Regulation of H2O2-induced cells injury through Nrf2 signaling pathway: An introduction of a novel cysteic acid-modified peptide.

A novel peptide (Cya-Phe-Leu-Ala-Pro, SCP) was formulated through non-protein amino acid-cysteic acid (Cya) modification of collagen peptide (Phe-Leu-Ala-Pro, CP) from Acaudina molpadioides. Introduction of this Cya showed remarkable improvement in the scavenging activities of OH·. SCP exhibited stronger effects than CP in preventing H2O2-induced oxidative damage due to lower levels of ROS and MDA, and higher activities of antioxidant enzymes, such as SOD, GSH-Px, HO-1, and NQO1. It was speculated that SCP could significantly increase the expression level of Nrf2 compared to CP, thereby activating the expression of downstream ARE genes. The expression levels of p38 in the upstream pathway to regulate Nrf2 content were significantly higher in both the CP and SCP-treated groups, while a higher level of JNK was observed only in the SCP-treated groups. The present study provided insights towards the application of cysteic acid modified peptide in protecting cell from oxidative damage through the JNK/Nrf2 pathway.

Simultaneous determination of l­DOPA, l­tyrosine and uric acid by cysteic acid - modified glassy carbon electrode.

Electrochemical oxidation of l­cysteine resulted in the formation of a film on glassy carbon electrode. In a solution of levodopa (l­DOPA), l­tyrosine (Tyr) and uric acid (UA), three separated intense anodic peaks were appeared in differential pulse voltammetry regime. Experimental conditions were optimized for simultaneous determination of the three compounds. All experiments were carried out in phosphate buffer solution (0.1 M, pH 8). Calibration curves were obtained in the presence of various concentrations of l­DOPA, Tyr, and UA. Linear concentration ranges were 0.65-22 µM for l­DOPA, 3.5-96 µM for Tyr, and 1.0-19 µM for UA. The limits of detection (LODs) were calculated as 0.2, 1.1 and 0.36 µM for l­DOPA, Tyr, and UA, respectively. The electrochemical sensor was used successfully for the simultaneous determination of l­DOPA, Tyr, and UA species in human blood serum samples.

Electrochemical fabrication of a novel ZnO/cysteic acid nanocomposite modified electrode and its application to simultaneous determination of sunset yellow and tartrazine.

A new nanocomposite (ZnO/Cysteic acid) was deposited on glassy carbon electrode by cyclic voltammetry. Uniform deposition of the nanocomposite was observed by scanning electron microscopy. The electron transfer characteristics of two food additives, sunset yellow and tartrazine, were greatly improved on the modified electrode. The prepared electrode was used in the sensitive simultaneous determination of sunset yellow and tartrazine by differential pulse voltammetry. Linear calibration curves were obtained in the concentration ranges of 0.1-3.0, and 0.07-1.86µM, and detection limits of 0.03 and 0.01µM for sunset yellow and tartrazine, respectively. The proposed method was evaluated by determination of the dyes in processed soft drinks with satisfactory results (recovery>95% and RSD%<5%).

Aza-BODIPY dyes with enhanced hydrophilicity.

Attempts to make a diamino disulfonic acid derivative of an aza-BODIPY showed it was difficult to add BF2 to a disulfonated azadipyrromethene, and sulfonation of an aza-BODIPY resulted in loss of the BF2 fragment. We conclude the electron-deficient character of aza-BODIPY dyes destabilizes them relative to BODIPY dyes. Consequently, sulfonation of the aza-BODIPY core is not a viable strategy to increase water solubility. This assertion was indirectly supported via stability studies of a BODIPY and an aza-BODIPY in aqueous media. To afford the desired compound type, an aza-BODIPY with two amino and two sulfonic acid groups was prepared via modification of the aryl substituents with cysteic acid.

Synthesis and characterization of cysteine functionalized silver nanoparticles for biomolecule immobilization.

A facile method for the aqueous phase synthesis of cysteine-functionalized silver nanoparticles by potato extract has been reported in the present work. These functionalized nanoparticles were then used for the covalent immobilization of a biomolecule, alkaline phosphatase, on its surface through carbodiimide coupling. Different reaction parameters such as cysteine concentration, reducing agent concentration, temperature, pH and reaction time were varied during the nanoparticles' formation, and their effects on plasmon resonance were studied using Ultraviolet-visible spectroscopy. Fourier transform infrared spectroscopy was used to confirm the surface modification of silver nanoparticles by cysteine and the particle size analysis was done using particle size analyzer, which showed the average nanoparticles' size of 61 nm for bare silver nanoparticles and 201 nm for the enzyme-immobilized nanoparticles. The synthesized nanoparticles were found to be highly efficient for the covalent immobilization of alkaline phosphatase on its surface and retained 67% of its initial enzyme activity (9.44 U/mg), with 75% binding efficiency. The shelf life of the enzyme-nanoparticle bioconjugates was found to be 60 days, with a 12% loss in the initial enzyme activity. With a simple synthesis strategy, high immobilization efficiency and enhanced stability, these enzyme-coated nanoparticles have the potential for further integration into the biosensor technology.

