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1.
Heliyon ; 7(8): e07816, 2021 Aug.
Article in English | MEDLINE | ID: mdl-34423146

ABSTRACT

Natural products have been used in the treatment of illnesses throughout the history of humankind. Exploitation of bioactive compounds from natural sources can aid in the discovery of new drugs, provide the scaffold of new medicines. In the face of challenging diseases, such as the COVID-19 pandemic, for which there was no effective treatment, nature could offer insights as to novel therapeutic options for control measures. However, the environmental impact and supply chain of bioactive production must be carefully evaluated to ensure the detrimental effects will not outweigh the potential benefits gained. History has already proven that highly bioactive compounds can be rare and not suitable for medicinal exploitation; therefore, the sustainability must be accessed before expensive, time-demanding, and large trials can be initialized. A sustainable option to readily produce a phytotherapy with minimal environmental stress is the use of agro-industry wastes, a by-product produced in high quantities. In this review we evaluate the sustainability issues associated with the production of phytotherapy as a readily available tool for pandemic control.

2.
Faraday Discuss ; 202: 31-42, 2017 09 21.
Article in English | MEDLINE | ID: mdl-28653735

ABSTRACT

Mango peel is the major by-product of mango processing, and compromises 7-24% of the total mango weight. In this study, pectin was extracted from mango peel waste by using subcritical water extraction (SWE) in the absence of mineral acid. A highest yield of 18.34% was achieved from the Kesar variety and the pectin was characterised using ATR-IR spectroscopy, TGA and 13C solid-state NMR spectroscopy to confirm the structure. The degree of esterification (DE) of the pectin was analysed with both titrimetry and 13C solid-state NMR spectroscopy, and a high DE (>70%) was observed for all three varieties (Keitt, Sindhri and Kesar). This is the first report on acid-free subcritical water extraction of pectin from mango peel, which provides a green route for the valorisation of mango peel waste and contributes to a source of biobased materials and chemicals for a sustainable 21st century.


Subject(s)
Food Supply , Mangifera/chemistry , Pectins/isolation & purification , Water/chemistry , Pectins/chemistry , Waste Products
3.
Br J Pharmacol ; 159(3): 518-33, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20128803

ABSTRACT

BACKGROUND AND PURPOSE: We have investigated the effect of deletions of a postsynaptic density, disc large and zo-1 protein (PDZ) motif at the end of the COOH-terminus of the rat A(2B) adenosine receptor on intracellular trafficking following long-term exposure to the agonist 5'-(N-ethylcarboxamido)-adenosine. EXPERIMENTAL APPROACH: The trafficking of the wild type A(2B) adenosine receptor and deletion mutants expressed in Chinese hamster ovary cells was studied using an enzyme-linked immunosorbent assay in combination with immunofluorescence microscopy. KEY RESULTS: The wild type A(2B) adenosine receptor and deletion mutants were all extensively internalized following prolonged treatment with NECA. The intracellular compartment through which the Gln(325)-stop receptor mutant, which lacks the Type II PDZ motif found in the wild type receptor initially trafficked was not the same as the wild type receptor. Expression of dominant negative mutants of arrestin-2, dynamin or Eps-15 inhibited internalization of wild type and Leu(330)-stop receptors, whereas only dominant negative mutant dynamin inhibited agonist-induced internalization of Gln(325)-stop, Ser(326)-stop and Phe(328)-stop receptors. Following internalization, the wild type A(2B) adenosine receptor recycled rapidly to the cell surface, whereas the Gln(325)-stop receptor did not recycle. CONCLUSIONS AND IMPLICATIONS: Deletion of the COOH-terminus of the A(2B) adenosine receptor beyond Leu(330) switches internalization from an arrestin- and clathrin-dependent pathway to one that is dynamin dependent but arrestin and clathrin independent. The presence of a Type II PDZ motif appears to be essential for arrestin- and clathrin-dependent internalization, as well as recycling of the A(2B) adenosine receptor following prolonged agonist addition.


Subject(s)
Arrestin/metabolism , Clathrin/metabolism , Dynamins/metabolism , Receptors, Purinergic P1/metabolism , Amino Acid Motifs , Animals , CHO Cells , Cell Membrane/metabolism , Cricetinae , Cricetulus , Enzyme-Linked Immunosorbent Assay , Female , Glutamine/metabolism , Leucine/metabolism , Microscopy, Fluorescence , Phenylalanine/metabolism , Protein Structure, Tertiary , Protein Transport , Rats , Receptor, Adenosine A2B/metabolism , Sequence Deletion
4.
Opt Lett ; 28(13): 1072-4, 2003 Jul 01.
Article in English | MEDLINE | ID: mdl-12879911

ABSTRACT

Polarization holographic and surface-relief gratings have been recorded in an amorphous azobenzene polyester by use of a frequency-doubled argon-ion laser beam at 257 nm. Higher excited states of azobenzene in the trans and cis configurations contribute to the formation of a diffraction grating in this experiment. A combination of right and left circularly polarized writing beams has been found to give the highest diffraction efficiency. The contributions to the total phase difference that arise from anisotropy and surface relief have been separated experimentally, and it is shown that the surface-relief grating contributes a larger phase difference than that which is due to anisotropy.

