Amperometric detection of stimulus-induced quantal release of catecholamines from cultured superior cervical ganglion neurons.

Amperometry has been used for real-time electrochemical detection of the quantal release of catecholamines and indolamines from secretory granules in chromaffin and mast cells. Using improved-sensitivity carbon fiber electrodes, we now report the detection of quantal catecholamine release at the surface of somas of neonatal superior cervical ganglion neurons that are studded with axon varicosities containing synaptic vesicles. Local application of a bath solution containing high K+ or black widow spider venom, each of which greatly enhances spontaneous quantal release of transmitter at synapses, evoked barrages of small-amplitude (2-20 pA), short-duration (0.5-2 ms) amperometric quantal "spikes". The median spike charge was calculated as 11.3 fC. This figure corresponds to 3.5 x 10(4) catecholamine molecules per quantum of release, or approximately 1% that evoked by the discharge of the contents of a chromaffin granule.

Toxin-induced pore formation is hindered by intermolecular hydrogen bonding in sphingomyelin bilayers

Sticholysin I and II (StnI and StnII) are pore-forming toxins that use sphingomyelin (SM) for membrane binding. We examined how hydrogen bonding among membrane SMs affected the StnI- and StnII-induced pore formation process, resulting in bilayer permeabilization. We compared toxin-induced permeabilization in bilayers containing either SM or dihydro-SM (lacking the trans 4 double bond of the long-chain base), since their hydrogen-bonding properties are known to differ greatly. We observed that whereas both StnI and StnII formed pores in unilamellar vesicles containing palmitoyl-SM or oleoyl-SM, the toxins failed to similarly form pores in vesicles prepared from dihydro-PSM or dihydro-OSM. In supported bilayers containing OSM, StnII bound efficiently, as determined by surface plasmon resonance. However, StnII binding to supported bilayers prepared from dihydro-OSM was very low under similar experimental conditions. The association of the positively charged StnII (at pH 7.0) with unilamellar vesicles prepared from OSM led to a concentration-dependent increase in vesicle charge, as determined from zeta-potential measurements. With dihydro-OSM vesicles, a similar response was not observed. Benzyl alcohol, which is a small hydrogen-bonding compound with affinity to lipid bilayer interfaces, strongly facilitated StnII-induced pore formation in dihydro-OSM bilayers, suggesting that hydrogen bonding in the interfacial region originally prevented StnII from membrane binding and pore formation. We conclude that interfacial hydrogen bonding was able to affect the membrane association of StnI- and StnII, and hence their pore forming capacity. Our results suggest that other types of protein interactions in bilayers may also be affected by hydrogen-bonding origination from SMs.

Study of the interface Pt(111)/ [Emmim][NTf2] using laser-induced temperature jump experiments

The interface between a Pt(111) electrode and a room temperature ionic liquid, 1-ethyl-2,3-dimethylimidazolium bis(trifluoromethylsulfonyl)imide, was investigated with the laser-induced temperature jump method. In this technique, the temperature of the interface is suddenly increased by applying short laser pulses. The change of the electrode potential caused by the thermal perturbation is measured under coulostatic conditions during the subsequent temperature relaxation. This change is mainly related to the reorganization of the solvent components near the electrode surface. The sign of the potential transient depends on the potential of the experiment. At high potential values, positive transients indicate a higher density of anions than cations close the surface, contributing negatively to the potential of the electrode. Decreasing the applied potential to sufficiently low values, the transient becomes negative, meaning that the density of cations becomes then higher at the surface of the electrode. The potential dependence of the interfacial response shows a marked hysteresis depending on the direction in which the applied potential is changed.

