuits in specific brain regions, as well as higher-order neural networks. Abnormal network activities may result in a vicious cycle, further impairing the integrity and functions of neurons and synapses, for example, through aberrant excitation or inhibition. The most common neurodegenerative disorders are Alzheimer’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis and Huntington’s disease. The molecular features of these disorders have been extensively researched and various unique neurotherapeutic interventions have been developed. However, there is an enormous coercion to integrate the existing knowledge in order to intensify the reliability with which neurodegenerative disorders can be diagnosed and treated. The objective of this review article is therefore to assimilate these disorders’ in terms of their neuropathology, neurogenetics, etiology, trends in pharmacological treatment, clinical management, and the use of innovative neurotherapeutic interventions. Full article

(This article belongs to the Special Issue Advances in Molecular Neuropathology)

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Schematic diagram outlining the pathogenesis of common neurodegenerative diseases (Adapted from Yuan and Yanker, [<a href="#b16-ijms-10-02510" class="html-bibr">16</a>]). Full article ">
The role of glial cells in central nervous system inflammation and neurodegeneration. BDNF = brain-derived neurotrophic factor, MP = macrophages, MMP = membrane metalloproteinase, TIMP = tissue inhibitors of metalloproteinase (Adapted from: Ghorpade et al. [<a href="#b113-ijms-10-02510" class="html-bibr">113</a>]). Full article ">
Molecular mechanisms leading to cell death in neurons and the yeast PD model (Adapted from: Winderickx et al. [<a href="#b164-ijms-10-02510" class="html-bibr">164</a>]). Full article ">
Depiction of adult neural stem demonstrating their intrinsic potential to generate cell types of the brain and spinal cord (Adapted from: Karimi and Eftekharpour, Fehlings lab, McEwan Centre for Regnerative Medicine; <a href="http://www.mcewencentre.com/res_prog_scnd.asp" target="_blank">www.mcewencentre.com/res_prog_scnd.asp</a>, [<a href="#b182-ijms-10-02510" class="html-bibr">182</a>]). Full article ">
Schematic of SOD-1 mutations activating cell death pathways in familial Amyotrophic Lateral Sclerosis (Source: Yuan and Yankner, [<a href="#b16-ijms-10-02510" class="html-bibr">16</a>]). Full article ">
Schematic depicting a pathway of oxidative damage in HD (Source: Trushina and McMurray, [<a href="#b232-ijms-10-02510" class="html-bibr">232</a>]). Full article ">
A minimally-invasive intrathecal drug delivery system for spinal cord injury repair (Source: Shoichet lab, McEwen Centre for Regenerative Medicine, [<a href="#b276-ijms-10-02510" class="html-bibr">276</a>]). Full article ">

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Open AccessArticle

Purification, Crystallization and Preliminary X-ray Crystallographic Studies of RAIDD Death-Domain (DD)

by Tae-ho Jang and Hyun Ho Park

Int. J. Mol. Sci. 2009, 10(6), 2501-2509; https://doi.org/10.3390/ijms10062501 - 3 Jun 2009

Cited by 8 | Viewed by 10597

Abstract

Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with [...] Read more.

Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3₁21 (or its enantiomorph P3₂21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 Å and γ = 120°. The crystals were obtained at room temperature and diffracted to 2.0 Å resolution. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

Association Study between BDNF Gene Polymorphisms and Autism by Three-Dimensional Gel-Based Microarray

by Lu Cheng, Qinyu Ge, Pengfeng Xiao, Beili Sun, Xiaoyan Ke, Yunfei Bai and Zuhong Lu

Int. J. Mol. Sci. 2009, 10(6), 2487-2500; https://doi.org/10.3390/ijms10062487 - 2 Jun 2009

Cited by 22 | Viewed by 17476

Abstract

Single nucleotide polymorphisms (SNPs) are important markers which can be used in association studies searching for susceptible genes of complex diseases. High-throughput methods are needed for SNP genotyping in a large number of samples. In this study, we applied polyacrylamide gel-based microarray combined [...] Read more.

