Stabilization of anionic and neutral forms of a fluorophoric ligand at the active site of human carbonic anhydrase I

We synthesized a fluorogenic dansylamide derivative (JB2-48), which fills the entire (15 Å deep) active site pocket of human carbonic anhydrase I, and investigated the contributions of sulfonamide and hydrophobic regions of the ligand structure on the spectral, kinetic, and thermodynamic properties of the enzyme–ligand complex. The steady-state and fluorescence lifetime data revealed that the deprotonation of the sulfonamide moiety of the enzyme bound ligand increases the fluorescence emission intensity as well as the lifetime of the fluorophores. This is manifested via the electrostatic interaction between the active site resident Zn²⁺ cofactor and the negatively charged sulfonamide group of the ligand, and such interaction contributes to about 2.2 kcal/mol (ΔΔG°) and 0.89 kcal/mol (ΔΔG^‡) energy in stabilizing the ground and the putative transition states, respectively. We provide evidence that the anionic and neutral forms of JB2-48 are stabilized by the complementary microscopic/conformational states of the enzyme. The implication of the mechanistic studies presented herein in rationale design of carbonic anhydrase inhibitors is discussed.     

The influence of niche and neutral processes on a neotropical anuran metacommunity

One of the most important questions in ecology is the relative importance of local conditions (niche processes) and dispersal ability (neutral processes) in driving metacommunity structure. Although many studies have been conducted in recent years, there is still much debate. We evaluated the processes (niche and neutral) responsible for variation in anuran composition in 28 lentic water bodies in southeastern Brazil. Because anurans depend heavily on environmental conditions, we hypothesized that environmental variables (niche processes) are the most important drivers of community composition. Additionally, as anurans have limited dispersal abilities, and the study region presents harsh conditions (high forest fragmentation, low rainfall and long dry season), we expected a lower, but significant, spatial signature in metacommunity structure, due to neutral dynamics. We used a partial redundancy analysis with variation partitioning to evaluate the relative influence of environmental and spatial variables as drivers of metacommunity structure. Additionally, we used a recently developed spatial autocorrelation analysis to test if neutral dynamics can be attributed to the pure spatial component. This analysis is based on predictions that species abundances are independent but similarly spatially structured, with correlograms similar in shape. Therefore, under neutral dynamics there is no expectation of a correlation between the pairwise distance of spatial correlograms and the pairwise correlation of species abundances predicted by the pure spatial component. We found that the environmental component explained 21.5%, the spatial component 10.2%, and the shared component 6.4% of the metacommunity structure. We found no correlation between correlograms and correlation of abundances predicted by the pure spatial component (Mantel test = ?0.109, P = 0.961). In our study, niche‐based processes are the dominant process that explained community composition. However, neutral processes are important because spatial variation can be attributed to pure neutral dynamics rather than to missing spatially structured environmental factors.     

Design and validation of a neutral protein scaffold for the presentation of peptide aptamers

Peptide aptamers are peptides constrained and presented by a scaffold protein that are used to study protein function in cells. They are able to disrupt protein-protein interactions and to constitute recognition modules that allow the creation of a molecular toolkit for the intracellular analysis of protein function. The success of peptide aptamer technology is critically dependent on the performance of the scaffold. Here, we describe a rational approach to the design of a new peptide aptamer scaffold. We outline the qualities that an ideal scaffold would need to possess to be broadly useful for in vitro and in vivo studies and apply these criteria to the design of a new scaffold, called STM. Starting from the small, stable intracellular protease inhibitor stefin A, we have engineered a biologically neutral scaffold that retains the stable conformation of the parent protein. We show that STM is able to present peptides that bind to targets of interest, both in the context of known interactors and in library screens. Molecular tools based on our scaffold are likely to be used in a wide range of studies of biological pathways, and in the validation of drug targets.     

Comparative studies of quantitative trait and neutral marker divergence: a meta-analysis

Comparative studies of quantitative genetic and neutral marker differentiation have provided means for assessing the relative roles of natural selection and random genetic drift in explaining among-population divergence. This information can be useful for our fundamental understanding of population differentiation, as well as for identifying management units in conservation biology. Here, we provide comprehensive review and meta-analysis of the empirical studies that have compared quantitative genetic (Q(ST)) and neutral marker (F(ST)) differentiation among natural populations. Our analyses confirm the conclusion from previous reviews - based on ca. 100% more data - that the Q(ST) values are on average higher than F(ST) values [mean difference 0.12 (SD 0.27)] suggesting a predominant role for natural selection as a cause of differentiation in quantitative traits. However, although the influence of trait (life history, morphological and behavioural) and marker type (e.g. microsatellites and allozymes) on the variance of the difference between Q(ST) and F(ST) is small, there is much heterogeneity in the data attributable to variation between specific studies and traits. The latter is understandable as there is no reason to expect that natural selection would be acting in similar fashion on all populations and traits (except for fitness itself). We also found evidence to suggest that Q(ST) and F(ST) values across studies are positively correlated, but the significance of this finding remains unclear. We discuss these results in the context of utility of the Q(ST)-F(ST) comparisons as a tool for inferring natural selection, as well as associated methodological and interpretational problems involved with individual and meta-analytic studies.     