Comparison of stress induced by manual restraint and immobilisation in the estuarine crocodile, Crocodylus porosus

This study compared the stress induced in captive estuarine crocodiles, Crocodylus porosus, by two different handling methods: manual restraint (noosing with ropes) and immobilization by electro-stunning. To stun, a short charge (approx. 6 s) at 110 V was delivered to the back of the necks of C. porosus using a custom-built device, which immobilized the animals for 5-10 min. Immobilized and restrained animals were measured and sexed, and the condition of the skin assessed. Blood samples were taken from some animals immediately after restraint or immobilization. Other animals were returned to their pens to recover for periods of 30 min, 1, 4, 12, 24 or 48 hours after which they were stunned and blood samples taken. Individual animals (mean body length 1.96 m, N=99) were bled only once. Haematocrit and haemoglobin concentrations were measured and plasma samples were analysed for corticosterone, glucose and lactate levels. Following restraint, there were significant increases in haematocrit, haemoglobin, glucose, lactate and corticosterone concentrations in C. porosus. For restrained animals, recovery to baseline levels occurred after approximately 8 hours. The stress response of stunned animals was significantly reduced compared to manually captured and restrained crocodiles. Both groups showed a significant increase in haematocrit, haemoglobin concentration and lactate levels, however the magnitude of change was significantly reduced, and recovery was faster in stunned animals. No increase in either glucose or corticosterone levels occurred with immobilisation. The results imply that immobilization by electro-stunning is much less stressful. (C) 2003 Wiley-Liss, Inc.

Development and evaluation of an ion induced x-ray emission system for the analysis of light and medium weight elements

The present work describes the development of a proton induced X-ray emission (PIXE) analysis system, especially designed and builtfor routine quantitative multi-elemental analysis of a large number of samples. The historical and general developments of the analytical technique and the physical processes involved are discussed. The philosophy, design, constructional details and evaluation of a versatile vacuum chamber, an automatic multi-sample changer, an on-demand beam pulsing system and ion beam current monitoring facility are described.The system calibration using thin standard foils of Si, P, S,Cl, K, Ca, Ti, V, Fe, Cu, Ga, Ge, Rb, Y and Mo was undertaken at proton beam energies of 1 to 3 MeV in steps of 0.5 MeV energy and compared with theoretical calculations. An independent calibration check using bovine liver Standard Reference Material was performed.  The minimum detectable limits have been experimentally determined at detector positions of 90° and 135° with respect to the incident beam for the above range of proton energies as a function of atomic number Z. The system has detection limits of typically 10- 7 to 10- 9 g for elements 14charge.  Computer programmes have been written for data analysis and calculations of areal density of thin foils using Rutherford backscattering data.  Amniotic fluid samples supplied by South Sefton Health Authority were successfully analysed for their low base line elemental concentrations. In conclusion the findings of this work are discussed with suggestions for further work .

Liposomal cationic charge and antigen adsorption are important properties for the efficient deposition of antigen at the injection site and ability of the vaccine to induce a CMI response

With respect to liposomes as delivery vehicles and adjuvants for vaccine antigens, the role of vesicle surface charge remains disputed. In the present study we investigate the influence of liposome surface charge and antigen-liposome interaction on the antigen depot effect at the site of injection (SOI). The presence of liposome and antigen in tissue at the SOI as well as the draining lymphatic tissue was quantified to analyse the lymphatic draining of the vaccine components. Furthermore investigations detailing cytokine production and T-cell antigen specificity were undertaken to investigate the relationship between depot effect and the ability of the vaccine to induce an immune response. Our results suggest that cationic charge is an important factor for the retention of the liposomal component at the SOI, and a moderate to high (>50%) level of antigen adsorption to the cationic vesicle surface was required for efficient antigen retention in the same tissue. Furthermore, neutral liposomes expressing poor levels of antigen retention were limited in their ability to mediate long term (14 days) antigen presentation to circulating antigen specific T-cells and to induce the Th1 and Th17 arms of the immune system, as compared to antigen adsorbing cationic liposomes. The neutral liposomes did however induce the production of IL-5 at levels comparable to those induced by cationic liposomes, indicating that neutral liposomes can induce a weak Th2 response.

Reentrant condensation of proteins in solution induced by multivalent counterions

Negatively charged globular proteins in solution undergo a condensation upon adding trivalent counterions between two critical concentrations C* and C**, C*charge on surface side chains through binding of the multivalent counterions.

