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1.
Biology (Basel) ; 11(8)2022 Jul 29.
Article in English | MEDLINE | ID: mdl-36009773

ABSTRACT

Microalgae have demonstrated a large potential in biotechnology as a source of various macromolecules (proteins, carbohydrates, and lipids) and high-added value products (pigments, poly-unsaturated fatty acids, peptides, exo-polysaccharides, etc.). The production of biomass at a large scale becomes more economically feasible when it is part of a biorefinery designed within the circular economy concept. Thus, the aim of this critical review is to highlight and discuss challenges and future trends related to the multi-product microalgae-based biorefineries, including both phototrophic and mixotrophic cultures treating wastewater and the recovery of biomass as a source of valuable macromolecules and high-added and low-value products (biofertilizers and biostimulants). The therapeutic properties of some microalgae-bioactive compounds are also discussed. Novel trends such as the screening of species for antimicrobial compounds, the production of bioplastics using wastewater, the circular economy strategy, and the need for more Life Cycle Assessment studies (LCA) are suggested as some of the future research lines.

2.
Molecules ; 25(14)2020 Jul 14.
Article in English | MEDLINE | ID: mdl-32674349

ABSTRACT

Curcumin, the most important secondary metabolite isolated from Curcuma longa, is known for its numerous purported therapeutic properties and as a natural dye. Herein, based on curcumin's intrinsic fluorescence, a search for improved curcumin-based fluorophores was conducted. Within the set of semi-synthetic curcumin derivatives i.e. mono (1), di (2), tri (3), tetra (4) benzylated and dibenzyl-fluoroborate (5), the fluorescence properties of 2 and 5 in solution outstood with a two-fold quantum yield compared to curcumin. Furthermore, all benzylated derivatives showed a favorable minimal cytotoxic activity upon screening at 25 µM against human cancer and non-tumoral COS-7 cell lines, with a reduction of its cytotoxic effect related to the degree of substitution. Fluorophores 2 and 5 are versatile bioimaging tools, as revealed by Confocal Fluorescence Microscopy (CFM), and showed permeation of living cell membranes of astrocytes and astrocytomas. When 2 is excited with a 405- (blue) or 543-nm (green) laser, it is possible to exclusively and intensively visualize the nucleus. However, the fluorescence emission fades as the laser wavelength moves towards the red region. In comparison, 5 allows selective visualization of cytoplasm when a 560-nm laser is used, showing emission in the NIR region, while it is possible to exclusively observe the nucleus at the blue region with a 405-nm laser.


Subject(s)
Cell Nucleus , Cytoplasm , Diarylheptanoids/chemistry , Diarylheptanoids/pharmacology , Fluorescent Dyes/chemistry , Fluorescent Dyes/pharmacology , Molecular Imaging , Cell Nucleus/metabolism , Curcumin/chemistry , Curcumin/pharmacology , Cytoplasm/metabolism , Models, Molecular , Molecular Conformation , Molecular Imaging/methods , Molecular Structure
3.
J Biomol Struct Dyn ; 34(1): 78-91, 2016.
Article in English | MEDLINE | ID: mdl-25702612

ABSTRACT

In order to contribute to the structural basis for rational design of calmodulin (CaM) inhibitors, we analyzed the interaction of CaM with 14 classic antagonists and two compounds that do not affect CaM, using docking and molecular dynamics (MD) simulations, and the data were compared to available experimental data. The Ca(2+)-CaM-Ligands complexes were simulated 20 ns, with CaM starting in the "open" and "closed" conformations. The analysis of the MD simulations provided insight into the conformational changes undergone by CaM during its interaction with these ligands. These simulations were used to predict the binding free energies (ΔG) from contributions ΔH and ΔS, giving useful information about CaM ligand binding thermodynamics. The ΔG predicted for the CaM's inhibitors correlated well with available experimental data as the r(2) obtained was 0.76 and 0.82 for the group of xanthones. Additionally, valuable information is presented here: I) CaM has two preferred ligand binding sites in the open conformation known as site 1 and 4, II) CaM can bind ligands of diverse structural nature, III) the flexibility of CaM is reduced by the union of its ligands, leading to a reduction in the Ca(2+)-CaM entropy, IV) enthalpy dominates the molecular recognition process in the system Ca(2+)-CaM-Ligand, and V) the ligands making more extensive contact with the protein have higher affinity for Ca(2+)-CaM. Despite their limitations, docking and MD simulations in combination with experimental data continue to be excellent tools for research in pharmacology, toward a rational design of new drugs.