5.
J Neurochem ; 78(3): 546-51, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11483657

ABSTRACT

At present, little is known regarding the mechanism of metabotropic glutamate receptor (mGluR) trafficking. To facilitate this characterization we inserted a haemagglutinin (HA) epitope tag in the extracellular N-terminal domain of the rat mGluR1a. In human embryonic kidney cells (HEK293), transiently transfected with HA-mGluR1a, the epitope-tagged receptor was primarily localized to the cell surface prior to agonist stimulation. Following stimulation with glutamate (10 microM; 30 min) the HA-mGluR1a underwent internalization to endosomes. Further quantification of receptor internalization was provided by ELISA experiments which showed rapid agonist-induced internalization of the HA-mGluR1a. To determine whether agonist-induced mGluR1a internalization is an arrestin- and dynamin-dependent process, cells were cotransfected with HA-mGluR1a and either of these dynamin-K44A or arrestin-2 (319-418). Expression of either dominant negative mutant constructs with receptor strongly inhibited glutamate-induced (10 microM; 30 min) HA-mGluR1a internalization. In addition, wild-type arrestin-2-green fluorescent protein (arrestin-2-GFP) or arrestin-3-GFP underwent agonist-induced translocation from cytosol to membrane in HEK293 cells coexpressing HA-mGluR1a. Taken together our observations demonstrate that agonist-induced internalization of mGluR1a is an arrestin- and dynamin-dependent process.


Subject(s)
Arrestin/metabolism , Endocytosis/physiology , GTP Phosphohydrolases/metabolism , Protein Transport/physiology , Receptors, Metabotropic Glutamate/metabolism , Animals , Arrestin/genetics , COS Cells , Cell Line , Dose-Response Relationship, Drug , Dynamins , Enzyme-Linked Immunosorbent Assay , GTP Phosphohydrolases/genetics , Genes, Reporter , Glutamic Acid/pharmacology , Humans , Microscopy, Fluorescence , Rats , Receptors, Metabotropic Glutamate/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Time Factors , Transfection
6.
J Biol Chem ; 276(32): 30199-207, 2001 Aug 10.
Article in English | MEDLINE | ID: mdl-11333255

ABSTRACT

The G(s)-coupled rat A(2B) adenosine receptor (A(2B)-AR) was epitope-tagged at the NH(2) terminus with hemagglutinin (HA) and subjected to progressive deletions or point mutations of the COOH terminus in order to determine regions of the receptor that contribute to agonist-induced desensitization and internalization. When expressed stably in Chinese hamster ovary cells, a mutant receptor in which the final 2 amino acids were deleted, the Leu(330)-stop mutant, underwent rapid agonist-induced desensitization and internalization as did the wild type (WT) receptor. However, the Phe(328) and the Gln(325)-stop mutants were resistant to rapid agonist-induced desensitization and internalization. Co-expression of arrestin-2-green fluorescent protein (arrestin-2-GFP) with WT receptor or Leu(330)-stop mutant resulted in rapid translocation of arrestin-2-GFP from cytosol to membrane upon agonist addition. On the other hand, agonist activation of the Phe(328)-stop or Gln(325)-stop mutant did not result in translocation of arrestin-2-GFP from cytosol. A COOH terminus point mutant, S329G, was also unable to undergo rapid agonist-induced desensitization and internalization, indicating that Ser(329) is a critical residue for these processes. A further deletion mutant (Ser(326)-stop) unexpectedly underwent rapid agonist-induced desensitization and internalization. However, activation of this mutant did not promote translocation of arrestin-2-GFP from cytosol to membrane. In addition, whereas WT receptor internalization was markedly inhibited by co-expression of dominant negative mutants of arrestin-2 (arrestin-2-(319-418)), dynamin (dynamin K44A), or Eps-15 (EDelta95-295), Ser(326)-stop receptor internalization was only inhibited by dominant negative mutant dynamin. Taken together these results indicate that Ser(329), close to the COOH terminus of the rat A(2B)-AR, is critical for the rapid agonist-induced desensitization and internalization of the receptor. However, deletion of the COOH terminus also uncovers a motif that is able to redirect internalization of the receptor to an arrestin- and clathrin-independent pathway.