Surface plasmon resonance-induced photocatalysis by Au nanoparticles decorated mesoporous g-C3N4 nanosheets under direct sunlight irradiation

In recent years, surface plasmon-induced photocatalytic materials with tunable mesoporous framework have attracted considerable attention in energy conversion and environmental remediation. Herein we report a novel Au nanoparticles decorated mesoporous graphitic carbon nitride (Au/mp-g-C3N4) nanosheets via a template-free and green in situ photo-reduction method. The synthesized Au/mp-g-C3N4 nanosheets exhibit a strong absorption edge in visible and near-IR region owing to the surface plasmon resonance effect of Au nanoparticles. More attractively, Au/mp-g-C3N4 exhibited much higher photocatalytic activity than that of pure mesoporous and bulk g-C3N4 for the degradation of rhodamine B under sunlight irradiation. Furthermore, the photocurrent and photoluminescence studies demonstrated that the deposition of Au nanoparticles on the surface of mesoporous g-C3N4 could effectively inhibit the recombination of photogenerated charge carriers leading to the enhanced photocatalytic activity. More importantly, the synthesized Au/mp-g-C3N4 nanosheets possess high reusability. Hence, Au/mp-g-C3N4 could be promising photoactive material for energy and environmental applications.

Experimental and Theoretical Models to Probe Mechanisms of Biological Charge Flow

Nature is challenged to move charge efficiently over many length scales. From sub-nm to μm distances, electron-transfer proteins orchestrate energy conversion, storage, and release both inside and outside the cell. Uncovering the detailed mechanisms of biological electron-transfer reactions, which are often coupled to bond-breaking and bond-making events, is essential to designing durable, artificial energy conversion systems that mimic the specificity and efficiency of their natural counterparts. Here, we use theoretical modeling of long-distance charge hopping (Chapter 3), synthetic donor-bridge-acceptor molecules (Chapters 4, 5, and 6), and de novo protein design (Chapters 5 and 6) to investigate general principles that govern light-driven and electrochemically driven electron-transfer reactions in biology. We show that fast, μm-distance charge hopping along bacterial nanowires requires closely packed charge carriers with low reorganization energies (Chapter 3); singlet excited-state electronic polarization of supermolecular electron donors can attenuate intersystem crossing yields to lower-energy, oppositely polarized, donor triplet states (Chapter 4); the effective static dielectric constant of a small (~100 residue) de novo designed 4-helical protein bundle can change upon phototriggering an electron transfer event in the protein interior, providing a means to slow the charge-recombination reaction (Chapter 5); and a tightly-packed de novo designed 4-helix protein bundle can drastically alter charge-transfer driving forces of photo-induced amino acid radical formation in the bundle interior, effectively turning off a light-driven oxidation reaction that occurs in organic solvent (Chapter 6). This work leverages unique insights gleaned from proteins designed from scratch that bind synthetic donor-bridge-acceptor molecules that can also be studied in organic solvents, opening new avenues of exploration into the factors critical for protein control of charge flow in biology.

Electrodeposited reduced-graphene oxide/cobalt oxide electrodes for charge storage applications

In the present work, electrochemically reduced-graphene oxide/cobalt oxide composites for charge storage electrodes were prepared by a one-step pulsed electrodeposition route on stainless steel current collectors and after that submitted to a thermal treatment at 200 degrees C. A detailed physico-chemical characterization was performed by X-ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and Raman spectroscopy. The electrochemical response of the composite electrodes was studied by cyclic voltammetry and charge-discharge curves and related to the morphological and phase composition changes induced by the thermal treatment. The results revealed that the composites were promising materials for charge storage electrodes for application in redox supercapacitors, attaining specific capacitances around 430 F g(-1) at 1 A g(-1) and presenting long-term cycling stability. (C) 2016 Elsevier B.V. All rights reserved.