Single nucleotide polymorphisms (SNPs) are important markers which can be used in association studies searching for susceptible genes of complex diseases. High-throughput methods are needed for SNP genotyping in a large number of samples. In this study, we applied polyacrylamide gel-based microarray combined with dual-color hybridization for association study of four BDNF polymorphisms with autism. All the SNPs in both patients and controls could be analyzed quickly and correctly. Among four SNPs, only C270T polymorphism showed significant differences in the frequency of the allele (χ² = 7.809, p = 0.005) and genotype (χ² = 7.800, p = 0.020). In the haplotype association analysis, there was significant difference in global haplotype distribution between the groups (χ² = 28.19，p = 3.44e-005). We suggest that BDNF has a possible role in the pathogenesis of autism. The study also show that the polyacrylamide gel-based microarray combined with dual-color hybridization is a rapid, simple and high-throughput method for SNPs genotyping, and can be used for association study of susceptible gene with disorders in large samples. Full article

(This article belongs to the Section Biochemistry)

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Open AccessArticle

Voltage-Dependent Anion Channel 2 of Arabidopsis thaliana (AtVDAC2) Is Involved in ABA-Mediated Early Seedling Development

by Jinping Yan, Han He, Shibo Tong, Wanrong Zhang, Jianmei Wang, Xufeng Li and Yi Yang

Int. J. Mol. Sci. 2009, 10(6), 2476-2486; https://doi.org/10.3390/ijms10062476 - 26 May 2009

Cited by 27 | Viewed by 14285

Abstract

The voltage-dependent anion channel (VDAC) is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and [...] Read more.

The voltage-dependent anion channel (VDAC) is the major transport protein in the outer membrane of mitochondria and plays crucial roles in energy metabolism, apoptosis, and metabolites transport. In plants, the expression of VDACs can be affected by different stresses, including drought, salinity and pathogen defense. In this study, we investigated the expression pattern of AtVDAC2 in A. thaliana and found ABA suppressed the accumulation of AtVDAC2 transcripts. Further, phenotype analysis of this VDAC deregulated-expression transgenic Arabidopsis plants indicated that AtVDAC2 anti-sense line showed an ABA-insensitivity phenotype during the early seedling development under ABA treatment. The results suggested that AtVDAC2 might be involved in ABA signaling in A. thaliana. Full article

(This article belongs to the Section Biochemistry)

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Graphical abstract
Full article ">
Effect of ABA on AtVDAC2 gene expression at the transcriptional level detected by semi-quantitative PCR. (a) The effect of ABA on AtVDAC2 mRNA level. Four-week old Arabidopsis seedlings were treated with 30μM ABA for 0, 2 h, 8 h, 16 h and 24 h, respectively. (b) The relative AtVDAC2 abundance in Arabidopsis mesophyll protoplasts under 5 μM, 50 μM ABA treatment. The quantitative analysis of the PCR signal performed with imaging software (Gel-Pro analyzer 3.0) and the bands intensities relative to their actin. Full article ">
The relative activity of 5′ upstream region of AtVDAC2 regulated by ABA. (a) Construction of the pBI221-pVDAC::LUC vector for the transient gene expression in Arabidopsis mesophyll protoplasts. (b) The luciferase activity of AtVDAC2 promoter in protoplasts was regulated by different level of ABA (0.1, 1, 10 and 100 μM). Luciferase activity was means ± SD (n = 3) from one of three independent experiments. * Significant at P<0.05 compared with the control (treated with 0 μM ABA) based on Student’s test. Full article ">
Identification of the AtVDAC2 transgenic Arabidopsis plants by semi-quantitative analysis. (a) The amplified DNA fragment of AtVDAC2 in different transgenic lines were stained by ethidium bromide in agarose gel and the ralative level in each sample was normalized for actin transcripts. (b) The relative amount of AtVDAC2 mRNA were quantified using a software Gel-Pro analyzer 3.0. OE, Dn, WT represent AtVDAC2 sense lines, anti-sense lines and wild type, respectively. Full article ">
Effect of exogenous ABA on seed germination of different AtVDAC2 tansgenic lines. After stratification, seeds of AtVDAC2 sense (OE) or antisense (Dn) lines and wild-type (WT) were grown with 0.7μM ABA and Germination was scored every 24 hour in two independent seed batches. Values are means ± SD (n=3) from one representative of three independent experiments with similar results. Asterisks indicate significant difference from wild type (P<0.05) based on Student’s test. Full article ">
The AtVDAC2 transgenic plants showed different sensitivity to ABA during the early seedling development. Early seedling of AtVDAC2 sense (OE) or antisense (Dn) lines and wild-type (WT) plants after 10 days of growth on control MS (a) or on MS media added with 0.7μM ABA (b). Similar results were obtained in three replicates. Full article ">

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Open AccessReview

An Updated Review of Tyrosinase Inhibitors

by Te-Sheng Chang

Int. J. Mol. Sci. 2009, 10(6), 2440-2475; https://doi.org/10.3390/ijms10062440 - 26 May 2009

Cited by 1266 | Viewed by 65306

Abstract

Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are [...] Read more.

