Inhibition of calcium-calmodulin complex formation by vasorelaxant basic dipeptides demonstrated by in vitro and in silico analyses

Background

Tryptophan-histidine (Trp-His) was found to suppress the activity of the Ca^2 +/calmodulin (CaM)-dependent protein kinases II (CaMKII), which requires the Ca^2 +-CaM complex for an initial activation. In this study, we attempted to clarify whether Trp-His inhibits Ca^2 +-CaM complex formation, a CaMKII activator.

Methods

The ability of Trp-His and other peptides to inhibit Ca^2 +-CaM complex formation was investigated by a Ca^2 +-encapsulation fluorescence assay. The peptide-CaM interactions were illustrated by molecular dynamic simulation.

Results

We showed that Trp-His inhibited Ca^2 +-CaM complex formation with a 1:1 binding stoichiometry of the peptide to CaM, considering that Trp-His reduced Hill coefficient of Ca^2 +-CaM binding from 2.81 to 1.92. His-Trp also showed inhibitory activity, whereas Trp + His, 3-methyl His-Trp, and Phe-His did not show significant inhibitory activity, suggesting that the inhibitory activity was due to a peptide skeleton (irrespective of the sequence), a basic amino acid, a His residue, the N hydrogen atom of its imidazole ring, and Trp residue. In silico studies suggested the possibility that Trp-His and His-Trp interacted with the Ca^2 +-binding site of CaM by forming hydrogen bonds with key Ca^2 +-binding residues of CaM, with a binding free energy of − 49.1 and − 68.0 kJ/mol, respectively.

Conclusions

This is the first study demonstrating that the vasoactive dipeptide Trp-His possesses inhibitory activity against Ca^2 +-CaM complex formation, which may elucidate how Trp-His inhibited CaMKII in a previous study.

General significance

The results provide a basic idea that could lead to the development of small peptides binding with high affinity to CaM and inhibiting Ca^2 +-CaM complex formation in the future.     

Glutaredoxin 1 is a major player in copper metabolism in neuroblastoma cells

Background

Glutaredoxin 1 (Grx1), a small protein belonging to the thioredoxin family, is involved in redox-regulation since it catalyzes the reduction of protein disulfides and that of mixed disulfides. It was reported to modulate active copper extrusion from cells, by affecting the function of the pumps ATP7A and B. These are components of the network of protein chaperones involved in the control of the homeostasis of copper, an essential, though harmful, metal. However, the effect of Grx1 on copper levels, copper chaperones and copper-elicited cell toxicity was never investigated.

Methods

In order to investigate the effect of Grx1 on copper metabolism, we constitutively overexpressed Grx1 in human neuroblastoma SH-SY5Y cells (SH-Grx1 cells) and assessed a number of copper-related parameters.

Results

SH-Grx1 cells show a basal intracellular copper level higher than control cells, accumulate more copper upon CuSO₄ treatment, but are more resistant to copper-induced toxicity. Grx1 shows copper-binding properties and copper overload produces a decrease of Grx1 enzyme activity in SH-Grx1 cells. Finally, Grx1 overexpression decreases copper accumulation in mitochondria upon copper overload and modulates the expression of copper transporter 1 (Ctr1).

Conclusion

Altogether, these data demonstrate that Grx1 is a major player in copper metabolism in neuronal cells.     

Crystal structure of a Bombyx mori sigma-class glutathione transferase exhibiting prostaglandin E synthase activity

Background

Glutathione transferases (GSTs) are members of a major family of detoxification enzymes. Here, we report the crystal structure of a sigma-class GST of Bombyx mori, bmGSTS1, to gain insight into the mechanism catalysis.

Methods

The structure of bmGSTS1 and its complex with glutathione were determined at resolutions of 1.9 Å and 1.7 Å by synchrotron radiation and the molecular replacement method.

Results

The three-dimensional structure of bmGSTS1 shows that it exists as a dimer and is similar in structure to other GSTs with respect to its secondary and tertiary structures. Although striking similarities to the structure of prostaglandin D synthase were also detected, we were surprised to find that bmGSTS1 can convert prostaglandin H₂ into its E₂ form. Comparison of bmGSTS1 with its glutathione complex showed that bound glutathione was localized to the glutathione-binding site (G-site). Site-directed mutagenesis of bmGSTS1 mutants indicated that amino acid residues Tyr8, Leu14, Trp39, Lys43, Gln50, Met51, Gln63, and Ser64 in the G-site contribute to catalytic activity.