Subject(s)
Receptors, Purinergic P1/chemistry , Receptors, Purinergic P1/metabolism , Serine/chemistry , Amino Acid Sequence , Animals , Arrestins/genetics , Arrestins/metabolism , Binding Sites , CHO Cells , Cell Membrane/metabolism , Clathrin/metabolism , Cloning, Molecular , Cricetinae , Cytosol/metabolism , DNA, Complementary/metabolism , Dose-Response Relationship, Drug , Dynamins , Enzyme-Linked Immunosorbent Assay , Epitopes , GTP Phosphohydrolases/genetics , Gene Deletion , Genes, Dominant , Glutamine/chemistry , Green Fluorescent Proteins , Leucine/chemistry , Luminescent Proteins/metabolism , Microscopy, Fluorescence , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Phenylalanine/chemistry , Phosphoproteins/genetics , Phosphoproteins/metabolism , Point Mutation , Rats , Receptor, Adenosine A2B , Receptors, Purinergic P1/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Time Factors , Transfection
7.
Eur J Biochem ; 268(6): 1640-5, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11248682

ABSTRACT

Crystallography shows that aspartate aminotransferase binds dicarboxylate substrate analogues by bonds to Arg292 and Arg386, respectively [Jager, J, Moser, M. Sauder, U. & Jansonius, J. N. (1994) J. Mol. Biol., 239, 285-305]. The contribution of each interaction to the conformational change that the enzyme undergoes when it binds ligands via these residues, is assessed by probing mutant forms of the enzyme lacking either or both arginines. The probes used are NaH(3)BCN which reduces the cofactor imine, the reactive substrate analogue, cysteine sulfinate and proteolysis by trypsin. The unreactive substrate analogue, maleate, is used to induce closure. Each single mutant reacted only 2.5-fold more slowly with NaH(3)BCN than the wild-type indicating that charge repulsion by the arginines contributes little to maintaining the open conformation. Maleate lowered the rate of reduction of the wild-type enzyme more than 300-fold but had little effect on the reaction of the mutant enzymes indicating that the ability of this dicarboxylate analogue to bridge the arginines precisely makes the major contribution to closure. The R292L mutant reacted 20 times more rapidly with cysteine sulfinate than R386L but 5 x 10(4) times more slowly than the wild-type enzyme, consistent with the proposal that enzyme's catalytic abilities are not developed unless closure is induced by bridging of the arginines. Proteolysis of the mutants with trypsin showed that, in the wild-type enzyme, the bonds most susceptible to trypsin are those contributed by Arg292 and Arg386. Proteolysis of the next most susceptible bond, at Arg25 in the double mutant, was protected by maleate demonstrating the presence of an additional site on the enzyme for binding dicarboxylates.


Subject(s)
Aspartate Aminotransferases/metabolism , Escherichia coli/enzymology , Maleates/metabolism , Amino Acid Sequence , Aspartate Aminotransferases/chemistry , Binding Sites , Borohydrides/chemistry , Cysteine/analogs & derivatives , Cysteine/chemistry , Hydrogen-Ion Concentration , Kinetics , Neurotransmitter Agents , Spectrometry, Mass, Electrospray Ionization , Spectrophotometry, Ultraviolet , Substrate Specificity , Trypsin/metabolism
8.
Biochemistry ; 39(42): 12828-36, 2000 Oct 24.
Article in English | MEDLINE | ID: mdl-11041847

ABSTRACT

Adenosine mediates the activation of adenylyl cyclase via its interaction with specific A(2A) and A(2B) adenosine receptors. Previously, we demonstrated that arrestins are involved in rapid agonist-promoted desensitization of the A(2B) adenosine receptor (A(2B)AR) in HEK293 cells. In the present study, we investigate the role of arrestins in A(2B)AR trafficking. Initial studies demonstrated that HEK293 cells stably expressing arrestin antisense constructs, which reduce endogenous arrestin levels, effectively reduced A(2B)AR internalization. A(2B)AR recycling after agonist-induced endocytosis was also significantly impaired in cells with reduced arrestin levels. Interestingly, while overexpression of arrestin-2 or arrestin-3 rescued A(2B)AR internalization and recycling, arrestin-3 promoted a significantly faster rate of recycling as compared to arrestin-2. The specificity of arrestin interaction with A(2B)ARs was further investigated using arrestins fused to the green fluorescent protein (arr-2-GFP and arr-3-GFP). Both arrestins underwent rapid translocation (<1 min) from the cytosol to the plasma membrane following A(2B)AR activation. However, longer incubations with agonist (>10 min) revealed that arr-2-GFP but not arr-3-GFP colocalized with the A(2B)AR in rab-5 and transferrin receptor containing early endosomes. At later times, the A(2B)AR but not arr-2-GFP was observed in an apparent endocytic recycling compartment. Thus, while arrestin-2 and arrestin-3 mediate agonist-induced A(2B)AR internalization with relative equal potency, arrestin isoform binding dictates the differential kinetics of A(2B)AR recycling and resensitization.


