Time-resolved FRET reveals the structural mechanism of SERCA–PLB regulation

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to characterize the interaction between phospholamban (PLB) and the sarcoplasmic reticulum (SR) Ca-ATPase (SERCA) under conditions that relieve SERCA inhibition. Unphosphorylated PLB inhibits SERCA in cardiac SR, but inhibition is relieved by either micromolar Ca²⁺ or PLB phosphorylation. In both cases, it has been proposed that inhibition is relieved by dissociation of the complex. To test this hypothesis, we attached fluorophores to the cytoplasmic domains of SERCA and PLB, and reconstituted them functionally in lipid bilayers. TR-FRET, which permitted simultaneous measurement of SERCA–PLB binding and structure, was measured as a function of PLB phosphorylation and [Ca²⁺]. In all cases, two structural states of the SERCA–PLB complex were resolved, probably corresponding to the previously described T and R structural states of the PLB cytoplasmic domain. Phosphorylation of PLB at S16 completely relieved inhibition, partially dissociated the SERCA–PLB complex, and shifted the T/R equilibrium within the bound complex toward the R state. Since the PLB concentration in cardiac SR is at least 10 times that in our FRET measurements, we calculate that most of SERCA contains bound phosphorylated PLB in cardiac SR, even after complete phosphorylation. 4 μM Ca²⁺ completely relieved inhibition but did not induce a detectable change in SERCA–PLB binding or cytoplasmic domain structure, suggesting a mechanism involving structural changes in SERCA’s transmembrane domain. We conclude that Ca²⁺ and PLB phosphorylation relieve SERCA–PLB inhibition by distinct mechanisms, but both are achieved primarily by structural changes within the SERCA–PLB complex, not by dissociation of that complex.     

Steady-state analysis of metabolic pathways: comparing the double modulation method and the lin-log approach

The increasing interest in studying enzyme kinetics under in vivo conditions requires practical methods to estimate control parameters from experimental data. In contrast to currently established approaches of dynamic modelling, this paper addresses the steady-state analysis of metabolic pathways. Within the framework of metabolic control analysis (MCA), elasticity coefficients are used to describe the control properties of a local enzyme reaction. The double modulation method is one of the first experimental approaches to estimate elasticity coefficients from measurements of steady-state flux rates and metabolite concentrations. We propose a generalized form of the double modulation method and compare it to the recently developed linear-logarithmic approach.     

Biological protection of hardwood logs destined for panel manufacturing using <Emphasis Type="Italic">Gliocladium roseum</Emphasis> against biodegradation

The objective of the present study was to develop a biological technology that would protect logs destined for oriented strand board (OSB) manufacturing from biodegradation. Aspen, red maple, and yellow birch trees were felled in one summer and the logs either debarked or not debarked, and either treated or not treated with a biological product of Gliocladium roseum. The logs were piled in different treatment groups and stored in a yard for 5 months and 1 year before evaluation. The results showed that all untreated logs, with or without bark, were seriously degraded by moulds, stain and decay fungi after a summer storage period of 5 months. The logs with bark were more degraded than the debarked logs, and the log ends were more degraded than the middle sections. After 5 months, 55–83% of the surface area of the wood discs was degraded in untreated logs. The biological treatment was effective, and only 4–16% of the surface area of the wood discs in treated logs was infected by various fungi. Strands cut from untreated logs consisted of 50–75% grey- or blue-stained strands, whereas those cut from biologically treated logs consisted of 10–25% such strands. Panels made using biologically treated logs had a lower thickness swelling and water absorption values compared to panels made using freshly cut logs and untreated stored logs. The other physical and mechanical properties of the various panels made in this test were comparable. In terms of mould resistance, all panels made from fungal-treated logs had a better mould resistance than those made from freshly cut and untreated logs.     

