Facile "stop codon" method reveals elevated neuronal toxicity by discrete S87p-α-synuclein oligomers.

Herein, a new method for preparing phosphorylated proteins at specific sites has been applied to α-synuclein (α-Syn). Three different α-Syn species phosphorylated at Serine 87 (S87p-α-Syn), Serine 129 (S129p-α-Syn) and Serine 87/129 (S87p,129p-α-Syn) were prepared through the 'stop codon' method and verified by LC/MS/MS and immunoblotting. Each type of phosphorylated α-Syn was tested for oligomerization trends and cellular toxicity with dopamine (DA), Cu(2+) ions and pyridoxal 5'-phosphate. Aggregation trends induced by DA or DA/Cu(2+) were similar between phosphorylated and non-phosphorylated α-Syn in SDS-PAGE. However, except for the monomer, phosphorylated oligomers showed higher toxicity than the non-phosphorylated α-Syn (Np-α-Syn) oligomers via WST-1 assays when tested on SH-SY5Y human neuroblastoma cells. In particular, S87p-α-Syn and S87p,129p-α-Syn oligomers induced by DA/Cu(2+), showed higher toxicity than did S129p-α-Syn. When α-Syn was treated with pyridoxal 5'-phosphate in the presence of DA or Cu(2+) to determine aggregation effects, high inhibition effects were shown in both non-phosphorylated and phosphorylated versions. α-Syn co-incubated with DA or DA/Cu(2+) showed less cellular toxicity upon pyridoxal 5'-phosphate treatment, especially in the case of DA-induced Np-α-syn. This study supports that phosphorylated oligomers of α-Syn at residue 87 can contribute to neuronal toxicity and the pyridoxal 5'-phosphate can be used as an inhibitor for α-Syn aggregation.

Dopamine and Cu+/2+ can induce oligomerization of α-synuclein in the absence of oxygen: Two types of oligomerization mechanisms for α-synuclein and related cell toxicity studies.

α-Synuclein oligomers can induce neurotoxicity and are implicated in Parkinson's disease etiology and disease progression. Many studies have reported α-synuclein oligomerization by dopamine (DA) and transition metal ions, but few studies provide insight into joint influences of DA and Cu2+ . In this study, DA and Cu2+ were coadministered aerobically to measure α-synuclein oligomerization under these conditions. In the presence of oxygen, DA induced α-synuclein oligomerization in a dose-dependent manner. Cu+/2+ did not effect oligomerization in such a manner in the presence of DA. By electrophoresis, Cu2+ was found easily to induce oligomerization with DA. This implies that oligomerization invoked by DA is reversible in the presence of Cu2+, which appears to be mediated by noncovalent bond interactions. In the absence of oxygen, DA induced less oligomerization of α-synuclein, whereas DA/Cu2+ induced aerobic-level amounts of oligomers, suggesting that DA/Cu2+ induces oligomerization independent of oxygen concentration. Radical species were detected through electron paramagnetic resonance (EPR) spectroscopic analysis arising from coincubation of DA/Cu2+ with α-synuclein. Redox reactions induced by DA/Cu2+ were observed in multimer regions of α-synuclein oligomers through NBT assay. Cellular toxicity results confirm that, for normal and hypoxic conditions, copper or DA/Cu2+ can induce cell death, which may arise from copper redox chemistry. From these results, we propose that DA and DA/Cu2+ induce different mechanisms of α-synuclein oligomerization, cross-linking with noncovalent (or reversible covalent) bonding vs. likely radical-mediated covalent modification.

Labile zinc-assisted biological phosphate chemosensing and related molecular logic gating interpretations.

Herein, molecular fluorescence 'OFF-ON' behavior with aqueous addition of biological phosphate and Zn(2+) is studied with Zn(2)(slys)(2)Cl(2) [H(2)slys = 6-amino-2-{(2-hydroxybenzylidene)amino}hexanoic acid], a fluorescent water-soluble complex, using various spectroscopic tools (e.g., (31)P NMR, UV-vis, emission, and CD spectroscopy) at the micromolar level. Adduct-dependent fluorescence intensity changes can be interpreted as a two-input (cation/anion) implication molecular logic gating system. A displacement study of PPi from the dizinc complex is also reported. Diphosphate and triphosphate addition/displacements were also studied. (31)P NMR spectroscopy shows gradual NMR peak shifts from bound ADP/GDP to free ADP/GDP with increasing [PPi]. In the emission spectrum, fluorescence quenching is shown: CD signal maxima decrease with addition of PPi. These displacement events are also tested with triphosphates (ATP, GTP), and their binding strength/displacement ability over ADP/GDP is quantified: PPi > ATP ≈ GTP (3.35 ± 0.77 × 10(4) M(-1) for PPi, 7.73 ± 1.79 × 10(3) M(-1) for ATP, 9.21 ± 2.88 × 10(3) M(-1) for GTP over 1·ADP). Many anions and cations were also screened for selectivity. Tubulin polymerization was assayed in the presence of 1 and its copper analogue which reflected a slight inhibition in polymerization.