Muller's ratchet and the pattern of variation at a neutral locus

The levels and patterns of variation at a neutral locus are analyzed in a haploid asexual population undergoing accumulation of deleterious mutations due to Muller's ratchet. We find that the movement of Muller's ratchet can be associated with a considerable reduction in genetic diversity below classical neutral expectation. The extent to which variability is reduced is a function of the deleterious mutation rate, the fitness effects of the mutations, and the population size. Approximate analytical expressions for the expected genetic diversity are compared with simulation results under two different models of deleterious mutations: a model where all deleterious mutations have equal effects and a model where there are two classes of deleterious mutations. We also find that Muller's ratchet can produce a considerable distortion in the neutral frequency spectrum toward an excess of rare variants.     

On the genealogy of a sample of neutral rare alleles

This paper concerns the genealogical structure of a sample of chromosomes sharing a neutral rare allele. We suppose that the mutation giving rise to the allele has only happened once in the history of the entire population, and that the allele is of known frequency q in the population. Within a coalescent framework C. Wiuf and P. Donnelly (1999, Theor. Popul. Biol. 56, 183-201) derived an exact analysis of the conditional genealogy but it is inconvenient for applications. Here, we develop an approximation to the exact distribution of the conditional genealogy, including an approximation to the distribution of the time at which the mutation arose. The approximations are accurate for frequencies q<5-10%. In addition, a simple and fast simulation scheme is constructed. We consider a demography parameterized by a d-dimensional vector alpha=(alpha(1), em leader, alpha(d)). It is shown that the conditional genealogy and the age of the mutation have distributions that depend on a=qalpha and q only, and that the effect of q is a linear scaling of times in the genealogy; if q is doubled, the lengths of all branches in the genealogy are doubled. The theory is exemplified in two different demographies of some interest in the study of human evolution: (1) a population of constant size and (2) a population of exponentially decreasing size (going backward in time).     

A complex speciation-richness relationship in a simple neutral model

Speciation is the "elephant in the room" of community ecology. As the ultimate source of biodiversity, its integration in ecology's theoretical corpus is necessary to understand community assembly. Yet, speciation is often completely ignored or stripped of its spatial dimension. Recent approaches based on network theory have allowed ecologists to effectively model complex landscapes. In this study, we use this framework to model allopatric and parapatric speciation in networks of communities. We focus on the relationship between speciation, richness, and the spatial structure of communities. We find a strong opposition between speciation and local richness, with speciation being more common in isolated communities and local richness being higher in more connected communities. Unlike previous models, we also find a transition to a positive relationship between speciation and local richness when dispersal is low and the number of communities is small. We use several measures of centrality to characterize the effect of network structure on diversity. The degree, the simplest measure of centrality, is the best predictor of local richness and speciation, although it loses some of its predictive power as connectivity grows. Our framework shows how a simple neutral model can be combined with network theory to reveal complex relationships between speciation, richness, and the spatial organization of populations.     

Community structure of vascular epiphytes: a neutral perspective

Vascular epiphytes form a diverse group of almost 30 000 species, yet theory concerning their community structure is still largely lacking. We therefore employed the simplest models of biodiversity, (near-)neutral models, to generate hypotheses concerning their community structure. With recently developed tools for (near-)neutral models we analyzed species abundance data from many samples in Central and South America which we divided into four metacommunities (Mesoamerica, Central America, Amazonia and Paraná), where for each metacommunity we considered two subsets differing in dispersal syndrome: an animal-dispersed guild and a wind-dispersed guild. We considered three models differing in the underlying speciation mode. Across all metacommunities, we found observed patterns to be indistinguishable from patterns generated by neutral or near-neutral processes. Furthermore, we found that subdivision in different dispersal guilds was often supported, with recruitment limitation being stronger for animal-dispersed species than for wind-dispersed species. This is the first time that (near-)neutral theory has been applied to epiphyte communities. Future efforts with additional data sets and more refined models are expected to further improve our understanding of community structure in epiphytes and will have to test the generality of our findings.     