A novel composite film derived from cysteic acid and PDDA-functionalized graphene: enhanced sensing material for electrochemical determination of metronidazole.

A novel composite film derived from cysteic acid and poly(diallydimethylammonium chloride)-functionalized graphene (PDDA-GN) was employed as an enhanced electrode material for ultrasensitive determination of metronidazole. The cysteic acid/PDDA-GN composite film was prepared by the electrochemical grafting of cysteic acid onto the PDDA-GN coated glassy carbon electrode (GCE). The cyclic voltammetry investigations reveal that the peak current of metronidazole reduction at the cysteic acid/PDDA-GN/GCE was remarkably enhanced compared to the bare GCE, the cysteic acid/GCE and the PDDA-GN/GCE. This result implies the synergistic electrocatalytic effect of cysteic acid and PDDA-GN. The fabricated sensor shows linear response to metronidazole in the ranges of 10 nM-1 µM and 70 µM-800 µM, with a detection limit of 2.3 nM (S/N=3). The heterogeneous electron transfer rate constant and the diffusion coefficient of metronidazole were further evaluated by rotating disk electrode experiments. Moreover, we applied the present method to the determination of metronidazole in urine and lake water with satisfactory results.

Simultaneous determination of ofloxacin and gatifloxacin on cysteic acid modified electrode in the presence of sodium dodecyl benzene sulfonate.

A novel cysteic acid modified carbon paste electrode (cysteic acid/CPE) based on electrochemical oxidation of L-cysteine was developed to simultaneously determine ofloxacin and gatifloxacin in the presence of sodium dodecyl benzene sulfonate (SDBS). Fourier transform infrared spectra (FTIR) indicated that L-cysteine was oxidated to cysteic acid. Electrochemical impedance spectroscopy (EIS) and cyclic voltammograms (CV) indicated that cysteic acid was successfully modified on electrode. The large peak separation (116 mV) between ofloxacin and gatifloxacin was obtained on cysteic acid/CPE while only one oxidation peak was found on bare electrode. And the peak currents increased 5 times compared to bare electrode. Moreover, the current could be further enhanced in the presence of an anionic surfactant, sodium dodecyl benzene sulfonate. The differential pulse voltammograms (DPV) exhibited that the oxidation peak currents were linearly proportional to their concentrations in the range of 0.06-10 µM for ofloxacin and 0.02-200 µM for gatifloxacin, and the detection limits of ofloxacin and gatifloxacin were 0.02 µM and 0.01 µM (S/N=3), respectively. This proposed method was successfully applied to determine ofloxacin and gatifloxacin in pharmaceutical formulations and human serum samples.

Alumoxane/ferroxane nanoparticles for the removal of viral pathogens: the importance of surface functionality to nanoparticle activity.

A bi-functional nano-composite coating has been created on a porous Nomex® fabric support as a trap for aspirated virus contaminated water. Nomex® fabric was successively dip-coated in solutions containing cysteic acid functionalized alumina (alumoxane) nanoparticles and cysteic acid functionalized iron oxide (ferroxane) nanoparticles to form a nanoparticle coated Nomex® (NPN) fabric. From SEM and EDX the nanoparticle coating of the Nomex® fibers is uniform, continuous, and conformal. The NPN was used as a filter for aspirated bacteriophage MS2 viruses using end-on filtration. All measurements were repeated to give statistical reliability. The NPN fabrics show a large decrease as compared to Nomex® alone or alumoxane coated Nomex®. An increase in the ferroxane content results in an equivalent increase in virus retention. This suggests that it is the ferroxane that has an active role in deactivating and/or binding the virus. Heating the NPN to 160 °C results in the loss of cysteic acid functional groups (without loss of the iron nanoparticle's core structure) and the resulting fabric behaves similar to that of untreated Nomex®, showing that the surface functionalization of the nanoparticles is vital for the surface collapse of aspirated water droplets and the absorption and immobilization of the MS2 viruses. Thus, for virus immobilization, it is not sufficient to have iron oxide nanoparticles per se, but the surface functionality of a nanoparticle is vitally important in ensuring efficacy.