Recent conjugation strategies of small organic fluorophores and ligands for cancer-specific bioimaging.

Conjugation between various small fluorophores and specific ligands has become one of the main strategies for bioimaging in disease diagnosis, medicinal chemistry, immunology, and fluorescence-guided surgery, etc. Herein, we present our review of recent studies relating to molecular fluorescent imaging techniques for various cancers in cell-based and animal-based models. Various organic fluorophores, especially near-infrared (NIR) probes, have been employed with specific ligands. Types of ligands used were small molecules, peptides, antibodies, and aptamers; each has specific affinities for cellular receptor proteins, cancer-specific antigens, enzymes, and nucleic acids. This review can aid in the selection of cancer-specific ligands and fluorophores, and may inspire the further development of new conjugation strategies in various cellular and animal models.

Sodium and Potassium Relating to Parkinson's Disease and Traumatic Brain Injury.

Alkali metals, especially sodium and potassium, are plentiful and vital in biological systems. They take on important roles in health and disease. Such roles include the regulation of homeostasis, osmosis, blood pressure, electrolytic equilibria, and electric current. However, there is a limit to our present understanding; the ions have a great ability and capacity for action in health and disease, much greater than our current understanding. For the regulation of physiological homeostasis, there is a crucial regulator (renin-angiotensin system, RAS), found at both peripheral and central levels. Misregulation of the Na(+)-K(+) pump, and sodium channels in RAS are important for the understanding of disease progression, hypertension, diabetes, and neurodegenerative diseases, etc. In particular, RAS displays direct or indirect interaction important to Parkinson's disease (PD). In this chapter, the relationship between the regulation of sodium/potassium concentration and PD was sought. In addition, some recent biochemical and clinical findings are also discussed that help describe sodium and potassium in the context of traumatic brain injury (TBI). TBI is caused from the heavy striking of the head; this strongly affects ion flux in the affected tissue (brain) and damages cellular regulation systems. Thus, inappropriate concentrations of ions (hyper- and hyponatremia, and hyper- and hypokalemia) will perturb homeostasis giving rise to important and far reaching effects. These changes also impact osmotic pressure and the concentration of other metal ions, such as the calcium(II) ion.

H(+)-Assisted fluorescent differentiation of Cu(+) and Cu(2+): effect of Al(3+)-induced acidity on chemical sensing and generation of two novel and independent logic gating pathways.

A novel Schiff base probe exhibited strong 'turn-ON' fluorescence for Cu(2+) at 345 nm, Al(3+) at 445 nm, and Cu(+) at 360 nm in the presence of Al(3+) in organic solvent (acetonitrile), which allowed for construction of molecular logic gates 'INH' and '1:2 DEMULTIPLEXING.' H(+) generated from Al(3+) contributed greatly to Cu(+) chemosensing based on a redox non-innocence mechanism.

A novel, selective, and extremely responsive thienyl-based dual fluorogenic probe for tandem superoxide and Hg2+ chemosensing.

Novel, high "turn-on" Hg(2+) and O(2)(-) fluorescence behaviour (∼25-fold) with probes bearing [S(thi)N(py)] and [S(thi)N(py)N(py)] binding receptors, joined by oxidizable sulphides, may involve S-bound transient ROS species; such optical O(2)(-) behaviour operates moderately in neuroblastoma.

Interplay of salicylaldehyde, lysine, and M2+ ions on α-synuclein aggregation: cancellation of aggregation effects and determination of salicylaldehyde neurotoxicity.

In this study, α-synuclein was treated in vitro with salicylaldehyde (SA), lysine (lys) and M(n+) (Cu(2+) or Zn(2+)) in various ratios. SA induced aggregation of α-syn in the ratio of 1:500 (α-syn:SA) after incubation (pH 7.4, PBS buffer, 16-24h). Free lys can thus scavenge SA, inhibiting the aggregation of α-syn up to ∼63% (α-syn:SA:lys=1:1000:5000). When Cu(2+) and Zn(2+) are added to SA and α-syn, protein aggregation is induced. In the case of Zn(2+), the aggregation of α-syn increased to 74% (ratio=1:1000:50). Fluorescence studies support the production of protein-bound Zn(2+)-salicylaldimine species. For Cu(2+), aggregation of α-syn was shown (138%). Thus, possible protective or inducing effects of lys, Cu(2+) and Zn(2+) may exist with α-syn. α-Syn, SA and Cu(2+) can undergo complexation (fluorescence, CD and MALDI data). Cellular toxicity of SA (700µM), Zn(2+) (700µM) and Cu(2+) (700µM) on SH-SY5Y (1×10(5) cells) showed 9.8%, 38.0% and 14.4% compared to control values. Combinations showed more severe toxicities: 71.9% and 93.1% for SA (70µM)+Cu(2+) (700µM) and SA (70µM)+Zn(2+) (700µM), respectively, suggesting complexation itself may be toxic.