Conclusion

We determined the tertiary structure of bmGSTS1 exhibiting prostaglandin E synthase activity.

General significance

These results are, to our knowledge, the first report of a prostaglandin synthase activity in insects.     

Allosteric modulation of zinc speciation by fatty acids

Background

Serum albumin is the major protein component of blood plasma and is responsible for the circulatory transport of a range of small molecules that include fatty acids, hormones, metal ions and drugs. Studies examining the ligand-binding properties of albumin make up a large proportion of the literature. However, many of these studies do not address the fact that albumin carries multiple ligands (including metal ions) simultaneously in vivo. Thus the binding of a particular ligand may influence both the affinity and dynamics of albumin interactions with another.

Scope of review

Here we review the Zn^2 + and fatty acid transport properties of albumin and highlight an important interplay that exists between them. Also the impact of this dynamic interaction upon the distribution of plasma Zn^2 +, its effect upon cellular Zn^2 + uptake and its importance in the diagnosis of myocardial ischemia are considered.

Major conclusions

We previously identified the major binding site for Zn^2 + on albumin. Furthermore, we revealed that Zn^2 +-binding at this site and fatty acid-binding at the FA2 site are interdependent. This suggests that the binding of fatty acids to albumin may serve as an allosteric switch to modulate Zn^2 +-binding to albumin in blood plasma.

General significance

Fatty acid levels in the blood are dynamic and chronic elevation of plasma fatty acid levels is associated with some metabolic disorders such as cardiovascular disease and diabetes. Since the binding of Zn^2 + to albumin is important for the control of circulatory/cellular Zn^2 + dynamics, this relationship is likely to have important physiological and pathological implications. This article is part of a Special Issue entitled Serum Albumin.     

Nucleic acid binding properties of allicin: Spectroscopic analysis and estimation of anti-tumor potential

Background

Allicin has received much attention due to its anti-proliferative activity and not-well elucidated underlying mechanism of action. This work focuses towards determining the cellular toxicity of allicin and understanding its interaction with nucleic acid at molecular level.

Methods

MTT assay was used to assess the cell viability of A549 lung cancer cells against allicin. Fourier transform infrared (FTIR) and UV-visible spectroscopy were used to study the binding parameters of nucleic acid-allicin interaction.

Results

Allicin inhibits the proliferation of cancer cells in a concentration dependent manner. FTIR spectroscopy exhibited that allicin binds preferentially to minor groove of DNA via thymine base. Analysis of tRNA allicin complex has also revealed that allicin binds primarily through nitrogenous bases. Some amount of external binding with phosphate backbone was also observed for both DNA and RNA. UV visible spectra of both DNA allicin and RNA allicin complexes showed hypochromic shift with an estimated binding constant of 1.2 × 10⁴ M^- 1 for DNA and 1.06 × 10³ M^− 1for RNA binding. No major transition from the B-form of DNA and A-form of RNA is observed after their interaction with allicin.

Conclusions

The results demonstrated that allicin treatment inhibited the proliferation of A549 cells in a dose-dependent manner. Biophysical outcomes are suggestive of base binding and helix contraction of nucleic acid structure upon binding with allicin.

General significance

The results describe cytotoxic potential of allicin and its binding properties with cellular nucleic acid, which could be helpful in deciphering the complete mechanism of cell death exerted by allicin.     

Spinal mechanism of micturition reflex inhibition by naftopidil in rats

Aim

We investigated the spinal mechanism through which naftopidil inhibits the micturition reflex by comparing the effects of noradrenaline and naftopidil in rats.

Methods

The following were investigated: the influence of oral naftopidil on plasma monoamine and amino acid levels, the distribution of oral 14C-naftopidil, the effects of intravenous (IV) or intrathecal (IT) injection of noradrenaline or naftopidil on isovolumetric bladder contractions, amino acid levels in the lumbosacral spinal cord after IT noradrenaline or naftopidil, and the effects of IT naftopidil and strychnine and/or bicuculline on isovolumetric bladder contractions.