Formation of the bisulfite anion (HSO(3) (-) , m/z 81) upon collision-induced dissociation of anions derived from organic sulfonic acids.

In the negative-ion collision-induced dissociation mass spectra of most organic sulfonates, the base peak is observed at m/z 80 for the sulfur trioxide radical anion (SO(3) (-·) ). In contrast, the product-ion spectra of a few sulfonates, such as cysteic acid, aminomethanesulfonate, and 2-phenylethanesulfonate, show the base peak at m/z 81 for the bisulfite anion (HSO(3) (-) ). An investigation with an extensive variety of sulfonates revealed that the presence of a hydrogen atom at the ß-position relative to the sulfur atom is a prerequisite for the formation of the bisulfite anion. The formation of HSO(3) (-) is highly favored when the atom at the ß-position is nitrogen, or the leaving neutral species is a highly conjugated molecule such as styrene or acrylic acid. Deuterium-exchange experiments with aminomethanesulfonate demonstrated that the hydrogen for HSO(3) (-) formation is transferred from the ß-position. The presence of a peak at m/z 80 in the spectrum of 2-sulfoacetic acid, in contrast to a peak at m/z 81 in that of 3-sulfopropanoic acid, corroborated the proposed hydrogen transfer mechanism. For diacidic compounds, such as 4-sulfobutanoic acid and cysteic acid, the m/z 81 ion can be formed by an alternative mechanism, in which the negative charge of the carboxylate moiety attacks the α-carbon relative to the sulfur atom. Experiments conducted with deuterium-exchanged and deuterium-labeled analogs of sulfocarboxylic acids demonstrated that the formation of the bisulfite anion resulted either from a hydrogen transfer from the ß-carbon, or from a direct attack by the carboxylate moiety on the α-carbon.

Isolated Hb Providence ß82Asn and ß82Asp fractions are more stable than native HbA(0) under oxidative stress conditions.

We have previously shown that hydrogen peroxide (H(2)O(2)) triggers irreversible oxidation of amino acids exclusive to the ß-chains of purified human hemoglobin (HbAo). However, it is not clear, whether α- or ß-subunit Hb variants exhibit different oxidative resistance to H(2)O(2) when compared to their native HbAo. Hb Providence contains two ß-subunit variants with single amino acid mutations at ßLys82→Asp (ßK82D) and at ßLys82→Asn (ßK82N) positions and binds oxygen at lower affinity than wild type HbA. We have separated Hb Providence into its 3 component fractions, and contrasted oxidative reactions of its ß-mutant fractions with HbAo. Relative to HbAo, both ßK82N and ßK82D fractions showed similar autoxidation kinetics and similar initial oxidation reaction rates with H(2)O(2). However, a more profound pattern of changes was seen in HbAo than in the two Providence fractions. The structural changes in HbAo include a collapse of ß-subunits, and α-α dimer formation in the presence of excess H(2)O(2). Mass spectrometric and amino acid analysis revealed that ßCys93 and ßCys112 were oxidized in the HbAo fraction, consistent with oxidative pathways driven by a ferrylHb and its protein radical. These amino acids were oxidized at a lesser extent in ßK82D fraction. While the 3 isolated components of Hb Providence exhibited similar ligand binding and oxidation reaction kinetics, the variant fractions were more effective in consuming H(2)O(2) and safely internalizing radicals through the ferric/ferryl pseudoperoxidase cycle.

Negative ion fragmentation of cysteic acid containing peptides: cysteic acid as a fixed negative charge.

We present here a study of the collision induced dissociation (CID) of deprotonated cysteic acid containing peptides produced by MALDI. The effect of cysteic acid (C(ox)) position is interrogated by considering the positional isomers, C(ox)LVINVLSQG, LVINVLSQGC(ox), and LVINVC(ox)LSQG. Although considerable variation between the CID spectra is observed, the mechanistic picture that emerges involves charge retention at the deprotonated cysteic acid side chain. Fragmentation occurs in the proximity of the cysteic acid group by charge directed mechanisms as well as remote from this group to form ions, which may be rationalized by charge remote mechanisms. Additionally, the formation of the SO(3)(-•) ion is observed in all cases. Fragmentation of C(ox)LVINVLSQC(ox) provides both N- and C-terminal, y and b ions, respectively indicating that the negative charge may be retained at either of the cysteic acids; however, there is some evidence that charge retention at the C-terminal cysteic acid may be preferred. Fragmentation of tryptic type peptides containing a C-terminal arginine or lysine residue is considered through comparison of three peptides C(ox)LVINKLSQG, C(ox)LVINVLSQK, and C(ox)LVINVLSQR. Lastly, we rationalize the formation of b(n-1)+ H(2)O and a(n-1) ions through a mechanism involving rearrangement of the C-terminal residue to form a mixed anhydride intermediate.

