Gas Exchange of In Vitro and Ex Vitro Grown Grapevine Plants

Net photosynthetic rate (P _N) and dark respiration rate (R _D) were measured in Vitis vinifera L. cvs. Dimiat 4/24 (23^rd subculture), Dimiat 4/38 (22^nd subculture), and Italian Riesling 3/47 (22^nd subculture) on days 3, 2, and 1 (1^st series) before transfer from the in vitro culture and on days 14, 15, 16 (2^nd series) and 28, 29, 30 (3^rd series) after the transfer. P _N of in vitro and ex vitro plants was strongly affected by irradiance. P _N and R _D of in vitro plantlets were lower and transpiration rate (E) was higher compared to those of ex vitro plantlets. P _N, R _D, and E changed in the course of acclimation.     

High-Throughput Profiling of Caenorhabditis elegans Starvation-Responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6–20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans.     

The morphology of single muscle fibre potentials – Part I: Simulation study of the distortion introduced by the distant-interfering potentials

Some morphologic aspects of human single fibre action potentials (SFAPs) are not sufficiently well-known. This uncertainty especially concerns the declining negative phase and the final positive phase (third phase) of SFAPs, as these parts are significantly affected by distant electrical activity. The incomplete characterisation of the SFAP shape is also explained by the limited knowledge of human intracellular action potentials (IAPs). The objectives of this study are to assess the morphologic features of human SFAPs and to derive information about the characteristics of human IAPs. To achieve this, the study has been divided into two parts. The present paper, Part I, aims to analyse the changes in the SFAP time-course introduced by distant-interfering potentials and to evaluate how these changes depend on the spike duration of the corresponding IAP. It was found that, for fibre-to-electrode distances shorter than about 0.2 mm, SFAPs generated by short-spike IAPs have a declining negative phase with a steep approximately constant slope that is largely unaffected by the potentials from distant fibres. For the same distances, SFAPs resulting from wide-spike IAPs have a declining negative phase with a slow return towards the baseline that is highly sensitive to distant-interfering potentials. The third phase of an SFAP is considerably distorted by distant potentials irrespective of the spike duration of the IAP.     

Chlorophyll fluorescence as a tool for nutrient status identification in rapeseed plants

In natural conditions, plants growth and development depends on environmental conditions, including the availability of micro- and macroelements in the soil. Nutrient status should thus be examined not by establishing the effects of single nutrient deficiencies on the physiological state of the plant but by combinations of them. Differences in the nutrient content significantly affect the photochemical process of photosynthesis therefore playing a crucial role in plants growth and development. In this work, an attempt was made to find a connection between element content in (i) different soils, (ii) plant leaves, grown on these soils and (iii) changes in selected chlorophyll a fluorescence parameters, in order to find a method for early detection of plant stress resulting from the combination of nutrient status in natural conditions. To achieve this goal, a mathematical procedure was used which combines principal component analysis (a tool for the reduction of data complexity), hierarchical k-means (a classification method) and a machine-learning method—super-organising maps. Differences in the mineral content of soil and plant leaves resulted in functional changes in the photosynthetic machinery that can be measured by chlorophyll a fluorescent signals. Five groups of patterns in the chlorophyll fluorescent parameters were established: the ‘no deficiency’, Fe-specific deficiency, slight, moderate and strong deficiency. Unfavourable development in groups with nutrient deficiency of any kind was reflected by a strong increase in F _o and ΔV/Δt ₀ and decline in φ _Po, φ _Eo δ _Ro and φ _Ro. The strong deficiency group showed the suboptimal development of the photosynthetic machinery, which affects both PSII and PSI. The nutrient-deficient groups also differed in antenna complex organisation. Thus, our work suggests that the chlorophyll fluorescent method combined with machine-learning methods can be highly informative and in some cases, it can replace much more expensive and time-consuming procedures such as chemometric analyses.     

The conduction of protons in different stereoisomers of dioxolane-linked gramicidin A channels

