Kinetic and proteolytic identification of heat-induced conformational changes in the urea herbicide binding site of isolated Phaseolus vulgaris chloroplast thylakoids

Kinetic parameters of 3-(3, 4-dichlorophenyl)-1, 1-dimethyl urea (DCMU)-induced inhibition of electron transport in chloroplast thylakoids isolated from Phaseolus vulgaris L. cv. Oregon 1604 were determined from analysis of a convergent, parallel electrical circuit. Through this analogue, the apparent affinity of the purported binding site for DCMU (K₁) and the relative amount of DCMU-insensitive electron transport (v^max₁/v_o) were obtained using a reiterative non-linear least squares curve-fitting procedure. Exposure of thylakoids to heat caused a gradual increase in K₁ (or decrease in the affinity of the thylakoid for DCMU) with an apparent activation energy of 134 kJ mol⁻¹. Tryptic susceptibility of a protein region regulating K₁ also decreased gradually with exposure to 45°C, suggesting that the heat-induced increase in K₁ might be due to a protein conformational change. On the other hand, thylakoid exposure to 45°C resulted in a rapid (<5 min) irreversible increase in v^max_I/v_o, which was also the apparent result of a conformational change in a region of the protein which regulates this function. These results are suggestive of the existence of differential thermal sensitivities of proteins within the thylakoids and, perhaps, of different regions within a single membrane protein.     

Exclosure fences around nests of imperiled Florida Grasshopper Sparrows reduce rates of predation by mammals

Increasing nest survival by excluding predators is a goal of many bird conservation programs. However, new exclosure projects should be carefully evaluated to assess the potential risks of disturbance. We tested the effectiveness of predator exclosure fences (hereafter, fences) for nests of critically endangered Florida Grasshopper Sparrows (Ammodramus savannarum floridanus) at a dry prairie site (Three Lakes; 2015–2018) and a pasture site (the Ranch; 2015–2016) in Osceola County, Florida, USA. We installed fences at nests an average of 8 days after the start of incubation, and nest abandonment after fence installation was rare (2 of 149 installations). Predation was the leading cause of failure for unfenced nests at both sites (48–73%). At Three Lakes, nest cameras revealed that mammals and snakes were responsible for 61.5% and 38.5% of predation events, respectively, at unfenced nests. Fences reduced the daily probability of predation (0.016 for fenced nests vs. 0.074 for unfenced nests). The probability that a fenced nest would survive from discovery to fledging was more than double that of unfenced nests (60.4% vs. 27.7%). However, we found no difference in daily nest survival at the Ranch between the year before nests were fenced (2015; 0.874) and the year when all but one nest were fenced (2016; 0.867) because red imported fire ants (Solenopsis invicta) were responsible for 86% of predation events at fenced nests at the Ranch. The use of cameras at fenced nests revealed that site‐specific differences in nest predators explained variation in fence efficiency between sites. Our fence design may be useful for other species of grassland birds, but site‐specific predator communities and species‐specific response of target bird species to fences should be assessed before installing fences at other sites.     

Membrane Binding and Homodimerization of Atg16 Via Two Distinct Protein Regions is Essential for Autophagy in Yeast

Macroautophagy is a bulk degradation mechanism in eukaryotic cells. Efficiency of an essential step of this process in yeast, Atg8 lipidation, relies on the presence of Atg16, a subunit of the Atg12–Atg5-Atg16 complex acting as the E3-like enzyme in the ubiquitination-like reaction. A current view on the functional structure of Atg16 in the yeast S. cerevisiae comes from the two crystal structures that reveal the Atg5-interacting α-helix linked via a flexible linker to another α-helix of Atg16, which then assembles into a homodimer. This view does not explain the results of previous in vitro studies revealing Atg16-dependent deformations of membranes and liposome-binding of the Atg12–Atg5 conjugate upon addition of Atg16. Here we show that Atg16 acts as both a homodimerizing and peripheral membrane-binding polypeptide. These two characteristics are imposed by the two distinct regions that are disordered in the nascent protein. Atg16 binds to membranes in vivo via the amphipathic α-helix (amino acid residues 113–131) that has a coiled-coil-like propensity and a strong hydrophobic face for insertion into the membrane. The other protein region (residues 64–99) possesses a coiled-coil propensity, but not amphipathicity, and is dispensable for membrane anchoring of Atg16. This region acts as a Leu-zipper essential for formation of the Atg16 homodimer. Mutagenic disruption in either of these two distinct domains renders Atg16 proteins that, in contrast to wild type, completely fail to rescue the autophagy-defective phenotype of atg16Δ cells. Together, the results of this study yield a model for the molecular mechanism of Atg16 function in macroautophagy.     