N-Fmoc-α-sulfo-ß-alanine: a versatile building block for the water solubilisation of chromophores and fluorophores by solid-phase strategy.

An easy and efficient solid-phase synthesis strategy to obtain rapidly water-soluble chromophores/fluorophores in highly pure form has been developed. This first successful use of N-Fmoc-α-sulfo-ß-alanine as a SPPS building block opens the way to the future development of promising direct "on-resin" peptide labelling and water-solubilising methods.

A phenylacetylated peptide, JBIR-96, isolated from Streptomyces sp. RI051-SDHV6.

Searching for metabolites from Streptomyces sp. RI051-SDHV6 resulted in the discovery of a novel peptide, JBIR-96 (1). The structure of 1 was established as an N-phenylacetylated pentapeptide involving a cysteic acid and a peptide lactone structure by extensive NMR and MS analyses. In addition, the absolute configuration of 1 was established by Marfey's and modified Mosher's methods.

Effect of cysteic acid position on the negative ion fragmentation of proteolytic derived peptides.

A study on the effect of cysteic acid position on the types of fragment ions formed by collision-induced dissociation (CID) of [M - H](-) ions is presented. Of particular note is the observation of d-type fragment ions for peptides that contain an N-terminal cysteic acid (fixed negative charge) and cleavable amino acid side chains possessing a ß-γ carbon-carbon bond. For example, the CID mass spectrum of oxidized cys-kemptide (C(ox)LRRASLG) [M - H + O(3)](-) ions contains abundant series of d-type fragment ions, and similar results are observed for oxidized cysteine-containing ribonuclease A proteolytic peptides. The d(i) fragment ions are assumed to arise by a charge-remote and/or charge-assisted fragmentation mechanism, which both occur at high collision energies and involve consecutive reactions (i.e., the formation of a(i) ions followed by the elimination of the side chain to form d(i) ions).

18F-labeled bombesin analog for specific and effective targeting of prostate tumors expressing gastrin-releasing peptide receptors.

UNLABELLED: Bombesin is a peptide exhibiting high affinity for the gastrin-releasing peptide receptor (GRPr), which is highly overexpressed on prostate cancer cells. In the present study, we developed an (18)F-labeled bombesin analog, (18)F-BAY 86-4367, which is currently being clinically tested for use in PET of prostate cancer. METHODS: In vitro pharmacologic studies were performed to characterize the nonradioactive ((19)F) standard of the bombesin analog for binding affinity and selectivity for GRPr. The stability of (18)F-BAY 86-4367 was determined in murine and human plasma. In vivo, the tumor-targeting potential and pharmacokinetic profile of the (18)F tracer were analyzed with biodistribution experiments and PET studies of prostate tumor-bearing mice. RESULTS: The nonradioactive ((19)F) standard of the bombesin analog showed subnanomolar and GRPr-selective binding affinity. The stability of the tracer in murine and human plasma was found to be high. In 2 prostate cancer xenograft models (PC-3 and LNCaP), (18)F-BAY 86-4367 showed more specific and effective GRPr-based targeting in vivo than the benchmark radiotracers (18)F-fluoroethylcholine and (18)F-FDG. In addition, rapid tumor targeting and fast renal excretion (∼70%) and hepatobiliary excretion (∼10%) were identified in both xenograft models. Furthermore, PET studies provided clear and specific visualization of PC-3 tumors in mice. CONCLUSION: Favorable preclinical data showing specific and effective tumor targeting by (18)F-BAY 86-4367 suggest that a clinical trial be undertaken to test its diagnostic utility in PET for prostate carcinoma patients.

Akt1 intramitochondrial cycling is a crucial step in the redox modulation of cell cycle progression.