Two different stereoisomers of the dioxolane-linked gramicidin A (gA) channels were individually synthesized (the SS and RR dimers;. Science. 244:813-817). The structural differences between these dimers arise from different chiralities within the dioxolane linker. The SS dimer mimics the helicity and the inter- and intramolecular hydrogen bonding of the monomer-monomer association of gA's. In contrast, there is a significant disruption of the helicity and hydrogen bonding pattern of the ion channel in the RR dimer. Single ion channels formed by the SS and RR dimers in planar lipid bilayers have different proton transport properties. The lipid environment in which the different dimers are reconstituted also has significant effects on single-channel proton conductance (g(H)). g(H) in the SS dimer is about 2-4 times as large as in the RR. In phospholipid bilayers with 1 M [H(+)](bulk), the current-voltage (I-V) relationship of the SS dimer is sublinear. Under identical experimental conditions, the I-V plot of the RR dimer is supralinear (S-shaped). In glycerylmonooleate bilayers with 1 M [H(+)](bulk), both the SS and RR dimers have a supralinear I-V plot. Consistent with results previously published (. Biophys. J. 73:2489-2502), the SS dimer is stable in lipid bilayers and has fast closures. In contrast, the open state of the RR channel has closed states that can last a few seconds, and the channel eventually inactivates into a closed state in either phospholipid or glycerylmonooleate bilayers. It is concluded that the water dynamics inside the pore as related to proton wire transfer is significantly different in the RR and SS dimers. Different physical mechanisms that could account for this hypothesis are discussed. The gating of the synthetic gA dimers seems to depend on the conformation of the dioxolane link between gA's. The experimental results provide an important framework for a detailed investigation at the atomic level of proton conduction in different and relatively simple ion channel structures.     

Allosteric activation mechanism of the alpha1beta2gamma2 gamma-aminobutyric acid type A receptor revealed by mutation of the conserved M2 leucine

A conserved leucine residue in the midpoint of the second transmembrane domain (M2) of the ligand-activated ion channel family has been proposed to play an important role in receptor activation. In this study, we assessed the importance of this leucine in the activation of rat alpha1beta2gamma2 GABA receptors expressed in Xenopus laevis oocytes by site-directed mutagenesis and two-electrode voltage clamp. The hydrophobic conserved M2 leucines in alpha1(L263), beta2(L259), and gamma2(L274) subunits were mutated to the hydrophilic amino acid residue serine and coexpressed in all possible combinations with their wild-type and/or mutant counterparts. The mutation in any one subunit decreased the EC(50) and created spontaneous openings that were blocked by picrotoxin and, surprisingly, by the competitive antagonist bicuculline. The magnitudes of the shifts in GABA EC(50) and picrotoxin IC(50) as well as the degree of spontaneous openings were all correlated with the number of subunits carrying the leucine mutation. Simultaneous mutation of the GABA binding site (beta2Y157S; increased the EC(50)) and the conserved M2 leucine (beta2L259S; decreased the EC(50)) produced receptors with the predicted intermediate agonist sensitivity, indicating the two mutations affect binding and gating independently. The results are discussed in light of a proposed allosteric activation mechanism.     

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV.

New tetrazolium method for the histochemical localization of dipeptidyl peptidase IV (DPP IV), based on a newly synthesized substrate Gly-L-Pro-1-hydroxy-4-naphthylamide is proposed. Upon the enzyme hydrolysis of the substrate a strong reducing agent, i.e. 4-amino-1-naphthol is released, which reduces tetrazolium salts to water-insoluble, deeply colored formazans, that precipitate on the sites of enzyme activity, marking them accurately. No auxiliary electron acceptor is needed for the redox reaction. The incubation is performed at the optimal pH of the enzyme. Precise enzyme localization is achieved in all organs studied. Thus, the new method avoids most of the disadvantages of the methods in use and might open new possibilities in peptidases histochemistry.     

Simulation analysis of interference EMG during fatiguing voluntary contractions. Part II--changes in amplitude and spectral characteristics.

Capabilities of amplitude and spectral methods for information extraction from interference EMG signals were assessed through simulation and preliminary experiment. Muscle was composed of 4 types of motor units (MUs). Different hypotheses on changes in firing frequency of individual MUs, intracellular action potential (IAP) and muscle fibre propagation velocity (MFPV) during fatigue were analyzed. It was found that changes in amplitude characteristics of interference signals (root mean square, RMS, or integrated rectified value, IEMG) detected by intramuscular and surface electrodes differed. RMS and IEMG of surface detected interference signals could increase even under MU firing rate reduction and without MU synchronisation. IAP profile lengthening can affect amplitude characteristics more significantly than MU firing frequency. Thus, an increase of interference EMG amplitude is unreliable to reflect changes in the neural drive. The ratio between EMG amplitude and contraction response can hardly characterise the so-called 'neuromuscular efficiency'. The recently proposed spectral fatigue indices can be used for quantification of interference EMG signals. The indices are practically insensitive to MU firing frequency. IAP profile lengthening and decrease in MFPV enhanced the index value, while recruitment of fast fatigable MUs reduced it. Sensitivity of the indices was higher than that of indices traditionally used.