Tyrosinase is a multifunctional, glycosylated, and copper-containing oxidase, which catalyzes the first two steps in mammalian melanogenesis and is responsible for enzymatic browning reactions in damaged fruits during post-harvest handling and processing. Neither hyperpigmentation in human skin nor enzymatic browning in fruits are desirable. These phenomena have encouraged researchers to seek new potent tyrosinase inhibitors for use in foods and cosmetics. This article surveys tyrosinase inhibitors newly discovered from natural and synthetic sources. The inhibitory strength is compared with that of a standard inhibitor, kojic acid, and their inhibitory mechanisms are discussed. Full article

(This article belongs to the Special Issue Inhibitors of Melanogenesis Related Processes: Application to Food and Cosmetic Industry)

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Biosynthetic pathway of melanin [<a href="#b1-ijms-10-02440" class="html-bibr">1</a>–<a href="#b4-ijms-10-02440" class="html-bibr">4</a>]. TYR, tyrosinase; TRP; tyrosinase related protein; dopa, 3,4-dihydroxyphenylalanine; DHICA, 5,6-dihydroxyindole-2-carboxylic acid; DHI, 5,6-dihydroxyindole; ICAQ, indole-2-carboxylic acid-5,6-quinone; IQ, indole-5,6-quinone; HBTA, 5-hydroxy-1,4-benzothiazinylalanine. Full article ">
Catalytic cycles of the hydroxylation of monophenol and oxidation of o-diphenol to o-quinone by tyrosinase [<a href="#b23-ijms-10-02440" class="html-bibr">23</a>–<a href="#b24-ijms-10-02440" class="html-bibr">24</a>]. Eoxy, Emet, and Edeoxy are the three types of tyrosinase, respectively. EoxyD, EoxyM, and EmetM are Eoxy-Diphenol, Eoxy-Monophenol, and Emet-Monophenol complexes, respectively. Full article ">
Chemical structures of selected tyrosinase inhibitors belonging to some standard ones (a), flavonoids (b–j) or N-benzylbenzamides analogs (k). RAa and RAb are the relative diphenolase and monophenolase inhibitory activity, respectively, against mushroom tyrosinase compared to the standard kojic acid, where 1.0F means one time activity of kojic acid. Full article ">
Chemical structures of selected tyrosinase inhibitors belonging to stilbenes (a), bibenzyl derivatives (b), coumarins (c), and benzaldehyde derivatives (d). RAa and RAb have the same meanings as those of <a href="#f3-ijms-10-02440" class="html-fig">Figure 3</a>. Full article ">
Chemical structures of selected tyrosinase inhibitors belonging to long-chain lipids (a) or steroids (b). RAa and RAb have the same meanings as those of <a href="#f3-ijms-10-02440" class="html-fig">Figure 3</a>. Full article ">
Chemical structures of other tyrosinase inhibitors from natural (a) or synthetic (b) sources. RAa and RAb have the same meanings as those of <a href="#f3-ijms-10-02440" class="html-fig">Figure 3</a>. Full article ">
Chemical structures of irreversible tyrosinase inhibitors. Full article ">
Molecular reaction mechanism of suicide inactivation of tyrosinase by the oxidation of an o-diphenol substrate. The curly arrows shows the effect of deprotonation leading to the reduction of copper from bivalent to zero-valent form, elimination of an o-quinone and inactivated tyrosinase [<a href="#b168-ijms-10-02440" class="html-bibr">168</a>]. Full article ">
Action mechanism of reversible inhibitors. E, S, I, and P are the enzyme, substrate, inhibitor, and product, respectively; ES is the enzyme-substrate complex, and EI and ESI are the enzyme-inhibitor and enzyme-substrate-inhibitor complexes, respectively. Full article ">

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Int. J. Mol. Sci., Volume 10, Issue 6 (June 2009) – 22 articles , Pages 2440-2872

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