Key findings

Oral naftopidil decreased the plasma adrenaline level, while it increased the serotonin and glycine levels. After oral administration, 14C-naftopidil was detected in the spinal cord and cerebrum, as well as in plasma and the prostate gland. When the bladder volume was below the threshold for isovolumetric reflex contractions, IV (0.1 mg) or IT (0.1 μg) noradrenaline evoked bladder contractions, but IV (1 mg) or IT (0.01–1 μg) naftopidil did not. When the bladder volume was above the threshold for isovolumetric reflex contractions, IV or IT noradrenaline transiently abolished bladder contractions. IT noradrenaline decreased the levels of glycine and gamma-aminobutyric acid (GABA) in the lumbosacral cord, while IT naftopidil increased the GABA level. IT strychnine and/or bicuculline blocked the inhibitory effect of IT naftopidil on bladder contractions.

Significance

Naftopidil inhibits the micturition reflex by blocking α1 receptors, as well as by the activation of serotonergic, glycinergic, and GABAergic neurons in the central nervous system.     

Novel complex Re-Arrangement of ARG1 commonly shared by unrelated patients with Hyperargininemia

Background

Hyperargininemia is a very rare progressive neurometabolic disorder caused by deficiency of hepatic cytosolic arginase I, resulting from mutations in the ARG1 gene. Until now, some mutations were reported worldwide and none of them were of Southeast Asian origins. Furthermore, most reported mutations were point mutations and a few others deletions or insertions.

Objective

This study aims at identifying the disease-causing mutation in the ARG1 gene of Malaysian patients with hyperargininemia.

Methodology

We employed a series of PCR amplifications and direct sequencing in order to identify the mutation. We subsequently used quantitative real-time PCR to determine the copy number of the exons flanking the mutation. We blasted our sequencing data with that of the reference sequence in the NCBI in order to obtain positional insights of the mutation.

Results

We found a novel complex re-arrangement involving insertion, inversion and gross deletion of ARG1 (designated g.insIVS1 + 1899GTTTTATCAT;g.invIVS1 + 1933_ + 1953;g.delIVS1 + 1954_IVS2 + 914;c.del116_188;p.Pro20SerfsX4) commonly shared by 5 patients with hyperargininemia, each originating from different family. None of the affected families share known relationship with each other, although four of the five patients were known to have first-cousin consanguineous parents.

Conclusion

This is the first report of complex re-arrangement in the ARG1. Further analyses showing that the patients have shared the same geographic origin within the northeastern part of Malaysia prompted us to suggest a simple molecular screening of hyperargininemia within related ethnicities using a long-range PCR.     

Structural studies of several clinically important oncology drugs in complex with human serum albumin

Background

Serum albumin is a major pharmacokinetic effector of drugs. To gain further insight into albumin binding chemistry, the crystal structures of six oncology agents were determined in complex with human serum albumin at resolutions of 2.8 to 2.0 Å: camptothecin, 9-amino-camptothecin, etoposide, teniposide, bicalutamide and idarubicin.

Methods

Protein crystal growth and low temperature X-ray crystallography

Results

These large, complex drugs are all bound within the subdomain IB binding region which can be described as a hydrophobic groove formed by α-helices h7, h8 and h9 covered by the extended polypeptide L1. L1 creates a binding cavity with two access sites, one between loop L1 and α-helices h7 and h8 (distal site: IB_d) and the other between L1 and α-helix h9 (proximal site: IB_p). Camptothecin (2.4 Å) and 9 amino camptothecin (2.0 Å) are clearly bound as the open lactone form (IB_p). Idarubicin (2.8 Å) binds in a DNA like dimer complex via an intermolecular π stacking arrangement in IB_d. Bicalutamide (2.4 Å) is bound in a folded intramolecular π stacking arrangement between two aromatic rings in IB_d similar to idarubicin. Teniposide (2.7 Å) and etoposide (2.7 Å), despite small chemical differences, are bound in two distinctly different sites at or near IB. Teniposide is internalized via primarily hydrophobic interactions and spans through both openings (IB_p-d). Etoposide is bound between the exterior of IB and IIA and exhibits an extensive hydrogen bonding network.

Conclusions

Subdomain IB is a major binding site for complex heterocyclic molecules.

General significance

The structures have important implications for drug design and development. This article is part of a Special Issue entitled Serum Albumin.     

Zn rather than Ca or Mg used as a cofactor in non-muscular actin from the oyster to control protein polymerization

Background

The major cytoskeletal protein of most cells is actin, which polymerizes to form actin filaments (F-actin). Each actin monomer (G-actin) contains a divalent alkaline earth metal ion (in vivo Mg^2 +; in vitro usually Ca^2 +) as a cofactor that is crucial for protein polymerization. Prior to this study, however, whether or not other types of metal ions can play the same role as Mg^2 + or Ca^2 + in actins remains unknown.