Microcantilever arrays functionalised with spiropyran photoactive moieties as systems to measure photo-induced surface stress changes

[EN] Herein we investigate the feasibility of detecting photo-induced surface stress changes using the deflection response of cantilevers. For this purpose, silicon microcantilevers have been functionalised with spiropyran photochromic molecules, using both a monolayer and a polymeric brushes approach. Uponultraviolet light irradiation, the spiropyran unit is converted to the merocyanine form due to the photo-induced cleavage of the Cspiro-O bond. The two forms of the molecule have dramatically different charge,polarity and molecular conformations. This makes spiropyrans an ideal system to study the correlation between photo-induced molecular changes and corresponding changes in surface stress. Our investigations include monitoring the changes in static cantilever deflection, and consequently, surface stress of the spiropyran functionalised cantilevers on exposure to ultraviolet light. Cantilever deflection data reveals that ultraviolet induced conformational changes in the spiropyran moiety cause a change incompressive surface stress and this varies with the type of functionalisation method implemented. The change in surface stress response from the spiropyran polymer brushes functionalised cantilevers gives an average surface stress change of 98 Nm−1(n = 24) while the spiropyran monolayer coated cantilevers have an average surface stress change of about 446 Nm−1(n = 8) upon irradiation with UV light.

An Isoflavone From Dipteryx Alata Vogel Is Active Against The In Vitro Neuromuscular Paralysis Of Bothrops Jararacussu Snake Venom And Bothropstoxin I, And Prevents Venom-induced Myonecrosis.

Snakebite is a neglected disease and serious health problem in Brazil, with most bites being caused by snakes of the genus Bothrops. Although serum therapy is the primary treatment for systemic envenomation, it is generally ineffective in neutralizing the local effects of these venoms. In this work, we examined the ability of 7,8,3'-trihydroxy-4'-methoxyisoflavone (TM), an isoflavone from Dipteryx alata, to neutralize the neurotoxicity (in mouse phrenic nerve-diaphragm preparations) and myotoxicity (assessed by light microscopy) of Bothrops jararacussu snake venom in vitro. The toxicity of TM was assessed using the Salmonella microsome assay (Ames test). Incubation with TM alone (200 μg/mL) did not alter the muscle twitch tension whereas incubation with venom (40 μg/mL) caused irreversible paralysis. Preincubation of TM (200 μg/mL) with venom attenuated the venom-induced neuromuscular blockade by 84% ± 5% (mean ± SEM; n = 4). The neuromuscular blockade caused by bothropstoxin-I (BthTX-I), the major myotoxic PLA2 of this venom, was also attenuated by TM. Histological analysis of diaphragm muscle incubated with TM showed that most fibers were preserved (only 9.2% ± 1.7% were damaged; n = 4) compared to venom alone (50.3% ± 5.4% of fibers damaged; n = 3), and preincubation of TM with venom significantly attenuated the venom-induced damage (only 17% ± 3.4% of fibers damaged; n = 3; p < 0.05 compared to venom alone). TM showed no mutagenicity in the Ames test using Salmonella strains TA98 and TA97a with (+S9) and without (-S9) metabolic activation. These findings indicate that TM is a potentially useful compound for antagonizing the neuromuscular effects (neurotoxicity and myotoxicity) of B. jararacussu venom.

Changes In Chromatin Structure In Nih 3t3 Cells Induced By Valproic Acid And Trichostatin A.

Valproic acid (VPA) and trichostatin A (TSA) are known histone deacetylase inhibitors (HDACIs) with epigenetic activity that affect chromatin supra-organization, nuclear architecture, and cellular proliferation, particularly in tumor cells. In this study, chromatin remodeling with effects extending to heterochromatic areas was investigated by image analysis in non-transformed NIH 3T3 cells treated for different periods with different doses of VPA and TSA under conditions that indicated no loss of cell viability. Image analysis revealed chromatin decondensation that affected not only euchromatin but also heterochromatin, concomitant with a decreased activity of histone deacetylases and a general increase in histone H3 acetylation. Heterochromatin protein 1-α (HP1-α), identified immunocytochemically, was depleted from the pericentromeric heterochromatin following exposure to both HDACIs. Drastic changes affecting cell proliferation and micronucleation but not alteration in CCND2 expression and in ratios of Bcl-2/Bax expression and cell death occurred following a 48-h exposure of the NIH 3T3 cells particularly in response to higher doses of VPA. Our results demonstrated that even low doses of VPA (0.05 mM) and TSA (10 ng/ml) treatments for 1 h can affect chromatin structure, including that of the heterochromatin areas, in non-transformed cells. HP1-α depletion, probably related to histone demethylation at H3K9me3, in addition to the effect of VPA and TSA on histone H3 acetylation, is induced on NIH 3T3 cells. Despite these facts, alterations in cell proliferation and micronucleation, possibly depending on mitotic spindle defects, require a longer exposure to higher doses of VPA and TSA.