Akt is a serine/threonine kinase involved in cell proliferation, apoptosis, and glucose metabolism. Akt is differentially activated by growth factors and oxidative stress by sequential phosphorylation of Ser(473) by mTORC2 and Thr(308) by PDK1. On these bases, we investigated the mechanistic connection of H(2)O(2) yield, mitochondrial activation of Akt1 and cell cycle progression in NIH/3T3 cell line with confocal microscopy, in vivo imaging, and directed mutagenesis. We demonstrate that modulation by H(2)O(2) entails the entrance of cytosolic P-Akt1 Ser(473) to mitochondria, where it is further phosphorylated at Thr(308) by constitutive PDK1. Phosphorylation of Thr(308) in mitochondria determines Akt1 passage to nuclei and triggers genomic post-translational mechanisms for cell proliferation. At high H(2)O(2), Akt1-PDK1 association is disrupted and P-Akt1 Ser(473) accumulates in mitochondria in detriment to nuclear translocation; accordingly, Akt1 T308A is retained in mitochondria. Low Akt1 activity increases cytochrome c release to cytosol leading to apoptosis. As assessed by mass spectra, differential H(2)O(2) effects on Akt1-PDK interaction depend on the selective oxidation of Cys(310) to sulfenic or cysteic acids. These results indicate that Akt1 intramitochondrial-cycling is central for redox modulation of cell fate.

En bloc elution of proteomes from combinatorial peptide ligand libraries.

In contrast to the three to four sequential elution steps routinely adopted for recovering proteomes adsorbed onto combinatorial peptide ligand libraries, we report here two en bloc elution systems, which are able to achieve recoveries in the order of 95% in a single step. One consists of TUC (7 M urea, 2 M thiourea, 3% CHAPS) added with 40 mM formic acid, the other of TUC added with 25 mM cysteic acid (Cys-A). Although both systems are almost equally performing, the formic acid eluant has as a drawback, namely the potential to modify proteins by formylation of Ser and Thr residues. On the contrary, the Cys-A system is unreactive towards proteins. Additionally, Cys-A, due to its very low pI value (1.80) does not interfere with subsequent 2D map analyses since, during the first isoelectric focusing step (in general performed in immobilized pH gradients), it migrates to the anodic compartment and thus vacates the gel. Conversely, formic acid would mostly collect around pH 3 and acetic or citric acid, formerly used in the UCA (9 M urea, 50 mM citric acid) eluant, would condense around pH 4 in the focusing step, interfering thus with 2D map analyses. Elution by boiling SDS of the small amount of protein left over after three sequential elution steps in TUC and 25 mM Cys-A and analysis by nanoLC-MS/MS has demonstrated that these residual proteins are indeed a residue left over from proteins already eluted in TUC-Cys-A and not new species absent in the latter eluate.

Free radical conformations and conversions in X-irradiated single crystals of L-cysteic acid by electron magnetic resonance and density functional theory studies.

Single crystals of L-cysteic acid monohydrate were X-irradiated and studied at 295 K using EPR, ENDOR, and EIE techniques. Three spectroscopically different radicals were observed. These were a deamination radical reduction product (R1), and two oxidation products formed by hydrogen abstraction (radicals R2, R3). R2 and R3 were shown to exhibit the same chemical structure while exhibiting very different geometrical conformations. Cluster DFT calculations at the 6-31G(d,p) level of theory supported the experimental observations for radicals R1 and R2. It was not possible to simulate the R3 radical in any attempted cluster; hence, for this purpose a single molecule approach was used. The precursor radicals for R1, R2, and R3, identified in the low-temperature work on L-cysteic acid monohydrate by Box and Budzinski, were also investigated using DFT calculations. The experimentally determined EPR parameters for the low-temperature decarboxylated cation could only be reproduced correctly within the cluster when the carboxyl group remained in the proximity of the radical. Only one of the two observed low-temperature carboxyl anions (stable at 4 and 48 K) could be successfully simulated by the DFT calculations. Evidence is presented in support of the conclusions that the carboxyl reduction product already is protonated at 4 K and that the irreversible conversion between the two reduction products is brought forward by an umbrella-type inversion of the carboxyl group.

Voltammetric determination of terbinafine in biological fluid at glassy carbon electrode modified by cysteic acid/carbon nanotubes composite film.

The electrochemical oxidation of L-cysteine (CySH) in presence of carbon nanotubes (CNTs) formed a composite film at a glassy carbon electrode (GCE) as a novel modifier for directly electroanalytical determination of terbinafine without sample pretreatment in biological fluid. The determination of terbinafine at the modified electrode with strongly accumulation was studied by differential pulse voltammetry (DPV). The peak current obtained at +1.156 V (vs. SCE) from DPV was linearly dependent on the terbinafine concentration in the range of 8.0 x 10(-8)-5.0 x 10(-5 )M in a B-R buffer solution (0.04 M, pH 1.81) with a correlation coefficient of 0.998. The detection limit (S/N=3) was 2.5 x 10(-8 )M. The low-cost modified electrode showed good sensitivity, selectivity, and stability. This developed method had been applied to the direct determination of terbinafine in human serum samples with satisfactory results. It is hopeful that the modified electrode will be applied for the medically clinical test and the pharmacokinetics in future.