Methods

A new actin from the gills of oyster (AGO) was prepared and characterized by protein purification techniques, SDS- and native-PAGE, and LC–MS\MS for the first time. The property of this protein was studied by CD, fluorescence and UV/vis spectroscopy, laser light scattering, and TEM.

Results

AGO is a monomer with a MW of ~ 42 kDa. AGO is unique among all known actins in that Zn^2 + is only a naturally binding metal in the protein, and that one native AGO molecule binds 8 zinc ions, which can be removed by EDTA treatment at pH 7.2. The presence of zinc has a great effect on the secondary and tertiary structure of the protein. Correlated with such effect is that these zinc ions in native AGO facilitate protein polymerization, whereas removal of zinc ions from native AGO results in a loss of such polymerization property.

Conclusions

The present work demonstrates that AGO is a novel zinc-binding protein with high capacity, and high selectivity.

General significance

This work extends an understanding of the function of zinc and actin.     

Calcium binding studies of peptides of human phospholipid scramblases 1 to 4 suggest that scramblases are new class of calcium binding proteins in the cell

Background

Phospholipid scramblases are a group of four homologous proteins conserved from C. elegans to human. In human, two members of the scramblase family, hPLSCR1 and hPLSCR3 are known to bring about Ca²⁺ dependent translocation of phosphatidylserine and cardiolipin respectively during apoptotic processes. However, affinities of Ca²⁺/Mg²⁺ binding to human scramblases and conformational changes taking place in them remains unknown.

Methods

In the present study, we analyzed the Ca²⁺ and Mg²⁺ binding to the calcium binding motifs of hPLSCR1–4 and hPLSCR1 by spectroscopic methods and isothermal titration calorimetry.

Results

The results in this study show that (i) affinities of the peptides are in the order hPLSCR1  > hPLSCR3 > hPLSCR2 > hPLSCR4 for Ca²⁺ and in the order hPLSCR1 > hPLSCR2 > hPLSCR3 > hPLSCR4 for Mg²⁺, (ii) binding of ions brings about conformational change in the secondary structure of the peptides. The affinity of Ca²⁺ and Mg²⁺ binding to protein hPLSCR1 was similar to that of the peptide I. A sequence comparison shows the existence of scramblase-like motifs among other protein families.

Conclusions

Based on the above results, we hypothesize that the Ca²⁺ binding motif of hPLSCR1 is a novel type of Ca²⁺ binding motif.

General significance

Our findings will be relevant in understanding the calcium dependent scrambling activity of hPLSCRs and their biological function.     

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy

Background

Human α₁-proteinase inhibitor (α₁-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α₁-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern.

Methods

Five variants of plant-expressed recombinant α₁-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α₁-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy.

Results

Urea-induced unfolding of recombinant α₁-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4 M to 4.3 M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~ 5-fold at 54 °C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α₁-PI but provided enhanced resistance to oxidative inactivation.

Conclusions

Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α₁-PI protein, without inflicting the inhibitory activity of the protein.

General significance

Our results demonstrate the significance of engineered modifications in plant-derived recombinant α₁-PI protein molecule for further therapeutic development.     

Mutational analysis of NHAoc/NHA2 in Saccharomyces cerevisiae

Background

NHAoc/NHA2 is highly and selectively expressed in osteoclasts and plays a role(s) in normal osteoclast differentiation, apoptosis and bone resorptive function in vitro. Extensive mutational analysis of a bacterial homologue, NhaA, has revealed a number of amino acid residues essential for its activity. Some of these residues are evolutionarily conserved and have been shown to be essential not only for activity of NhaA in bacteria, but also of NHAoc/NHA2 in eukaryotes.

Methods

The salt-sensitive Saccharomyces cerevisiae strain BW31a was used for heterologous expression of mutants of NHAoc/NHA2. Membrane expression of NHAoc/NHA2 was confirmed by confocal microscopy. Intracellular concentration of Na+ (a measure of Na+ antiporter activity) was estimated by atomic absorption spectroscopy. The growth phenotypes of cells expressing NHAoc/NHA2 mutants were studied on YNB agar supplemented with NaCl and by growth curves in YNB broth.