Mechanistic studies on carboxypeptidase a from goat pancreas — part II: Evidence for carboxyl group

Studies with substrate analogues and the pH optimum indicated the involvement of carboxyl group in the active site of goat carboxypeptidase A. Chemical modification of the enzyme with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methoI -p-toluene sulphonate, a carboxyl specific reagent, led to loss of both esterase and peptidase activities. Protection studies showed that this carboxyl group was in the active site and was protected by Βp-phenylpropionic acid and glycyl-L-tyrosine. Kinetic studies also confirmed the involvement of carboxylic group because the enzyme modification with water soluble carbodiimide was a two step reaction which excluded the possibility of tyrosine or lysine which are known to give a one step reaction with this reagent     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Spatial and temporal factors associated with nest survival of Gray Flycatchers in managed ponderosa pine forests

Gray Flycatchers (Empidonax wrightii) breed in a variety of habitats in the arid and semi‐arid regions of the western United States, but little is known about their breeding biology, especially in the northern portion of their range where they nest in ponderosa pine (Pinus ponderosa) forests. From May to July 2014 and 2015, we conducted surveys for singing male Gray Flycatchers along the eastern slope of the Cascade Range in Washington, U.S.A, monitored flycatcher nests, and quantified nest‐site vegetation. We used a logistic‐exposure model fit within a Bayesian framework to model the daily survival probability of flycatcher nests. During the 2 yr of our study, we monitored 141 nests, with 93% in ponderosa pines. Mean clutch size was 3.6 eggs and the mean number of young fledged per nest was 3.2. Predation accounted for 90% of failed nests. We found a positive association between daily nest survival and both nest height and distance of nest substrates from the nearest tree. Flycatchers that locate their nests higher above the ground and further from adjacent trees may be choosing the safest alternative because higher nests may be less exposed to terrestrial predators and nests in trees that are farther from other trees may be less exposed to arboreal predators such as jays (Corvidae) that may forage in patches with connected canopies. Nests in trees farther from other trees may also allow earlier detection of approaching predators and thus aid in nest defense.     

Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Correlations between the psychological peculiarities of an individual and the efficacy of a single neurofeedback session (by the EEG characteristics)

Modifications of different EEG rhythms induced by a single neurofeedback session (by the EEG characteristics) directed toward an increase in the ratio of the spectral powers (SPs) of the α vs θ oscillations were compared with the psychological characteristics of the tested subjects (the group included 30 persons). A generally accepted neurofeedback technique was used; the intensity of acoustic white noise served as the feedback signal. EEG potentials were recorded from the C3 and C4 leads. Psychological testing was carried out using Eysenck’s (EPQ), Rusalov’s (OST), and (16 PF) questionnaires. The directions of changes in the SPs of EEG frequency components were found to significantly correlate with some individuality-related peculiarities of the tested subjects. The SP of the δ rhythm correlated with the EPQ scale “neuroticism,” OST scale “social plasticity,” and 16 PF factors H (“parmia”), I (“premsia”), and Q₃ (“self-control of behavior”). The SP of the θ component demonstrated correlations with the OST scales “ergisity,” “plasticity,” and “social temp” and with 16 PF factors M (“autia”), Q₄ (“frustration”), and Q1 (“exvia”). The SP of the α rhythm correlated with 16 PF factors Q₃ (“self-control of behavior”), G (“strength of superEgo”), O (“hypothymia”), L (“protension”), and N (“shrewdness”). The SP of the β rhythm correlated with the OST scale “emotionality,” while that of the γ rhythm showed correlations with the 16 PF indices L (“protension”) and M (“autia”). Changes in the ratio of the α vs θ SPs correlated with the EPQ scale “neuroticism.” Thus, our data confirm the statement that a high individual variability of the results of a single (first in the series) neurofeedback session is to a great extent related to peculiarities of the individual psychological pattern of the subject. Neirofiziologiya/Neurophysiology, Vol. 38, No. 3, pp. 239–247, May–June, 2006.     

Stereological Determination of Orientations,Second-Order Quantities and Correlations for Random Spatial Fibre Systems

This paper presents methods for the stereological analysis of spatial fibre systems on the base of planar or thin sections. Under the assumption that the cross-section figures of the tubular fibres can be measured, the orientation distribution of the fibre system and its line density L_v can be determined from one section only and without distributional assumptions. A simple way to study the degree of randomness of fibre systems consists in the statistical analysis of the point pattern of centres of intersection figures. More sophisticated methods are of stereological nature and yield the spatial reduced second moment measure. Similarly also correlations between two fibre systems can be quantified. The methods are demonstrated by two examples concerning samples of human brain.     

Influence of rainfall on sleeping site choice by a group of anubis baboons (Papio anubis)

For diurnal nonhuman primates, shifting among different sleeping sites may provide multiple benefits such as better protection from predators, reduced risk of parasitic infection, and closer proximity to spatially and temporally heterogeneous food and water. This last benefit may be particularly important in sleeping site selection by primates living in savanna‐woodlands where rainfall is more limited and more seasonally pronounced than in rainforests. Here, we examined the influence of rainfall, a factor that affects food and water availability, on the use of sleeping sites by anubis baboons (Papio anubis) over two 13‐month study periods that differed in rainfall patterns. We predicted that during wet periods, when food and water availability should be higher, the study group would limit the number of sleeping sites and would stay at each one for more consecutive nights than during dry periods. Conversely, we predicted that during dry periods the group would increase the number of sleeping sites and stay at each one for fewer consecutive nights as they searched more widely for food and water. We also predicted that the group would more often choose sleeping sites closer to the center of the area used during daytime (between 07:00 and 19:00) during wet months than during dry months. Using Global Positioning System data from collared individuals, we found that our first prediction was not supported on either monthly or yearly timescales, although past monthly rainfall predicted the use of the main sleeping site in the second study period. Our second prediction was supported only on a yearly timescale. This study suggests that baboons’ choice of sleeping sites is fluid over time while being sensitive to local environmental conditions, one of which may be rainfall.