Firing synchronization and temporal order in noisy neuronal networks

Noise-induced complete synchronization and frequency synchronization in coupled spiking and bursting neurons are studied firstly. The effects of noise and coupling are discussed. It is found that bursting neurons are easier to achieve firing synchronization than spiking ones, which means that bursting activities are more important for information transfer in neuronal networks. Secondly, the effects of noise on firing synchronization in a noisy map neuronal network are presented. Noise-induced synchronization and temporal order are investigated by means of the firing rate function and the order index. Firing synchronization and temporal order of excitatory neurons can be greatly enhanced by subthreshold stimuli with resonance frequency. Finally, it is concluded that random perturbations play an important role in firing activities and temporal order in neuronal networks.     

The role of tetrahydrobiopterin in superoxide generation from eNOS: enzymology and physiological implications

Tetrahydrobiopterin (BH₄) is a ubiquitous pteridine metabolite that serves as a NOS cofactor. Recently, we showed that BH₄ efficiently inhibits superoxide generation from the heme group at the oxygenase domain of eNOS. This role indicates that BH₄ acts as a redox switch in the catalytic mechanism of the enzyme, which may have important consequences in the physiology of the endothelium. Here the mechanism by which BH₄ inhibits superoxide release from eNOS and the "uncoupling" effects of oxidized BH₄ metabolites are presented. The implications of the disparate actions of fully reduced and oxidized BH 4 metabolites in the control of eNOS biochemistry are discussed in the light of clinical data indicating that BH 4 levels are important in the regulation of superoxide levels and of endothelial reactivity.     

Direct detection of nitric oxide and its roles in maintaining gastric mucosal integrity following ethanol-induced injury in rats

In gastric mucosal injury, nitric oxide (NO) plays both cytoprotective and cytotoxic roles, and the NO level is one determinant of these dual roles. We employed electron paramagnetic resonance (EPR)-spectrometry combined with an NO-trapping technique to directly evaluate NO production in ethanol-induced gastric injury in rats. The rat stomach, mounted on an ex vivo chamber, was perfused with ethanol (12.5 and 43%), and NO levels in mucosal tissues were measured during perfusion. Luminal nitrite/nitrate (NO_x) content, mucosal blood flow, area of mucosal injury, transmucosal potential difference (PD), and luminal pH were simultaneously monitored with/without preadministration of the NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (L-NAME). NO levels in the gastric tissue increased during ethanol perfusion, and luminal NO_x levels increased after the perfusion, accompanying an increase in the area of mucosal injury and changes in physiological parameters. Preadministration of L-NAME aggravated the gastric mucosal damage and suppressed increases in mucosal blood flow in a dose-dependent manner. These results demonstrate that endogenous NO produced in ethanol-induced gastric injury contributes to maintenance of mucosal integrity via regulation of mucosal blood flow.     

Resonance Raman and crystallographic studies on the complex [Fe2(bbpnol)2]·2DMF (bbpnol=N,N′-bis(2-hydroxybenzyl)-2-ol-1,3-propanediamine)

The ligand N,N′-bis(2-hydroxybenzyl)-2-ol-1,3-propanediamine (H₃bbpnol) reacts with iron(III) perchlorate forming two dinuclear bis-μ-alkoxo complexes, a ‘cis-trans’ isomer (complex 1) previously reported and a ‘cis-cis’ isomer (complex 2) characterized in this work. The main differences found in complex 2 structure are, (a) the four phenolic oxygens are trans to the alkoxo bridges; (b) each ligand is shared by the two Fe(III) ions occupying two coordination positions at each center. The Fe(III) centers in the resulting centrosymmetric structure in complex 2 have a distorted-octahedral geometry with the equatorial plane occupied by the phenolic and alcoholic oxygen atoms and the apical positions are filled by the aminic nitrogen atoms. The resonance Raman (RR) spectra of these two isomeric complexes are somewhat different with the intensity of some low-frequency modes increasing in the less symmetric core. The electronic spectra of both complexes are similar, but the molar absorptivities are substantially increased in complex 2, indicating the presence of an electronic coupling between the phenolate moieties trans in relation to the alkoxo bridge, and that phenolates coordinated cis to the alkoxo bridge do not seem to contribute to LMCT oscillator strength. This is directly reflected in the Raman excitation profiles (REP) of the chromophore modes, with the vibrational modes of the ‘cis-cis’ isomer showing a greater intensification compared with the ‘cis-trans’ isomer.     