Mark‐resight abundance and survival estimation of Indo‐Pacific bottlenose dolphins,Tursiops aduncus,in the Swatch‐of‐No‐Ground,Bangladesh

A mark‐resight analysis under Pollock's robust design was applied to Indo‐Pacific bottlenose dolphins Tursiops aduncus in the Swatch‐of‐No‐Ground (SoNG) submarine canyon, Bangladesh, during the winter seasons of 2005–2009. Information from sightings of photo‐identified individuals (1,144) and unmarked individuals generated abundance estimates of 1,701 (95% confidence interval [CI]= 1,533–1,888), 1,927 (95% CI = 1,851–2,006), 2,150 (95% CI = 1,906–2,425), and 2,239 (95% CI = 1,985–2,524) individuals for seasons 1–4, respectively. This makes the population among the largest assessed of the species. Overall apparent survival was estimated as 0.958 (95% CI = 0.802–0.992). Interseasonal probabilities of transitioning to an unobservable state were estimated as 0.045, 0.363, and 0.300 for years 1–2, 2–3, and 3–4, respectively, and the overall probability of remaining in an unobservable state was 0.688. These probabilities, together with an apparent increase in abundance during the study period, indicate that the identified dolphins are part of a larger superpopulation moving throughout a more extensive geographic area. Of the photo‐identified dolphins, 28.2% exhibited injuries related to entanglements with fishing gear. This implies a strong potential for fatal interactions that could jeopardize the conservation status of the population, which otherwise appears favorable.     

The relation between the neutrality index for mitochondrial genes and the distribution of mutational effects on fitness

We explore factors affecting patterns of polymorphism and divergence (as captured by the neutrality index) at mammalian mitochondrial loci. To do this, we develop a population genetic model that incorporates a fraction of neutral amino acid sites, mutational bias, and a probability distribution of selection coefficients against new nonsynonymous mutations. We confirm, by reanalyzing publicly available datasets, that the mitochondrial cyt-b gene shows a broad range of neutrality indices across mammalian taxa, and explore the biological factors that can explain this observation. We find that observed patterns of differences in the neutrality index, polymorphism, and divergence are not caused by differences in mutational bias. They can, however, be explained by a combination of a small fraction of neutral amino acid sites, weak selection acting on most amino acid mutations, and differences in effective population size among taxa.     

Forecasting Environmental Responses to Restoration of Rivers Used as Log Floatways: An Interdisciplinary Challenge

Log floating in the 19th to mid 20th centuries has profoundly changed the environmental conditions in many northern river systems of the world. Regulation of flow by dams, straightening and narrowing of channels by various piers and wing dams, and homogenization of bed structure are some of the major impacts. As a result, the conditions for many riverine organisms have been altered. Removing physical constructions and returning boulders to the channels can potentially restore conditions for these organisms. Here we describe the history of log driving, review its impact on physical and biological conditions and processes, and predict the responses to restoration. Reviewing the literature on comparable restoration efforts and building upon this knowledge, using boreal Swedish rivers as an example, we address the last point. We hypothesize that restoration measures will make rivers wider and more sinuous, and provide rougher bottoms, thus improving land-water interactions and increasing the retention capacity of water, sediment, organic matter and nutrients. The geomorphic and hydraulic/hydrologic alterations are supposed to favor production, diversity, migration and reproduction of riparian and aquatic organisms. The response rates are likely to vary according to the types of processes and organisms. Some habitat components, such as beds of very large boulders and bedrock outcrops, and availability of sediment and large woody debris are believed to be extremely difficult to restore. Monitoring and evaluation at several scales are needed to test our predictions.     

Imidacloprid and Related Compounds: Structure and Water Solubility of N-Alkyl Derivatives of Imidacloprid

An intramolecular hydrogen bond between NH???O₂N in insecticide, imidacloprid (1), and its nitromethylene analog 15 was proved by NMR and IR spectra. That electron delocalization over their planar moieties was disrupted by alkylation at the imidazolidine nitrogen atom is demonstrated by the hypsochromic shifts in UV and deshielding effect in NMR spectra. Interestingly, the N-alkyl derivatives (C1-5) had greater water solubility than 1, although increasing alkyl chain length decreased the solubility. The hydrophilicity of the alkyl derivatives would result from remote charge heads being formed as a result of the conjugation disruption by alkylation, while the hydrophobicity of 1 could be ascribed to the charge distribution over the conjugated system coupled with the intramolecular H-bonding. The greater water solubility of 15 than 1 and contrastively small solubility of the cyanoimine analogue are discussed based on the difference in their steric crowding.