New insights into the organisation of the oxidative phosphorylation system in the example of pea shoot mitochondria.

The physical and functional organisation of the OXPHOS system in mitochondria in vivo remains elusive. At present, different models of OXPHOS arrangement, representing either highly ordered respiratory strings or, vice versa, a set of randomly dispersed supercomplexes and respiratory complexes, have been suggested. In the present study, we examined a supramolecular arrangement of the OXPHOS system in pea shoot mitochondria using digitonin solubilisation of its constituents, which were further analysed by classical BN-related techniques and a multidimensional gel electrophoresis system when required. As a result, in addition to supercomplexes I1III2, I1III2IVn and III2IV1-2, dimer V2, and individual complexes I-V previously detected in plant mitochondria, new OXPHOS structures were also revealed. Of them, (1) a megacomplex (IIxIIIyIVz)n including complex II, (2) respirasomes I2III4IVn with two copies of complex I and dimeric complex III2, (3) a minor new supercomplex IV1Va2 comigrating with I1III2, and (4) a second minor form of ATP synthase, Va, were found. The activity of singular complexes I, IV, and V was higher than the activity of the associated forms. The detection of new supercomplex IV1Va2, along with assemblies I1III2 and I1-2III2-4IVn, prompted us to suggest the occurrence of in vivo oxphosomes comprising complexes I, III2, IV, and V. The putative oxphosome's stoichiometry, historical background, assumed functional significance, and subcompartmental location are discussed herein.

Facile chemical routes to mesoporous silver substrates for SERS analysis.

Mesoporous silver nanoparticles were easily synthesized through the bulk reduction of crystalline silver(I) oxide and used for the preparation of highly porous surface-enhanced Raman scattering (SERS)-active substrates. An analogous procedure was successfully performed for the production of mesoporous silver films by chemical reduction of oxidized silver films. The sponge-like silver blocks with high surface area and the in-situ-prepared mesoporous silver films are efficient as both analyte adsorbents and Raman signal enhancement mediators. The efficiency of silver reduction was characterized by X-ray diffraction and X-ray photoelectron spectroscopy. The developed substrates were applied for SERS detection of rhodamine 6G (enhancement factor of about 1-5 × 105) and an anti-ischemic mildronate drug (meldonium; enhancement factor of ≈102) that is known for its ability to increase the endurance performance of athletes.

Novel Multilayer Nanostructured Materials for Recognition of Polycyclic Aromatic Sulfur Pollutants and Express Analysis of Fuel Quality and Environmental Health by Surface Enhanced Raman Spectroscopy.

A novel concept of advanced SERS (surface enhanced Raman spectroscopy) planar sensors is suggested for fast analysis of sulfur-containing hazardous oil components and persistent pollutants. The main advantage of the proposed sensors is the utilization of an additional preconcentrating layer of optically transparent chitosan gel, which is chemically modified with appropriate π-acceptor compounds to selectively form charge-transfer complexes (CTCs) at the interface with nanostructured silver coatings. The CTCs shift absorption bands of polycyclic aromatic sulfur heterocycles (PASHs) and other important analytes in a controllable way and thus provide a surplus enhancement of vibration modes due to resonant Raman scattering. This novel indicator system provides multiplex determination of PASHs in different forms in a small volume of oil without any tedious sample pretreatment steps. This approach opens new possibilities of increasing either spectral and concentration sensitivity or specificity of SERS-based sensors, allowing for new developments in the fields of ecology, advanced fuel analysis, and other prospective applications.

Effect of hydrogen peroxide on electrical coupling between identified Lymnaea neurons.

The pair of giant reciprocally coupled neurons VD1 and RPaD2 within the CNS of the freshwater pond snail Lymnaea stagnalis was used to analyse the effect of hydrogen peroxide on gap-junction connection. Electrical activity of VD1/RPaD2 was recorded with intracellular microelectrodes in order to analyse gap-junction signalling. Hydrogen peroxide application (1 × 10⁻4 M) results in a rapid, 1.3-fold, increase in VD1/RPaD2 spiking frequency within 30 s after application. This was accompanied by a slight reduction in action potential amplitude. In addition, H2O2 induced a significant reduction in the steady-state bidirectional coupling ratio between the neurons. The maximal reduction in the coupling ratio, 1.8-1.9 fold, was measured 3 min after H2O2 application. However, the network input resistance did not undergo a detectable change. The voltage-gated Ca²âº channel blocker, nifedipine (1 × 10⁻4 M), abolished the effect of H2O2 on the coupling ratio and firing frequency. All the effects of H2O2 were reversible, that is, washing the preparation with standard physiological saline restored the properties of the neuronal coupling to the pre-treatment value. These data are consistent with a dynamic modulation of the gap-junction properties by H2O2 between these two neurons.

Sequence-specific cleavage of RNA in the absence of divalent metal ions by a DNAzyme incorporating imidazolyl and amino functionalities.

Two modified 2'-deoxynucleoside 5'-triphosphates have been used for the in vitro selection of a modified deoxyribozyme (DNAzyme) capable of the sequence-specific cleavage of a 12 nt RNA target in the absence of divalent metal ions. The modified nucleotides, a C5-imidazolyl-modified dUTP and 3-(aminopropynyl)-7-deaza-dATP were used in place of TTP and dATP during the selection and incorporate two extra protein-like functionalities, namely, imidazolyl (histidine analogue) and primary amino (lysine analogue) into the DNAzyme. The functional groups are analogous to the catalytic Lys and His residues employed during the metal-independent cleavage of RNA by the protein enzyme RNaseA. The DNAzyme requires no divalent metal ions or other cofactors for catalysis, remains active at physiological pH and ionic strength and can recognize and cleave a 12 nt RNA substrate with sequence specificity. This is the first example of a functionalized, metal-independent DNAzyme that recognizes and cleaves an all-RNA target in a sequence-specific manner. The selected DNAzyme is two orders of magnitude more efficient in its cleavage of RNA than an unmodified DNAzyme in the absence of metal ions and represents a rate enhancement of 10(5) compared with the uncatalysed hydrolysis of RNA.

Effect of temperature on synaptic transmission between identified neurones of the mollusc Lymnaea stagnalis.

The mollusc, Lymnaea stagnalis, has been used as a model to study the mechanisms of temperature-dependent processes in the central nervous system. Effects of temperature changes on transmission in monosynaptic connections, made by the FMRFamide-containing neurone VD4 and the giant dopaminergic neurone RPeD1 with follower neurones, were recorded with intracellular microelectrodes. In the temperature range of 4-6 degrees C, inhibitory postsynaptic potentials (IPSP) in response to VD4 stimulation were not observed in postsynaptic cells while the IPSPs persisted in the RPeD1 followers. A temperature rise resulted in a sharp increase in the IPSP amplitude in followers of both VD4 and RPeD1. In isolated nervous systems taken from molluscs which have been kept at 4-6 degrees C for 2 weeks and more, no coupling between VD4, RPeD1 and synaptically connected cells was seen in the full experimental temperature range. The synaptic coupling recovered only after maintaining the molluscs at a water temperature of 14-16 degrees C for at least 2 days. The changes observed in synaptic responses to temperature alterations correspond to the behaviour of the molluscs.