RT-PCR analysis and stress response capacity of transgenic gshI-poplar clones (Populus x canescens) in response to paraquat exposure

Stress response capacity (Fv/Fm at 690 nm and F690/F735 at Fmax) of untransformed hybrid poplar, Populus x canescens (P tremula x P alba), and two transgenic lines overexpressing gamma-ECS (gamma-glutamylcysteine synthetase) either in the cytosol (cyt-ECS) or in the chloroplast (chl-ECS) was studied in response to the herbicide paraquat (4.0 x 10(-9) to 4.0 x 10(-6) M) for 21 days. Significant differences at sublethal (4.0 x 10(-7) M) and bleaching (4.0 x 10(-6) M) concentrations of paraquat were observed with about a two-fold and eight-fold decrease in the photosynthetic activity (Fv/Fm at 690 nm and F690/F735 at Fmax), respectively. None of the gshI transgenic lines (cyt-ECS, chl-ECS) with elevated GSH content exhibited significant tolerance to paraquat. Semiquantitative RT-PCR of the cyt-ECS clone was used for gene expression analysis of the nuclear encoded rbcS gene and the stress responsive gst gene. Expression of the constitutively expressed 26SrRNA ribosomal gene was probed as a control for all RT-PCR reactions. The relative intensities of gene expressions normalized to the level of 26SrRNA intensity showed a 50% decrease in the nuclear encoded rbcS expression and a 120% increase in the stress responsive gst gene expression of the paraquat treated (4.0 x 10(-7) M) samples of the transgenic poplar line (cyt-ECS).     

The effects of training and detraining on memory, neurotrophins and oxidative stress markers in rat brain

In the current investigation we tested how swimming training (T) (8 week, 5 times/week, 2 h/day), and detraining (DT) affects brain functions and oxidative stress markers in rat brain. The free radical concentration, measured by electron paramagnetic resonance, decreased in brain of T and DT rats compared to controls (C). The level of brain-derived neurotrophic factor (BDNF) increased as a result of training, but decreased below the control level after 6 weeks of detraining. In addition, the concentration of nerve growth factor (NGF) also declined with DT. The passive avoidance test was used to assess the memory of rats, and training-induced improvement was observed but the enhancement disappeared with detraining. When the content of mitochondrial electron transport complexes, as a potent free radical generator, was evaluated by the blue native gel method, no significant alterations were observed. The repair of nuclear and mitochondrial 8-oxodeoxyguanosine, as measured by the activity of OGG1, showed no significant difference. Therefore, the results suggest that regular exercise training improves memory, decreases the level of reactive oxygen species, and increase the production of BDNF and NGF. On the other hand, it appears that the beneficial effects of training are reversible in the brain, since detraining down-regulates the neurotrophin level, and memory. It is suggested that exercise training is more likely to beneficially effect the production of reactive oxygen species and the related oxidative damage.     

Interactions of granisetron with an agonist-free 5-HT3A receptor model

A new homology model of type-3A serotonin receptors (5-HT(3A)Rs) was built on the basis of the electron microscopic structure of the nicotinic acetylcholine receptor and with an agonist-free binding cavity. The new model was used to re-evaluate the interactions of granisetron, a 5-HT(3A)R antagonist. Docking of granisetron identified two possible binding modes, including a newly identified region for antagonists formed by loop B, C, and E residues. Amino acid residues L184-D189 in loop B were mutated to alanine, while Y143 and Y153 in loop E were mutated to phenylalanine. Mutation H185A resulted in no detectable granisetron binding, while D189A resulted in a 22-fold reduction in affinity. Y143F and Y153F decreased granisetron affinity to the same extent as Y143A and Y153A mutations, supporting the role of the OH groups of these tyrosines in loop E. Modeling and mutation studies suggest that granisetron plays its antagonist role by hindering the closure of the back wall of the binding cavity.     