Vibrational spectroscopy to study the properties of redox-active tyrosines in photosystem II and other proteins

Tyrosine radicals play catalytic roles in essential metalloenzymes. Their properties—midpoint potential, stability…—or environment varies considerably from one enzyme to the other. To understand the origin of these properties, the redox tyrosines are studied by a number of spectroscopic techniques, including Fourier transform infrared (FTIR) and resonance Raman (RR) spectroscopy. An increasing number of vibrational data are reported for the (modified-) redox active tyrosines in ribonucleotide reductases, photosystem II, heme catalase and peroxidases, galactose and glyoxal oxidases, and cytochrome oxidase. The spectral markers for the tyrosinyl radicals have been recorded on models of (substituted) phenoxyl radicals, free or coordinated to metals. We review these vibrational data and present the correlations existing between the vibrational modes of the radicals and their properties and interactions formed with their environment: we present that the ν_7a(C-O) mode of the radical, observed both by RR and FTIR spectroscopy at 1480-1515 cm⁻¹, is a sensitive marker of the hydrogen bonding status of (substituted)-phenoxyl and Tyr, while the ν_8a(C-C) mode may probe coordination of the Tyr to a metal. For photosystem II, the information obtained by light-induced FTIR difference spectroscopy for the two redox tyrosines Tyr_D and Tyr_Z and their hydrogen bonding partners is discussed in comparison with those obtained by other spectroscopic methods.     

Successful resonance Raman study of cresolase activity of mushroom tyrosinase

Mono-oxygenase (cresolase) activity of mushroom tyrosinase (MT) in the presence of 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide (HPABS) was successfully studied by resonance Raman (rR) spectroscopy. HPABS is a synthetic competitive inhibitor (K(i)=7.17 x 10(-6)M) for the cresolase activity with a large extinction coefficient at 365 nm. Upon reacting with MT, HPABS produced an enzyme-inhibitor (EI) complex with sufficiently long life span. Analyzing the ensuing spectrum indicates that the azo tautomer of HPABS binds to the enzyme and retains its geometrical isomeric form in the EI complex. The observed changes in the rR spectrum of HPABS after binding to MT support the idea that an electrophilic attack on the inhibitor has happened. Similar experiments were designed for studying the oxidase activity of MT. However, the enzymatic reaction, even in the presence of 4-[(2,4-dinitrophenyl)azo]-1,2-benzenediols was still fast enough to tan the reaction solution quickly and render its rR spectrum impregnable background.     

Structural insights into a StART-like domain in Lam4 and its interaction with sterol ligands

Sterols are essential components of cellular membranes and shape their biophysical properties. The recently discovered family of Lipid transfer proteins Anchored at Membrane contact sites (LAMs) has been suggested to carry out intracellular sterol traffic using StART-like domains. Here, we studied the second StART-like domain of Lam4p from S. cerevisiae by NMR. We show that NMR data are consistent with the StART-like domain structure, and that several functionally important regions within the domain exhibit significant conformational dynamics. NMR titration experiments confirm sterol binding to the canonical sterol-binding site and suggest a role of membrane interactions on the thermodynamics and kinetics of sterol binding.     

An easy-to-use FRET protein substrate to detect calpain cleavage in vitro and in vivo

Calpain-1 and -2 are Ca^2 +-activated intracellular cysteine proteases that regulate a wide range of cellular functions through the cleavage of their protein substrates. Unlike degradative proteases, calpains make limited, transformative cleavages, typically in accessible sequences linking discrete subdomains, to irreversibly alter substrate functions. The biological roles of calpain and their interplay with calcium signaling are of significant biomedical interest as biomarkers and potential therapeutic targets in a growing number of diseases including Alzheimer's, cancer and fibrosis. Unfortunately, many of the colorimetric and fluorimetric assays that have been developed to study calpain activity suffer from low sensitivity and/or poor calpain specificity. To address the need for a highly sensitive and calpain-specific substrate suitable for in vitro and in vivo calpain activity analysis, we have developed a protein FRET probe. We inserted the optimized calpain cleavage sequence PLFAAR between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) and modulated its flanking sequences for optimal calpain cleavage. We demonstrate greater sensitivity and calpain-specificity of an optimal 16-residue PLFAAR-based FRET substrate compared to a standard α-spectrin-based probe. The 16-residue PLFAAR protein FRET substrate is not significantly cleaved by trypsin, chymotrypsin, cathepsin-L or caspase-3, and is highly sensitive to both calpain-1 and -2. After transfection of the substrate gene into breast cancer cells the PLFAAR protein FRET product was cut in lysed wild-type cells but not in those with a calpain knock-out phenotype. Blockage of substrate cleavage in the lysates by endogenous and exogenous calpastatin was observed, and was overcome by adding extra calpain.     

Optical fiber tips for biological applications: From light confinement,biosensing to bioparticles manipulation

Background

The tip of an optical fiber has been considered an attractive platform in Biology. The simple cleaved end of an optical fiber can be machined, patterned and/or functionalized, acquiring unique properties enabling the exploitation of novel optical phenomena. Prompted by the constant need to measure and manipulate nanoparticles, the invention of the Scanning Near-field Optical Microscopy (SNOM) triggered the optimization and development of novel fiber tip microfabrication methods. In fact, the fiber tip was soon considered a key element in SNOM by confining light to sufficiently small extensions, challenging the diffraction limit. As result and in consequence of the newly proposed “Lab On Tip” concept, several geometries of fiber tips were applied in three main fields: imaging (in Microscopy/Spectroscopy), biosensors and micromanipulation (Optical Fiber Tweezers, OFTs). These are able to exert forces on microparticles, trap and manipulate them for relevant applications, as biomolecules mechanical study or protein aggregates unfolding.