Low-density Lipoprotein Cholesterol And Radiotherapy-induced Carotid Atherosclerosis In Subjects With Head And Neck Cancer.

Radiotherapy (RT) is a risk factor for accelerated carotid artery atherosclerotic disease in subjects with head and neck cancer. However, the risk factors of RT-induced carotid artery remodeling are not established. This study aimed to investigate the effects of RT on carotid and popliteal arteries in subjects with head and neck cancer and to evaluate the relationship between baseline clinical and laboratory features and the progression of RT-induced atherosclerosis. Eleven men (age = 57.9 ± 6.2years) with head and neck cancer who underwent cervical bilateral irradiation were prospectively examined by clinical and laboratory analysis and by carotid and popliteal ultrasound before and after treatment (mean interval between the end of RT and the post-RT assessment = 181 ± 47 days). No studied subject used hypocholesterolemic medications. Significant increases in carotid intima-media thickness (IMT) (0.95 ± 0.08 vs. 0.87 ± 0.05 mm; p < 0.0001) and carotid IMT/diameter ratio (0.138 ± 0.013 vs. 0.129 ± 0.014; p = 0.001) were observed after RT, while no changes in popliteal structural features were detected. In addition, baseline low-density lipoprotein cholesterol levels showed a direct correlation with RT-induced carotid IMT change (r = 0.66; p = 0.027), while no other studied variable exhibited a significant relationship with carotid IMT change. These results indicate that RT-induced atherosclerosis is limited to the irradiated area and also suggest that it may be predicted by low-density lipoprotein cholesterol levels in subjects with head and neck cancer.

Peripheral P2x7 Receptor-induced Mechanical Hyperalgesia Is Mediated By Bradykinin.

P2X7 receptors play an important role in inflammatory hyperalgesia, but the mechanisms involved in their hyperalgesic role are not completely understood. In this study, we hypothesized that P2X7 receptor activation induces mechanical hyperalgesia via the inflammatory mediators bradykinin, sympathomimetic amines, prostaglandin E2 (PGE2), and pro-inflammatory cytokines and via neutrophil migration in rats. We found that 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate triethylammonium salt (BzATP), the most potent P2X7 receptor agonist available, induced a dose-dependent mechanical hyperalgesia that was blocked by the P2X7 receptor-selective antagonist A-438079 but unaffected by the P2X1,3,2/3 receptor antagonist TNP-ATP. These findings confirm that, although BzATP also acts at both P2X1 and P2X3 receptors, BzATP-induced hyperalgesia was mediated only by P2X7 receptor activation. Co-administration of selective antagonists of bradykinin B1 (Des-Arg(8)-Leu(9)-BK (DALBK)) or B2 receptors (bradyzide), β1 (atenolol) or β2 adrenoceptors (ICI 118,551), or local pre-treatment with the cyclooxygenase inhibitor indomethacin or the nonspecific selectin inhibitor fucoidan each significantly reduced BzATP-induced mechanical hyperalgesia in the rat hind paw. BzATP also induced the release of the pro-inflammatory cytokines tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6 and cytokine-induced neutrophil chemoattractant-1 (CINC-1), an effect that was significantly reduced by A-438079. Co-administration of DALBK or bradyzide with BzATP significantly reduced BzATP-induced IL-1β and CINC-1 release. These results indicate that peripheral P2X7 receptor activation induces mechanical hyperalgesia via inflammatory mediators, especially bradykinin, which may contribute to pro-inflammatory cytokine release. These pro-inflammatory cytokines in turn may mediate the contributions of PGE2, sympathomimetic amines and neutrophil migration to the mechanical hyperalgesia induced by local P2X7 receptor activation.