Subject(s)
Arrestin/physiology , Receptors, Purinergic P1/metabolism , Adenosine-5'-(N-ethylcarboxamide)/agonists , Animals , Arrestin/genetics , Cell Line , Genetic Vectors/metabolism , Green Fluorescent Proteins , Humans , Kinetics , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Protein Isoforms/genetics , Protein Isoforms/physiology , Purinergic P1 Receptor Agonists , Rats , Receptor, Adenosine A2B , Transfection , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism
9.
Nature ; 401(6752): 502-5, 1999 Sep 30.
Article in English | MEDLINE | ID: mdl-10519556

ABSTRACT

Antibiotic-producing polyketide synthases (PKSs) are enzymes responsible for the biosynthesis in Streptomyces and related filamentous bacteria of a remarkably broad range of bioactive metabolites, including antitumour aromatic compounds such as mithramycin and macrolide antibiotics such as erythromycin. The molecular basis for the selection of the starter unit on aromatic PKSs is unknown. Here we show that a component of aromatic PKS, previously named 'chain-length factor', is a factor required for polyketide chain initiation and that this factor has decarboxylase activity towards malonyl-ACP (acyl carrier protein). We have re-examined the mechanism of initiation on modular PKSs and have identified as a specific initiation factor a domain of previously unknown function named KSQ, which operates like chain-length factor. Both KSQ and chain-length factor are similar to the ketosynthase domains that catalyse polyketide chain extension in modular multifunctional PKSs and in aromatic PKSs, respectively, except that the ketosynthase domain active-site cysteine residue is replaced by a highly conserved glutamine in KSQ and in chain-length factor. The glutamine residue is important both for decarboxylase activity and for polyketide synthesis.


Subject(s)
Macrolides/metabolism , Multienzyme Complexes/metabolism , Acyl Carrier Protein/metabolism , Anthraquinones/metabolism , Binding Sites , Carboxy-Lyases/metabolism , Cloning, Molecular , Glutamine/metabolism , Multienzyme Complexes/chemistry , Multienzyme Complexes/genetics , Mutation
10.
Chem Biol ; 5(12): 699-711, 1998 Dec.
Article in English | MEDLINE | ID: mdl-9862793

ABSTRACT

BACKGROUND: It has been proposed that Streptomyces malonyl CoA: holo acyl carrier protein transacylases (MCATs) provide a link between fatty acid and polyketide biosynthesis. Two recent studies have provided evidence that the presence of MCAT is essential for polyketide synthesis to proceed in reconstituted minimal polyketide synthases (PKSs). In contrast to this, we previously showed that the holo acyl carrier proteins (ACPs) from type II PKSs are capable of catalytic self-malonylation in the presence of malonyl CoA, which suggests that MCAT might not be necessary for polyketide biosynthesis. RESULTS: We reconstituted a homologous actinorhodin (act) type II minimal PKS in vitro. When act holo-ACP is present in limiting concentrations, MCAT is required by the synthase complex in order for polyketide biosynthesis to proceed. When holo-ACP is present in excess, however, efficient polyketide synthesis proceeds without MCAT. The rate of polyketide production increases with holo-ACP concentration, but at low ACP concentration or equimolar AC:KS:CLF (KS, ketosynthase; CLF, chain length determining factor) concentrations this rate is significantly lower than expected, indicating that free holo-ACP is sequestered by the KS/CLF complex. CONCLUSIONS: The rate of polyketide biosynthesis is dictated by the ratio of holo-ACP to KS and CLF, as well as by the total protein concentration. There is no absolute requirement for MCAT in polyketide biosynthesis in vitro, although the role of MCAT during polyketide synthesis in vivo remains an open question. MCAT might be responsible for the rate enhancement of malonyl transfer at very low free holo-ACP concentrations or it could be required to catalyse the transfer of malonyl groups from malonyl CoA to sequestered holo-ACP.


Subject(s)
Acyltransferases/metabolism , Bacterial Proteins , Streptomyces/enzymology , Acyl-Carrier Protein S-Malonyltransferase , Acyltransferases/genetics , Acyltransferases/isolation & purification , Catalysis , Chromatography, High Pressure Liquid , Cloning, Molecular , Escherichia coli , Escherichia coli Proteins , Fatty Acid Synthase, Type II , Kinetics , Malonates/metabolism , Malonyl Coenzyme A/metabolism , Polyketide Synthases
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