Results

Mutations in amino acid residues V161 and F357 reduced the ability of transfected BW31a cells to remove intracellular sodium and to grow in NaCl-containing medium. Yeast expressing the double mutant F357 F437 cannot grow in 0.4 M NaCl, suggesting that these residues are also essential for antiporter activity.

Conclusions

Evolutionarily conserved amino acids are required for full antiporter function.

General Significance

Mutations in these amino acid residues may impact NHAoc activity and therefore osteoclast function in vitro and in vivo.     

Glutamate transporter type 3 regulates mouse hippocampal GluR1 trafficking

Background

Rapid trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) to the plasma membrane is considered a fundamental biological process for learning and memory. GluR1 is an AMPAR subunit. We have shown that mice with knockout of excitatory amino acid transporter type 3 (EAAT3), a neuronal glutamate transporter, have impaired learning and memory. The mechanisms for this impairment are not known and may be via regulation of AMPAR trafficking.

Methods

Freshly prepared 300 μm coronal hippocampal slices from wild-type or EAAT3 knockout mice were incubated with or without 25 mM tetraethylammonium for 10 min. The trafficking of GluR1, an AMPAR subunit, to the plasma membrane and its phosphorylation were measured.

Results

Tetraethylammonium increased the trafficking of GluR1 and EAAT3 to the plasma membrane in the wild-type mouse hippocampal slices but did not cause GluR1 trafficking in the EAAT3 knockout mice. Tetraethylammonium also increased the phosphorylation of GluR1 at S845, a protein kinase A (PKA) site, in the wild-type mice but not in the EAAT3 knockout mice. The PKA antagonist KT5720 attenuated tetraethylammonium-induced GluR1 phosphorylation and trafficking in the wild-type mice. The PKA agonist 6-BNz-cAMP caused GluR1 trafficking to the plasma membrane in the EAAT3 knockout mice. In addition, EAAT3 was co-immunoprecipitated with PKA.

Conclusions

These results suggest that EAAT3 is upstream of PKA in a pathway to regulate GluR1 trafficking.

General significance

Our results provide initial evidence for the involvement of EAAT3 in the biochemical cascade of learning and memory.     

A conserved isoleucine in the LOV1 domain of a novel phototropin from the marine alga Ostreococcus tauri modulates the dark state recovery of the domain

Background

Phototropins are UV-A/blue light receptor proteins with two LOV (Light-Oxygen-Voltage) sensor domains at their N terminus and a kinase domain at the C-terminus in photoautotrophic organisms. This is the first research report of a canonical phototropin from marine algae Ostreococcus tauri.

Methods

We synthesized core LOV1 (OtLOV1) domain-encoding portion of the phototropin gene of O. tauri, the domain was heterologously expressed, purified and assessed for its spectral properties and dark recovery kinetics by UV–Visible, fluorescence spectroscopy and mutational studies. Quaternary structure characteristics were studied by SEC and glutaraldehyde crosslinking.

Results

The absorption spectrum of OtLOV1 lacks the characteristic 361 nm peak shown by other LOV1 domains. It undergoes a photocycle with a dark state recovery time of approximately 30 min (τ = 300.35 s). Native OtLOV1 stayed as dimer in aqueous solution and the dimer formation was light and concentration independent. Mutating isoleucine at 43rd position to valine accelerated the dark recovery time by more than 10-fold. Mutating it to serine reduced sensitivity to blue light, but the dark recovery time remained unaltered. I43S mutation also destabilized the FMN binding to a great extent.

Conclusion

The OtLOV1 domain of the newly identified OtPhot is functional and the isoleucine at position 43 of OtLOV1 is the key residue responsible for fine-tuning the domain properties.

General significance

This is the first characterized LOV1 domain of a canonical phototropin from a marine alga and spectral properties of the domain are similar to that of the LOV1 domain of higher plants.     

Site directed processing: Role of amino acid sequences and glycosylation of acceptor glycopeptides in the assembly of extended mucin type O-glycan core 2

Background

The assembly of Ser/Thr-linked O-glycans of mucins with core 2 structures is initiated by polypeptide GalNAc-transferase (ppGalNAc-T), followed by the action of core 1 β3-Gal-transferase (C1GalT) and core 2 β6-GlcNAc-transferase (C2GnT). β4-Gal-transferase (β4GalT) extends core 2 and forms the backbone structure for biologically important epitopes. O-glycan structures are often abnormal in chronic diseases. The goal of this work is to determine if the activity and specificity of these enzymes are directed by the sequences and glycosylation of substrates.