Electrical properties and cell-to-cell communication of the salivary gland cells of the snail, Helix pomatia

The aim of the present study was to assess the cellular mechanism of secretion in the salivary gland of the snail, Helix pomatia, using electrophysiological, electron microscopic and immunohistochemical techniques. A homogeneously distributed membrane potential (-56.6 +/- 9.8 mV) was determined mainly by a K+ -electrochemical gradient and partly by the contribution of the electrogenic Na+ -pump and Cl- conductance. Low resistance electrical coupling sites were identified physiologically. Transmission electron microscopy and innexin 2 antibody revealed the presence of gap-junction-like membrane structures between gland cells. It is suggested that gap-junctions are sites of electrotonic intercellular communication, which integrate the gland cells into a synchronized functional unit in the acinus. Stimulation of the salivary nerve elicited secretory potentials (depolarization) which could be mimicked by local application of acetylcholine, dopamine or serotonin. In voltage-clamp experiments four major conductances were identified: a delayed rectifier (IK), a transient (IA) and a Ca2+ -activated outward K+ current (IK(Ca)) and Ca2+ -inward currents (ICa). It is suggested that one or more of these conductances may give rise to a stimulus activated secretory potential leading to excitation-secretion coupling and subsequent the release of the mucus from the gland cells.     

The effects of corticotropin-releasing factor and the urocortins on hypothalamic gamma-amino butyric acid release--the impacts on the hypothalamic-pituitary-adrenal axis

Corticotropin-releasing factor (CRF) and the urocortins (UCNs) are structurally and pharmacologically related neuropeptides which regulate the endocrine, autonomic, emotional and behavioral responses to stress. CRF and UCN1 activate both CRF receptors (CRFR1 and CRFR2) with CRF binding preferentially to CRFR1 and UCN1 binding equipotently to both receptors. UCN2 and UCN3 activate selectively CRFR2. Previously an in vitro study demonstrated that superfusion of both CRF and UCN1 elevated the GABA release elicited by electrical stimulation from rat amygdala, through activation of CRF1 receptors. In the present experiments, the same in vitro settings were used to study the actions of CRF and the urocortins on hypothalamic GABA release. CRF and UCN1 administered in equimolar doses increased significantly the GABA release induced by electrical stimulation from rat hypothalamus. The increasing effects of CRF and UCN1 were inhibited considerably by the selective CRFR1 antagonist antalarmin, but were not influenced by the selective CRFR2 antagonist astressin 2B. UCN2 and UCN3 were ineffective. We conclude that CRF1 receptor agonists induce the release of GABA in the hypothalamus as well as previously the amygdala. We speculate that CRF-induced GABA release may act as a double-edged sword: amygdalar GABA may disinhibit the hypothalamic CRF release, leading to activation of the hypothalamic-pituitary-adrenal axis, whereas hypothalamic GABA may inhibit the hypothalamic CRF release, terminating this activation.     

Brief communication: New Y‐chromosome binary markers improve phylogenetic resolution within haplogroup R1a1

Haplogroup R1a1‐M198 is a major clade of Y chromosomal haplogroups which is distributed all across Eurasia. To this date, many efforts have been made to identify large SNP‐based subgroups and migration patterns of this haplogroup. The origin and spread of R1a1 chromosomes in Eurasia has, however, remained unknown due to the lack of downstream SNPs within the R1a1 haplogroup. Since the discovery of R1a1‐M458, this is the first scientific attempt to divide haplogroup R1a1‐M198 into multiple SNP‐based sub‐haplogroups. We have genotyped 217 R1a1‐M198 samples from seven different population groups at M458, as well as the Z280 and Z93 SNPs recently identified from the “1000 Genomes Project”. The two additional binary markers present an effective tool because now more than 98% of the samples analyzed assign to one of the three sub‐haplogroups. R1a1‐M458 and R1a1‐Z280 were typical for the Hungarian population groups, whereas R1a1‐Z93 was typical for Malaysian Indians and the Hungarian Roma. Inner and Central Asia is an overlap zone for the R1a1‐Z280 and R1a1‐Z93 lineages. This pattern implies that an early differentiation zone of R1a1‐M198 conceivably occurred somewhere within the Eurasian Steppes or the Middle East and Caucasus region as they lie between South Asia and Eastern Europe. The detection of the Z93 paternal genetic imprint in the Hungarian Roma gene pool is consistent with South Asian ancestry and amends the view that H1a‐M82 is their only discernible paternal lineage of Indian heritage. © 2012 Wiley Periodicals, Inc.