Purification and characterization of a magnesium-dependent neutral sphingomyelinase from bovine brain

The magnesium-dependent, plasma membrane-associated neutral sphingomyelinase (N-SMase) catalyzes hydrolysis of membrane sphingomyelin to form ceramide, a lipid signaling molecule implied in intracellular signaling. We report here the biochemical purification to apparent homogeneity of N-SMase from bovine brain. Proteins from Nonidet P-40 extracts of brain membranes were subjected to four purification steps yielding a N-SMase preparation that exhibited a specific enzymatic activity 23,330-fold increased over the brain homogenate. When analyzed by two-dimensional gel electrophoresis, the purified enzyme presented as two major protein species of 46 and 97 kDa, respectively. Matrix-assisted laser desorption/ionization-mass spectrometry analysis of tryptic peptides revealed at least partial identity of these two proteins. Amino acid sequencing of tryptic peptides showed no apparent homologies of bovine N-SMase to any known protein. Peptide-specific antibodies recognized a single 97-kDa protein in Western blot analysis of cell lysates. The purified enzyme displayed a K(m) of 40 microM for sphingomyelin with an optimal activity at pH 7-8. Bovine brain N-SMase was strictly dependent on Mg(2+), whereas Zn(2+) and Ca(2+) proved inhibitory. The highly purified bovine N-SMase was effectively blocked by glutathione and scyphostatin. Scyphostatin proved to be a potent inhibitor of N-SMase with 95% inhibition observed at 20 microM scyphostatin. The results of this study define a N-SMase that fulfills the biochemical and functional criteria characteristic of the tumor necrosis factor-responsive membrane-bound N-SMase.     

The HKA test revisited: a maximum-likelihood-ratio test of the standard neutral model

We present a maximum-likelihood-ratio test of the standard neutral model, using multilocus data on polymorphism within species and divergence between species. The model is based on the Hudson-Kreitman-Aguade (HKA) test, but allows for an explicit test of selection at individual loci in a multilocus framework. We use coalescent simulations to show that the likelihood-ratio test statistic is conservative, particularly when the assumption of no recombination is violated. Application of the method to polymorphism data from 18 loci from a population of Arabidopsis lyrata provides significant evidence for a balanced polymorphism at a candidate locus thought to be linked to the centromere. The method is also applied to polymorphism data in maize, providing support for the hypothesis of directional selection on genes in the starch pathway.     

Thermodynamics of neutral protein evolution

Naturally evolving proteins gradually accumulate mutations while continuing to fold to stable structures. This process of neutral evolution is an important mode of genetic change and forms the basis for the molecular clock. We present a mathematical theory that predicts the number of accumulated mutations, the index of dispersion, and the distribution of stabilities in an evolving protein population from knowledge of the stability effects (delta deltaG values) for single mutations. Our theory quantitatively describes how neutral evolution leads to marginally stable proteins and provides formulas for calculating how fluctuations in stability can overdisperse the molecular clock. It also shows that the structural influences on the rate of sequence evolution observed in earlier simulations can be calculated using just the single-mutation delta deltaG values. We consider both the case when the product of the population size and mutation rate is small and the case when this product is large, and show that in the latter case the proteins evolve excess mutational robustness that is manifested by extra stability and an increase in the rate of sequence evolution. All our theoretical predictions are confirmed by simulations with lattice proteins. Our work provides a mathematical foundation for understanding how protein biophysics shapes the process of evolution.     

Modes of speciation and the neutral theory of biodiversity

Hubbell's neutral theory of biodiversity has generated much debate over the need for niches to explain biodiversity patterns. Discussion of the theory has focused on its neutrality assumption, i.e. the functional equivalence of species in competition and dispersal. Almost no attention has been paid to another critical aspect of the theory, the assumptions on the nature of the speciation process. In the standard version of the neutral theory each individual has a fixed probability to speciate. Hence, the speciation rate of a species is directly proportional to its abundance in the metacommunity. We argue that this assumption is not realistic for most speciation modes because speciation is an emergent property of complex processes at larger spatial and temporal scales and, consequently, speciation rate can either increase or decrease with abundance. Accordingly, the assumption that speciation rate is independent of abundance (each species has a fixed probability to speciate) is a more natural starting point in a neutral theory of biodiversity. Here we present a neutral model based on this assumption and we confront this new model to 20 large data sets of tree communities, expecting the new model to fit the data better than Hubbell's original model. We find, however, that the data sets are much better fitted by Hubbell's original model. This implies that species abundance data can discriminate between different modes of speciation, or, stated otherwise, that the mode of speciation has a large impact on the species abundance distribution. Our model analysis points out new ways to study how biodiversity patterns are shaped by the interplay between evolutionary processes (speciation, extinction) and ecological processes (competition, dispersal).     