Subject(s)
Calcium/chemistry , Calmodulin/chemistry , Ligands , Protein Conformation/drug effects , Benzoxazoles , Binding Sites , Calcium/metabolism , Calmodulin/antagonists & inhibitors , Calmodulin/metabolism , Maleimides , Molecular Docking Simulation , Molecular Dynamics Simulation , Protein Binding , Thermodynamics
4.
Channels (Austin) ; 7(3): 225-8, 2013.
Article in English | MEDLINE | ID: mdl-23584197

ABSTRACT

We have recently examined slow inactivation of Shab channels. Here we extend our characterization of Shab slow inactivation by presenting the properties of recovery from inactivation. The observations support our proposal that Shab reaches the same inactivated state either from open or closed states and suggest that closed and open state inactivation share the same mechanism. Regarding the latter, we also show that external K (+) and TEA slow down recovery from inactivation in agreement with the hypothesis that the mechanism of Shab inactivation qualitatively differs from C-type inactivation.


Subject(s)
Calcium/metabolism , Nitric Oxide/metabolism , Purkinje Cells/metabolism , Ryanodine Receptor Calcium Release Channel/metabolism , Animals
5.
Channels (Austin) ; 7(2): 97-108, 2013.
Article in English | MEDLINE | ID: mdl-23419584

ABSTRACT

Herein, we report the first characterization of Shab slow inactivation. Open Shab channels inactivate within seconds, with two voltage-independent time constants. Additionally, Shab presents significant closed-state inactivation. We found that with short depolarizing pulses, shorter than the slowest inactivation time constant, the resulting inactivation curve has a marked U-shape, but as pulse duration increases, approaching steady-state conditions, the U-shape vanishes, and the resulting inactivation curves converge to the classical Boltzmann h∞ curve. Regarding the mechanism of inactivation, we found that external K (+) and TEA facilitate both open- and closed-state inactivation, while the cavity blocker quinidine hinders inactivation. These results together with our previous observations regarding the K (+) -dependent stability of the K (+) conductance, suggest the novel hypothesis that inactivation of Shab channels, and possibly that of other Kv channels whose inactivation is facilitated by K (+) , does not involve a significant narrowing of the extracellular entry of the pore. Instead, we hypothesize that there is only a rearrangement of a more internal segment of the pore that affects the central cavity and halts K (+) conduction.


Subject(s)
Drosophila Proteins/physiology , Ion Channel Gating , Potassium/pharmacology , Shab Potassium Channels/physiology , Animals , Drosophila Proteins/antagonists & inhibitors , Drosophila Proteins/chemistry , Drosophila melanogaster/chemistry , Drosophila melanogaster/physiology , Potassium/metabolism , Potassium Channel Blockers/pharmacology , Protein Structure, Tertiary , Quinidine/pharmacology , Sf9 Cells , Shab Potassium Channels/antagonists & inhibitors , Shab Potassium Channels/chemistry , Spodoptera , Tetraethylammonium/pharmacology
6.
PLoS One ; 7(5): e38187, 2012.
Article in English | MEDLINE | ID: mdl-22666479

ABSTRACT

The Arc two-component system modulates the expression of numerous genes in response to respiratory growth conditions. This system comprises ArcA as the response regulator and ArcB as the sensor kinase. ArcB is a tripartite histidine kinase whose activity is regulated by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation. Here, we report that the ArcB protein segment covering residues 70-121, fulfills the molecular characteristics of a leucine zipper containing coiled coil structure. Also, mutational analyses of this segment reveal three different phenotypical effects to be distributed along the coiled coil structure of ArcB, demonstrating that this motif is essential for proper ArcB signaling.


Subject(s)
Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Leucine Zippers , Membrane Proteins/chemistry , Membrane Proteins/metabolism , Protein Kinases/chemistry , Protein Kinases/metabolism , Amino Acid Sequence , Escherichia coli/enzymology , Escherichia coli Proteins/genetics , Membrane Proteins/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Phosphoric Monoester Hydrolases/metabolism , Protein Kinases/genetics , Sequence Analysis
7.
Channels (Austin) ; 5(1): 56-64, 2011.
Article in English | MEDLINE | ID: mdl-21084865

ABSTRACT

Celecoxib is a drug designed to selectively inhibit COX-2, an inflammation-inducible cyclooxygenase isoform, over the constitutively expressed COX-1 isoform. In addition to this selective inhibition it is now known that celecoxib exerts a variety of effects on several types of ion channels, thus producing secondary physiological effects. In this work we demonstrate that at therapeutically relevant concentrations celecoxib interacts with Shab K(+) channels specifically promoting a fast inactivation gating (without blocking the pore or significantly affecting other gating processes). At least two celecoxib molecules bind to each channel promoting a fast inactivation that develops from both open and closed states. Channel inactivation in turn causes a reduction of the size of I(K). Taken together, our observations show that in addition to its intended therapeutic target celecoxib is a useful tool to further study the mechanism of Shab channel inactivation.