Methods

We studied the specificities of four enzymes that synthesize extended O-glycan core 2 using as acceptor substrates synthetic mucin derived peptides and glycopeptides, substituted with GalNAc or O-glycan core structures 1, 2, 3, 4 and 6.

Results

Specific Thr residues were found to be preferred sites for the addition of GalNAc, and Pro in the + 3 position was found to especially enhance primary glycosylation. An inverse relationship was found between the size of adjacent glycans and the rate of GalNAc addition. All four enzymes could distinguish between substrates having different amino acid sequences and O-glycosylated sites. A short glycopeptide Galβ1–3GalNAcα-TAGV was identified as an efficient C2GnT substrate.

Conclusions

The activities of four enzymes assembling the extended core 2 structure are affected by the amino acid sequence and presence of carbohydrates on nearby residues in acceptor glycopeptides. In particular, the sequences and O-glycosylation patterns direct the addition of the first and second sugar residues by ppGalNAc-T and C1GalT which act in a site directed fashion.

General significance

Knowledge of site directed processing enhances our understanding of the control of O-glycosylation in normal cells and in disease.     

Synthesis of 3-O-methylgallic acid a powerful antioxidant by electrochemical conversion of syringic acid

Background

A kinetic study of the electrochemical oxidation of syringic acid (3,5-dimethoxy-4-hydroxybenzoic acid) by cyclic voltammetry at treated gold disk was combined with results of electrolyses at Ta/PbO₂ anode in order to convert it into potentially high-added-value product.

Methods

The electrochemical oxidation of syringic acid was carried out in order to convert this compound to 3-O-methylgallic acid. This latter was identified by mass spectrophotometry using LC-MS/MS apparatus. The 3-O-methylgallic acid synthesis was controlled by cyclic volammetry, Ortho-diphenolicdeterminations and DPPH radical-scavenging activity.

Results

The proposed mechanism is based on the hypothesis of a bielectronic discharge of syringic acid molecule under free and adsorbed form involving two intermediate cation mesomers. Hydrolysis of the more stable of this last one leads to the formation of the 3,4-dihydroxy-5-methoxybenzoic acid (3-O-methylgallic acid) as a major product. The latter aromatic compound was synthesized by anodic oxidation of syringic acid at PbO2 electrode. The cyclic voltammogram of the electrolysis bath of syringic acid shows that the anodic peak potential of 3-O-methylgallic acid was lower (E_pa = 128 mV) than that of SA (E_pa = 320 mV). And the strongest antiradical activity was detected when the 3-O-methylgallic acid concentration was higher".

Conclusion

The electrochemical oxidation using PbO₂ anode is a rapid, simple and efficient method tool for a conversion of SA into 3-O-methylgallic acid, a potent antioxidant derivative

General Significance

The electrochemical process consists in a simple transformation of the syringic acid into 3-O-methylgallic acid having a better antioxidant capacity. This result has been justified by cyclic voltametry which shows that anodic peak of 3-O-methylgallic acid is reversible. Furthermore, its potential is lower than that of the irreversible anodic peak of syringic acid to 3-O-methylgallic acid.     

A new β-chain haemoglobin variant with increased oxygen affinity: Hb Roma [β115(g17)Ala → Val]

Background

Haemoglobin Roma [β115(G17)Ala → Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala → Val substitution at the α1β1 interface.

Methods and results

The variant chain was identified by direct DNA sequencing of the β-globin gene, which revealed a GCC → GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the βG17 position, situated at the α₁β₁ interface, has been examined using computational models of haemoglobin Roma and other known βG17 variants, in comparison with normal haemoglobin A.

Conclusions

This study suggests that the β115(G17)Ala → Val substitution at the α1β1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma.

General significance

An amino acid substitution at the G17 position of the α1β1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.     

Inhibition by polyamines of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid

Background

Viroids are the smallest pathogens known to date. They infect plants and cause considerable economic losses. The members of the Avsunviroidae family are known for their capability to form hammerhead ribozymes (HHR) that catalyze self-cleavage during their rolling circle replication.

Methods

In vitro inhibition assays, based on the self-cleavage kinetics of the hammerhead ribozyme from a Chrysanthemum chlorotic mottle viroid (CChMVd-HHR) were performed in the presence of various putative inhibitors.