Energetics of a hydrogen bond (charged and neutral) and of a cation-pi interaction in apoflavodoxin.

Anabaena apoflavodoxin contains a single histidine residue (H34) that interacts with two aromatic residues (F7 and Y47). The histidine and phenylalanine rings are almost coplanar and they can establish a cation-pi interaction when the histidine is protonated. The histidine and tyrosine side-chains are engaged in a hydrogen bond, which is their only contact. We analyse the energetics of these interactions using p Ka-shift analysis, double-mutant cycle analysis at two pH values, and X-ray crystallography. The H/F interaction is very weak when the histidine is neutral, but it is strengthened by 0.5 kcal mol-1on histidine protonation. Supporting this fact, the histidine p Kain a F7L mutant is 0.4 pH units lower than in wild-type. The strength of the H/Y hydrogen bond is 0.7 kcal mol-1when the histidine is charged, and it becomes stronger (1.3 kcal mol-1) when the histidine is neutral. This is consistent with our observation that the (H34)Nepsilon2-OH(Y47) distance is slightly shorter in the apoflavodoxin structure at pH 9.0 than in the previously reported structure at pH 6.0. It is also consistent with a histidine p Kavalue 0.6 pH units higher in a Y47F mutant than in the wild-type protein. We suggest that the higher stability of the neutral hydrogen bond could be due to a higher desolvation penalty of the charged hydrogen bond that would offset its more favourable enthalpy of formation. The relationship between hydrogen bond strength and the contribution of hydrogen bonds to protein stability is discussed.     

Activation of myocardial neutral triglyceride lipase and neutral cholesterol esterase by cAMP-dependent protein kinase

Lipolysis of intracellular triglycerides in the heart has been shown to be regulated by hormones. However, activation of myocardial triglyceride lipase in a cell-free system has not been directly demonstrated. In the present studies, initial attempts to demonstrate cAMP-dependent activation of triglyceride lipase using the 1,000 X g supernatant fraction (S1) of mouse heart homogenate were unsuccessful, presumably due to the masking effects of high levels of lipoprotein lipase activity even when assayed at pH 7.4 and in the absence of apolipoprotein C-II. Myocardial lipoprotein lipase in the 40,000 X g supernatant fraction was then removed by heparin-Sepharose affinity chromatography. The lipoprotein lipase-free fractions were shown to contain neutral triglyceride lipase and neutral cholesterol esterase of about equal activities. The triglyceride lipase and cholesterol esterase activities fell progressively during preincubation in the presence of 5 mM Mg2+. Additions of cAMP and ATP resulted in 40-70% activation of both triglyceride lipase and cholesterol esterase. The activation was blocked by protein kinase inhibitor and was restored by the addition of exogenous cAMP-dependent protein kinase. Since lipoprotein lipase has no activity toward cholesteryl oleate, activation of cholesterol esterase in untreated S1 was readily demonstrable. Both triglyceride lipase and cholesterol esterase activities were present in homogenates prepared from isolated rat heart myocytes. We conclude that the myocardium contains a hormone-sensitive lipase that is regulated in a fashion similar to that of the adipose tissue enzyme.     

Coflowering invasive plants and a congener have neutral effects on fitness components of a rare endemic plant

Network analyses rarely include fitness components, such as germination, to tie invasive plants to population‐level effects on the natives. We address this limitation in a previously studied network of flower visitors around a suite of native and invasive plants that includes an endemic plant at Badlands National Park, South Dakota, USA. Eriogonum visheri coflowers with two abundant invasive plants, Salsola tragus and Melilotus officinalis, as well as a common congener, E. pauciflorum. Network analyses had suggested strong linkages between E. visheri and S. tragus and E. pauciflorum, with a weaker link to M. officinalis. We measured visitation, pollen deposited on stigmas, achene weight and germination over three field seasons (two for germination) in four populations (two in the final season) of E. visheri and applied in situ pollen treatments to E. visheri, adding pollen from other flowers on the same plant; flowers on other E. visheri plants; S. tragus, M. officinalis, or E. pauciflorum; open pollination; or excluding pollinators. Insect visitation to E. visheri was not affected by floral abundance of any of the focal species. Most visitors were halictid bees; one of these (Lasioglossum packeri) was the only identified species to visit E. visheri all three years. Ninety‐seven percent of pollen on collected E. visheri stigmas was conspecific, but 22% of flowers had >1 grain of E. pauciflorum pollen on stigmas and 7% had >1 grain of S. tragus pollen; <1% of flowers had M. officinalis pollen on stigmas. None of the pollen treatments produced significant differences in weight or germination of E. visheri achenes. We conclude that, in contrast to the results of the network analysis, neither of the invasive species poses a threat, via heterospecific pollen deposition, to pollination of the endemic E. visheri, and that its congener provides alternative pollen resources to its pollinators.     

