Catalysis by dienelactone hydrolase: A variation on the protease mechanism

Dienelactone hydrolase (DLH), an enzyme from the β-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation. © 1993 Wiley-Liss, Inc.     

Canopy structure measurement by gap fraction analysis using commercial instrumentation

In the past few years, a number of systems for making indirectcanopy structural estimates have become commercially available.These include linear sensors, such as the DEMON, line quantumsensors, and hemispherical sensors, such as the LAI-2000, theLeaf Laser, and the Cl-100. The performance of these instrumentsas reported in the literature is reviewed for forests, row crops,and individual trees. Key words: Leaf area index, LAI-2000, DEMON, SunLink, SunScan, Ceptometer, AccuPAR, Cl-100, gap fraction, sunflecks, hemispherical photography     

Metastable behavior in Markov processes with internal states

A perturbation framework is developed to analyze metastable behavior in stochastic processes with random internal and external states. The process is assumed to be under weak noise conditions, and the case where the deterministic limit is bistable is considered. A general analytical approximation is derived for the stationary probability density and the mean switching time between metastable states, which includes the pre exponential factor. The results are illustrated with a model of gene expression that displays bistable switching. In this model, the external state represents the number of protein molecules produced by a hypothetical gene. Once produced, a protein is eventually degraded. The internal state represents the activated or unactivated state of the gene; in the activated state the gene produces protein more rapidly than the unactivated state. The gene is activated by a dimer of the protein it produces so that the activation rate depends on the current protein level. This is a well studied model, and several model reductions and diffusion approximation methods are available to analyze its behavior. However, it is unclear if these methods accurately approximate long-time metastable behavior (i.e., mean switching time between metastable states of the bistable system). Diffusion approximations are generally known to fail in this regard.     

Can dominance genetic variance be ignored in evolutionary quantitative genetic analyses of wild populations?

Accurately estimating genetic variance components is important for studying evolution in the wild. Empirical work on domesticated and wild outbred populations suggests that dominance genetic variance represents a substantial part of genetic variance, and theoretical work predicts that ignoring dominance can inflate estimates of additive genetic variance. Whether this issue is pervasive in natural systems is unknown, because we lack estimates of dominance variance in wild populations obtained in situ. Here, we estimate dominance and additive genetic variance, maternal variance, and other sources of nongenetic variance in eight traits measured in over 9000 wild nestlings linked through a genetically resolved pedigree. We find that dominance variance, when estimable, does not statistically differ from zero and represents a modest amount (2-36%) of genetic variance. Simulations show that (1) inferences of all variance components for an average trait are unbiased; (2) the power to detect dominance variance is low; (3) ignoring dominance can mildly inflate additive genetic variance and heritability estimates but such inflation becomes substantial when maternal effects are also ignored. These findings hence suggest that dominance is a small source of phenotypic variance in the wild and highlight the importance of proper model construction for accurately estimating evolutionary potential.     

Developing a culture of safety in biomedical research training

The National Institute of General Medical Sciences (NIGMS) at the U.S. National Institutes of Health (NIH) is committed to supporting the safety of the nation’s biomedical research and training environments. Institutional training grants affect many trainees and can have a broad influence across their parent institutions, making them good starting points for our initial efforts to promote the development and maintenance of robust cultures of safety at U.S. academic institutions. In this Perspective, we focus on laboratory safety, although many of the strategies we describe for improving laboratory safety are also applicable to other forms of safety including the prevention of harassment, intimidation, and discrimination. We frame the problem of laboratory safety using a number of recent examples of tragic accidents, highlight some of the lessons that have been learned from these and other events, discuss what NIGMS is doing to address problems related to laboratory safety, and outline steps that institutions can take to improve their safety cultures.

All new funding opportunity announcements (FOAs) for training programs supported by the National Institute of General Medical Sciences (NIGMS) contain the expectation that the programs will promote “inclusive, safe and supportive scientific and training environments.” In this context, the word “safe” refers to several aspects of safety. First, we mean an environment free from harassment and intimidation, in which everyone participating is treated in a respectful and supportive manner, optimized for productive learning and research. We also mean that institutions should ensure that their campuses are as safe as possible so that individuals can focus on their studies and research. Finally, we mean safety in the laboratory and clinical spaces. In this Perspective, we focus on this last issue and describe some of the approaches NIGMS is taking to help the biomedical research community move toward an enhanced culture of safety in which core values and the behaviors of leadership, principal investigators (PIs), research staff, and trainees emphasize safety over competing goals.     

Orthosteric Binding of ρ-Da1a,a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α_1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of ³H-prazosin or ¹²⁵I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α_1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α_1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α_1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of ³H-prazosin or ¹²⁵I-HEAT, and the IC₅₀ of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca²⁺ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α_1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106^3.32A and the S188^5.42A/S192^5.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86^2.64A, F288^6.51A and F312^7.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86^2.64 was identified as a key interaction point for ¹²⁵I-HEAT, as the variant F86^2.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α_1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86^2.64, F288^6.51 and F312^7.39) and agonist (F288^6.51 and F312^7.39) ligands. Its selectivity for the α_1A-adrenoceptor may result, at least partly, from its interaction with the residue F86^2.64, which appears to be important also for HEAT binding.