Subject(s)
Cyclooxygenase 2 Inhibitors/pharmacology , Ion Channel Gating/drug effects , Potassium/metabolism , Pyrazoles/pharmacology , Shab Potassium Channels/drug effects , Sulfonamides/pharmacology , Animals , Baculoviridae/genetics , Celecoxib , Cell Line , Cyclooxygenase 2 Inhibitors/metabolism , Kinetics , Membrane Potentials , Protein Binding , Pyrazoles/metabolism , Recombinant Proteins/drug effects , Recombinant Proteins/metabolism , Shab Potassium Channels/genetics , Shab Potassium Channels/metabolism , Sulfonamides/metabolism , Transfection
8.
Nat Struct Mol Biol ; 16(7): 704-10, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19561608

ABSTRACT

Transient receptor potential vanilloid 1 (TRPV1) channels mediate several types of physiological responses. Despite the importance of these channels in pain detection and inflammation, little is known about how their structural components convert different types of stimuli into channel activity. To localize the activation gate of these channels, we inserted cysteines along the S6 segment of mutant TRPV1 channels and assessed their accessibility to thiol-modifying agents. We show that access to the pore of TRPV1 is gated by S6 in response to both capsaicin binding and increases in temperature, that the pore-forming S6 segments are helical structures and that two constrictions are present in the pore: one that impedes the access of large molecules and the other that hampers the access of smaller ions and constitutes an activation gate of these channels.


Subject(s)
Models, Molecular , Protein Structure, Secondary , Protein Structure, Tertiary , TRPV Cation Channels/chemistry , TRPV Cation Channels/metabolism , Amino Acid Sequence , Animals , Capsaicin/metabolism , Cysteine/chemistry , Ion Channel Gating/physiology , Ions/metabolism , Leucine/chemistry , Mesylates/chemistry , Molecular Sequence Data , Rats , Sensory System Agents/metabolism , TRPV Cation Channels/genetics , Temperature , Tyrosine/chemistry
9.
PLoS One ; 3(10): e3560, 2008.
Article in English | MEDLINE | ID: mdl-18958281

ABSTRACT

BACKGROUND: The Ca(v)beta subunits of high voltage-activated Ca(2+) channels control the trafficking and biophysical properties of the alpha(1) subunit. The Ca(v)beta-alpha(1) interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that betas regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the alpha-interaction domain (AID) be a rigid structure. METHODOLOGY/PRINCIPAL FINDINGS: The present study tests this hypothesis by altering the flexibility and orientation of this region in alpha(1)2.2, then testing for Ca(v)beta regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished beta2a and beta3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of beta2a to produce non-inactivating currents. Orientation of Ca(v)beta with respect to alpha(1)2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca(v)beta subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca(v)beta subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms beta-sheet. The orientation of beta with respect to alpha was confirmed by the bimolecular fluorescence complementation assay. CONCLUSIONS/SIGNIFICANCE: These results show that the orientation of the Ca(v)beta subunit relative to the alpha(1)2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca(v)beta to regulate channel activity.


Subject(s)
Calcium Channels, L-Type/chemistry , Calcium Channels, L-Type/physiology , Calcium Channels, N-Type/physiology , Cell Polarity/physiology , Calcium Channels, L-Type/genetics , Calcium Channels, N-Type/chemistry , Calcium Channels, N-Type/genetics , Cells, Cultured , Electrophysiology , Homeostasis/physiology , Humans , Ion Channel Gating/physiology , Models, Biological , Models, Molecular , Mutagenesis/physiology , Patch-Clamp Techniques , Protein Folding , Protein Structure, Tertiary/physiology , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/physiology
10.
J Biol Chem ; 283(13): 8136-44, 2008 Mar 28.
Article in English | MEDLINE | ID: mdl-18218623

ABSTRACT

Mutations in the I-II loop of Ca(v)3.2 channels were discovered in patients with childhood absence epilepsy. All of these mutations increased the surface expression of the channel, whereas some mutations, and in particular C456S, altered the biophysical properties of channels. Deletions around C456S were found to produce channels that opened at even more negative potentials than control, suggesting the presence of a gating brake that normally prevents channel opening. The goal of the present study was to identify the minimal sequence of this brake and to provide insights into its structure. A peptide fragment of the I-II loop was purified from bacteria, and its structure was analyzed by circular dichroism. These results indicated that the peptide had a high alpha-helical content, as predicted from secondary structure algorithms. Based on homology modeling, we hypothesized that the proximal region of the I-II loop may form a helix-loop-helix structure. This model was tested by mutagenesis followed by electrophysiological measurement of channel gating. Mutations that disrupted the helices, or the loop region, had profound effects on channel gating, shifting both steady state activation and inactivation curves, as well as accelerating channel kinetics. Mutations designed to preserve the helical structure had more modest effects. Taken together, these studies showed that any mutations in the brake, including C456S, disrupted the structural integrity of the brake and its function to maintain these low voltage-activated channels closed at resting membrane potentials.


Subject(s)
Calcium Channels, T-Type/chemistry , Calcium Channels, T-Type/metabolism , Ion Channel Gating , Amino Acid Sequence , Calcium Channels, T-Type/genetics , Cell Line , Conserved Sequence , Electrophysiology , Humans , Models, Molecular , Molecular Sequence Data , Mutation/genetics , Patch-Clamp Techniques , Protein Structure, Secondary , Sequence Alignment
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