Gender-specific heat-shock tolerance of hydrated leaves in the desert moss Syntrichia caninervis

Female plants of the moss Syntrichia caninervis are more common than male plants and are found in more thermally stressful habitats than male plants. We hypothesized that this distribution pattern is due to a more favorable regeneration response of females to thermal stress compared to that of males. Hydrated leaves from four genotypes of both sexes of S. caninervis were exposed for 60 min under lighted conditions to temperatures of 20, 30, 35, 40, and 45°C. Leaves were allowed to regenerate on native sand under recovery conditions (12-h photoperiod, 20°C lighted, and 8°C dark) for 56 days, and over this period, protonemal emergence, growth rate, and shoot production were assessed. Leaves exposed to higher heat shocks produced protonemata significantly later, exhibited significantly reduced growth rates over the course of the experiment, and produced fewer shoots but did not differ in the probability of producing a shoot. Males tended to produce protonemata earlier than females at the highest thermal stress, whereas females tended to produce protonemata earlier under control conditions. Female leaves regenerated at twice the rate of male leaves, producing twice the area of protonemal cover; this gender difference was lessened at the highest thermal stress. Female leaves regenerated significantly more shoots than males, with each sex exhibiting different peaks of shoot production depending on the thermal stress. No interaction effect was detectable between gender and stress treatment. While females had a more favorable regeneration response relative to males, thermal stress diminished this difference, thus suggesting that our hypothesis was not supported.     

Soil-mixing effects on inorganic nitrogen production and consumption in forest and shrubland soils

Soils that are physically disturbed are often reported to show net nitrification and NO₃⁻ loss. To investigate the response of soil N cycling rates to soil mixing, we assayed gross rates of mineralization, nitrification, NH₄⁺ consumption, and NO₃⁻ consumption in a suite of soils from eleven woody plant communities in Oregon, New Mexico, and Utah. Results suggest that the common response of net NO₃⁻ flux from disturbed soils is not a straightforward response of increased gross nitrification, but instead may be due to the balance of several factors. While mineralization and NH₄⁺ assimilation were higher in mixed than intact cores, NO₃⁻ consumption declined. Mean net nitrification was 0.12 mg N kg⁻¹ d⁻¹ in disturbed cores, which was significantly higher than in intact cores (−0.19 mg N kg⁻¹ d⁻¹). However, higher net nitrification rates in disturbed soils were due to the suppression of NO₃⁻ consumption, rather than an increase in nitrification. Our results suggest that at least in the short term, disturbance may significantly increase NO₃⁻ flux at the ecosystem level, and that N cycling rates measured in core studies employing mixed soils may not be representative of rates in undisturbed soils.     

Protein composition and electron microscopy structure of affinity-purified human spliceosomal B complexes isolated under physiological conditions

The spliceosomal B complex is the substrate that undergoes catalytic activation leading to catalysis of pre-mRNA splicing. Previous characterization of this complex was performed in the presence of heparin, which dissociates less stably associated components. To obtain a more comprehensive inventory of the B complex proteome, we isolated this complex under low-stringency conditions using two independent methods. MS2 affinity-selected B complexes supported splicing when incubated in nuclear extract depleted of snRNPs. Mass spectrometry identified over 110 proteins in both independently purified B complex preparations, including approximately 50 non-snRNP proteins not previously found in the spliceosomal A complex. Unexpectedly, the heteromeric hPrp19/CDC5 complex and 10 additional hPrp19/CDC5-related proteins were detected, indicating that they are recruited prior to spliceosome activation. Electron microscopy studies revealed that MS2 affinity-selected B complexes exhibit a rhombic shape with a maximum dimension of 420 A and are structurally more homogeneous than B complexes treated with heparin. These data provide novel insights into the composition and structure of the spliceosome just prior to its catalytic activation and suggest a potential role in activation for proteins recruited at this stage. Furthermore, the spliceosomal complexes isolated here are well suited for complementation studies with purified proteins to dissect factor requirements for spliceosome activation and splicing catalysis.     