Results

Aminated compounds appear to be inhibitors of the self-cleavage activity of the CChMVd HHR. Surprisingly the spermine, a known activator of the autocatalytic activity of another hammerhead ribozyme in the presence or absence of divalent cations, is a potent inhibitor of the CChMVd-HHR with K_i of 17 ± 5 μM. Ruthenium hexamine and TMPyP4 are also efficient inhibitors with K_i of 32 ± 5 μM and IC₅₀ of 177 ± 5 nM, respectively.

Conclusions

This study shows that polyamines are inhibitors of the CChMVd-HHR self-cleavage activity, with an efficiency that increases with the number of their amino groups.

General significance

This fundamental investigation is of interest in understanding the catalytic activity of HHR as it is now known that HHR are present in the three domains of life including in the human genome. In addition these results emphasize again the remarkable plasticity and adaptability of ribozymes, a property which might have played a role in the early developments of life and must be also of significance nowadays for the multiple functions played by non-coding RNAs.     

Organic bioelectronics for electronic-to-chemical translation in modulation of neuronal signaling and machine-to-brain interfacing

Background

A major challenge when creating interfaces for the nervous system is to translate between the signal carriers of the nervous system (ions and neurotransmitters) and those of conventional electronics (electrons).

Scope of review

Organic conjugated polymers represent a unique class of materials that utilizes both electrons and ions as charge carriers. Based on these materials, we have established a series of novel communication interfaces between electronic components and biological systems. The organic electronic ion pump (OEIP) presented in this review is made of the polymer–polyelectrolyte system poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS). The OEIP translates electronic signals into electrophoretic migration of ions and neurotransmitters.

Major conclusions

We demonstrate how spatio-temporally controlled delivery of ions and neurotransmitters can be used to modulate intracellular Ca^2 + signaling in neuronal cells in the absence of convective disturbances. The electronic control of delivery enables strict control of dynamic parameters, such as amplitude and frequency of Ca^2 + responses, and can be used to generate temporal patterns mimicking naturally occurring Ca^2 + oscillations. To enable further control of the ionic signals we developed the electrophoretic chemical transistor, an analog of the traditional transistor used to amplify and/or switch electronic signals. Finally, we demonstrate the use of the OEIP in a new “machine-to-brain” interface by modulating brainstem responses in vivo.

General significance

This review highlights the potential of communication interfaces based on conjugated polymers in generating complex, high-resolution, signal patterns to control cell physiology. We foresee widespread applications for these devices in biomedical research and in future medical devices within multiple therapeutic areas. This article is part of a Special Issue entitled Organic Bioelectronics—Novel Applications in Biomedicine.     

Purification,characterization, sequencing and molecular cloning of a novel cysteine methyltransferase that regulates trehalose-6-phosphate synthase from Saccharomyces cerevisiae

Background

In Saccharomyces cerevisiae methylation at cysteine residue displayed enhanced activity of trehalose-6-phosphate synthase (TPS).

Methods

The cysteine methyltransferase (CMT) responsible for methylating TPS was purified and characterized. The amino acid sequence of the enzyme protein was determined by a combination of N-terminal sequencing and MALDI-TOF/TOF analysis. The nucleotide sequence of the CMT gene was determined, isolated from S. cerevisiae and expressed in E. coli. Targeted disruption of the CMT gene by PCR based homologous recombination in S. cerevisiae was followed by metabolite characterization in the mutant.

Results

The purified enzyme was observed to enhance the activity of TPS by a factor of 1.76. The 14 kDa enzyme was found to be cysteine specific. The optimum temperature and pH of enzyme activity was calculated as 30 °C and 7.0 respectively. The K_m V_max and K_cat against S-adenosyl-l-methionine (AdoMet) were 4.95 μM, 3.2 U/mg and 6.4 s^− 1 respectively. Competitive inhibitor S-Adenosyl-l-homocysteine achieved a K_i as 10.9 μM against AdoMet. The protein sequence contained three putative AdoMet binding motifs. The purified recombinant CMT activity exhibited similar physicochemical characteristics with the native counterpart. The mutant, Mataα, cmt:: kan^r exhibited almost 50% reduction in intracellular trehalose concentration.

Conclusion

A novel cysteine methyltransferase is purified, which is responsible for enhanced levels of trehalose in S. cerevisiae.

General significance

This is the first report about a cysteine methyltransferase which performs S methylation at cysteine residue regulating TPS activity by 50%, which resulted in an increase of the intercellular stress sugar, trehalose.