How a neutral evolutionary ratchet can build cellular complexity

Complex cellular machines and processes are commonly believed to be products of selection, and it is typically understood to be the job of evolutionary biologists to show how selective advantage can account for each step in their origin and subsequent growth in complexity. Here, we describe how complex machines might instead evolve in the absence of positive selection through a process of "presuppression," first termed constructive neutral evolution (CNE) more than a decade ago. If an autonomously functioning cellular component acquires mutations that make it dependent for function on another, pre-existing component or process, and if there are multiple ways in which such dependence may arise, then dependence inevitably will arise and reversal to independence is unlikely. Thus, CNE is a unidirectional evolutionary ratchet leading to complexity, if complexity is equated with the number of components or steps necessary to carry out a cellular process. CNE can explain "functions" that seem to make little sense in terms of cellular economy, like RNA editing or splicing, but it may also contribute to the complexity of machines with clear benefit to the cell, like the ribosome, and to organismal complexity overall. We suggest that CNE-based evolutionary scenarios are in these and other cases less forced than the selectionist or adaptationist narratives that are generally told.     

Herbivory by a Phloem-feeding insect inhibits floral volatile production

There is extensive knowledge on the effects of insect herbivory on volatile emission from vegetative tissue, but little is known about its impact on floral volatiles. We show that herbivory by phloem-feeding aphids inhibits floral volatile emission in white mustard Sinapis alba measured by gas chromatographic analysis of headspace volatiles. The effect of the Brassica specialist aphid Lipaphis erysimi was stronger than the generalist aphid Myzus persicae and feeding by chewing larvae of the moth Plutella xylostella caused no reduction in floral volatile emission. Field observations showed no effect of L. erysimi-mediated floral volatile emission on the total number of flower visits by pollinators. Olfactory bioassays suggested that although two aphid natural enemies could detect aphid inhibition of floral volatiles, their olfactory orientation to infested plants was not disrupted. This is the first demonstration that phloem-feeding herbivory can affect floral volatile emission, and that the outcome of interaction between herbivory and floral chemistry may differ depending on the herbivore's feeding mode and degree of specialisation. The findings provide new insights into interactions between insect herbivores and plant chemistry.     

Simultaneous fractionation of four placental neutral glycosphingolipids with a continuous gradient

We describe a method for isolating milligram quantities of the four neutral glycosphingolipids, glucocerebroside, lactosylceramide, traiosylceramide, and globoside, from human placental tissue. This procedure is carried out on a silicic acid column eluted with a continuous chloroform-methanol gradient (19:1 to 4:1); the four glycosphingolipids elute as separate fractions with no need for further separation. The method is simple, rapid, and yields sufficient material to use as analytical standards for several hundred runs. The lipids have been identified by NMR spectroscopy. Placental tissue is freely available in most centers and is an excellent untapped source for these compounds. Given that lactosylceramide is not commercially available and that triaosylceramide (ceramide trihexoside) cannot be obtained in a reliable state, this technique represents an effective solution to this dilemma.     

Network isolation and local diversity in neutral metacommunities

Biologists seek an understanding of the biological and environmental factors determining local community diversity. Recent advances in metacommunity ecology, and neutral theory in particular, highlight the importance of dispersal processes interacting with the spatial structure of a landscape for generating spatial patterns and maintaining biodiversity. The relative spatial isolation of a community is traditionally thought to have a large influence on local diversity. However, isolation remains an elusive concept to quantify, particularly in metacommunities with complex spatial structure. We represent the metacommunity as a network of local communities, and use network centrality measures to quantify the isolation of a local community. Using spatially explicit neutral theory, we examine how node position predicts variation in alpha diversity across a metacommunity. We find that diversity increases with node centrality in the network, but only when centrality is measured on a given scale in the network that widens with increasing dispersal rates and narrows with increasing evolutionary rates. More generally, complex biodiversity patterns form only when the underlying geography has structure on this critical scale. This provides a framework for understanding the influence of spatial geographic structure on global biodiversity patterns.