A Binocular Pupil Model for Simulation of Relative Afferent Pupil Defects and the Swinging Flashlight Test

Many important intracranial neural pathways are involved in the control of the two muscles of the human pupil and the observation and analysis of pupil responses to light or other stimuli is of great interest in many clinical procedures. The binocular pupil model presented in this document has a topology encompassing much of the complexity of the pupil system neurophysiology. The dynamic parameters of the model were matched against pupil experiments under multiple conditions. It is employed here to simulate responses to the swinging flashlight test, a procedure which is routinely practiced in ophthalmology to diagnose different degrees of relative afferent pupil defects often a consequence of severe optic nerve diseases or retinal dysfunctions. Other, not light-dependent, pupil stimuli are briefly discussed     

Astrocyte control of fetal cortical neuron glutathione homeostasis: up-regulation by ethanol

Ethanol increases apoptotic neuron death in the developing brain and at least part of this may be mediated by oxidative stress. In cultured fetal rat cortical neurons, Ethanol increases levels of reactive oxygen species (ROS) within minutes of exposure and reduces total cellular glutathione (GSH) shortly thereafter. This is followed by onset of apoptotic cell death. These responses to Ethanol can be blocked by elevating neuron GSH with N-acetylcysteine or by co-culturing neurons with neonatal cortical astrocytes. We describe here mechanisms by which the astrocyte-neuron gamma-glutamyl cycle is up-regulated by Ethanol, enhancing control of neuron GSH in response to the pro-oxidant, Ethanol. Up to 6 days of Ethanol exposure had no consistent effects on activities of gamma-glutamyl cysteine ligase or glutathione synthetase, and GSH content remained unchanged (p < 0.05). However, glutathione reductase was increased with 1 and 2 day Ethanol exposures, 25% and 39% for 2.5 and 4.0 mg/mL Ethanol by 1 day, and 11% and 16% for 2.5 and 4.0 mg/mL at 2 days, respectively (p < 0.05). A 24 h exposure to 4.0 mg/mL Ethanol increased GSH efflux from astrocyte up to 517% (p < 0.05). Ethanol increased both gamma-glutamyl transpeptidase expression and activity on astrocyte within 24 h of exposure (40%, p = 0.05 with 4.0 mg/mL) and this continued for at least 4 days of Ethanol treatment. Aminopeptidase N activity on neurons increased by 62% and 55% within 1 h of Ethanol for 2.5 and 4.0 mg/mL concentration, respectively (p < 0.05), remaining elevated for 24 h of treatment. Thus, there are at least three key points of the gamma-glutamyl cycle that are up-regulated by Ethanol, the net effect being to enhance neuron GSH homeostasis, thereby protecting neurons from Ethanol-mediated oxidative stress and apoptotic death.     

External radiation doses from <Superscript>137</Superscript>Cs to frog phantoms in a wetland area: in situ measurements and dose model calculations

For assessment of external radiation doses to frogs in a wetland area contaminated with ¹³⁷Cs, frog phantoms were constructed from polymethyl methacrylate (PMMA). The frog phantoms contained thermoluminescence (TL) chips and were used in situ at two study sites to measure doses. To test if higher doses are received by the sensitive skin of frogs, extra-thin TL chips were applied close to the surface of the frog phantoms. In addition, the measured doses were compared with those calculated on the basis of soil sample data from the wetland multiplied with dose-conversion coefficients from the US Department of Energy’s RESRAD-BIOTA code and from the ERICA assessment tool. Measured doses were generally lower than those calculated to ellipsoids used to model frogs. Higher doses were measured at the frog phantoms’ surfaces in comparison to inner parts at one of the two sites indicating that the frogs’ thin skin could receive a higher radiation dose than expected. In the efforts to assure protection of non-human biota, in situ measurements with phantoms provide valuable dose information and input to dose models in site-specific risk assessments of areas contaminated with radionuclides.     

Synapsis and catalysis by activated Tn3 resolvase mutants

The serine recombinase Tn3 resolvase catalyses recombination between two 114 bp res sites, each of which contains binding sites for three resolvase dimers. We have analysed the in vitro properties of resolvase variants with ‘activating’ mutations, which can catalyse recombination at binding site I of res when the rest of res is absent. Site I × site I recombination promoted by these variants can be as fast as res × res recombination promoted by wild-type resolvase. Activated variants have reduced topological selectivity and no longer require the 2–3′ interface between subunits that is essential for wild-type resolvase-mediated recombination. They also promote formation of a stable synapse comprising a resolvase tetramer and two copies of site I. Cleavage of the DNA strands by the activated mutants is slow relative to the rate of synapsis. Stable resolvase tetramers were not detected in the absence of DNA or bound to a single site I. Our results lead us to conclude that the synapse is assembled by sequential binding of resolvase monomers to site I followed by interaction of two site I-dimer complexes. We discuss the implications of our results for the mechanisms of synapsis and regulation in recombination by wild-type resolvase.