A new approach for plant proteomics: characterization of chloroplast proteins of Arabidopsis thaliana by top-down mass spectrometry

A recently developed methodology for the characterization of complex proteomes, top-down Fourier transform mass spectrometry (FTMS), is applied for the first time to a plant proteome, that of the model plant Arabidopsis thaliana. Of the 3000 proteins predicted by the genome sequence, 97 were recently identified in two separate "bottom-up" mass spectrometry studies in which the proteins were purified and digested and in which the mass spectrometry-measured mass values of the resulting peptides matched against those expected from the DNA-predicted proteins. In the top-down approach applied here, molecular ions from a protein mixture are purified, weighed exactly (+/-1 Da), and fragmented in the FTMS. Of the 22 molecular weight values found in three isolated mixtures, 7 were chosen, and their primary structures were fully characterized; in only one case was the bottom-up structure in full agreement. The top-down technique is not only efficient for identification of the DNA-predicted precursors, such as that of a protein present as a 5% mixture component, but also for characterization of the primary structure of the final protein. For two proteins the previously predicted cleavage site for loss of the signal peptide was found to be incorrect. Two 27-kDa proteins are fully characterized, although they are found to differ by only 12 residues and 6 Da in mass in a 3:1 ratio; the bottom-up studies did not distinguish these proteins. Direct tandem mass spectrometry dissociation of two 15-kDa molecular ions showed >90% sequence similarity, whereas three-stage mass spectrometry traced their +14-Da molecular mass discrepancies to an unusual N-methylation on the N-terminal amino group; the bottom-up approach identified only one precursor protein. The high potential of the top-down FTMS approach for characterization as well as identification of complex plant proteomes should provide a real incentive for its further automation.     

天然活性多肽的发掘策略和生产技术

活性多肽可以参与生命机体的多种生理活动,对促进人体健康发挥着重要的作用,如降血压、降血糖、降血脂和抗癌等,其创制技术也逐渐成为重要的研究和应用转化方向。本综述旨在总结天然活性多肽的发掘策略和生产技术的研究进展。目前,天然活性多肽的发掘与生产技术主要包括自上而下和自下而上两种方法,其中自上而下方法在多肽发掘方面主要为直接提取鉴定法,在生产技术方面主要包括直接提取法、酶解法和微生物发酵法;自下而上方法在多肽发掘方面包括天然活性多肽改造和数据库发掘方法,在生产技术方面主要方法包括化学合成法、酶合成法、基因重组表达法和无细胞合成法。自上而下的天然多肽制备与功能验证方法存在步骤烦琐、耗费时间长、功能不确定性大、实验与生产成本高以及质量控制难度大等问题;而自下而上的活性多肽合成与功能验证方法适合多肽药物的开发,而难以用于功能食品。随着测序和质谱技术的发展,人们更容易从分子水平获取物种蛋白组信息。以此蛋白组信息为根据,将自上而下和自下而上两种方法结合,可以克服单独使用这两种方法存在的问题,从而为快速开发和生产天然活性多肽提供新的策略。     

The effect of the Holling type II functional response on apparent competition

This article analyzes the classical 2-resource-1-consumer apparent competition community module with the Holling type II functional response. Two types of resource regulation (top-down vs. combined top-down and bottom-up) and two types of consumer behaviors (inflexible consumers with fixed preferences for resources vs. adaptive consumers) are considered. When resources grow exponentially and consumers are inflexible foragers, one resource is always outcompeted due to strong apparent competition. Density dependent resource growth relaxes apparent competition so that resources can coexist. As multiple attractors (either equilibria or limit cycles) coexist, population dynamics and community composition depend on initial population densities. Population dynamics change dramatically when consumers forage adaptively. In this case, the results both for top-down, and combined top-down and bottom-up regulation are similar and they show that species persistence occurs for a much larger set of parameter values when compared with inflexible consumers. Moreover, population dynamics will be chaotic when resource carrying capacities are high enough. This shows that adaptive consumer switching can destabilize population dynamics.     

Nonadditive interactions between trophic levels bias the appraisal of the strength of mortality factors

The role of bottom-up and top-down factors in determining the abundance of organisms is still often evaluated in terms of the nominal (i.e., apparent) cause of death. To determine whether estimates of mortality can be influenced by nonadditive multitrophic interactions, we generated both marginal (i.e., the entirety of mortality attributable to a given factor, including its obscured fraction) and nominal estimates of the trophic forces acting on an herbivore. This was accomplished by comparing mortality rates of the willow leaf beetle Phratora vulgatissima on willows affected or relatively unaffected by local growing conditions, in the presence or absence of natural enemies. Marginal estimates indicated that bottom-up and top-down mortality factors interacted in a synergistic or compensatory way, with the nature of the interaction varying in response to the time elapsed since the last harvest of willow plantations. In contrast, nominal estimates could not discriminate synergistic and compensatory interactions from top-down influence. Combined with recent evidence of compensatory mortality between trophic forces in another system, this study suggests that nonadditive effects between bottom-up and top-down mortality factors may be common, offering an explanation through which the contrasting evidences previously presented by proponents of the bottom-up and top-down views can be understood.     

Integrating Top-Down and Bottom-Up Sensory Processing by Somato-Dendritic Interactions

The classical view of cortical information processing is that of a bottom-up process in a feedforward hierarchy. However, psychophysical, anatomical, and physiological evidence suggests that top-down effects play a crucial role in the processing of input stimuli. Not much is known about the neural mechanisms underlying these effects. Here we investigate a physiologically inspired model of two reciprocally connected cortical areas. Each area receives bottom-up as well as top-down information. This information is integrated by a mechanism that exploits recent findings on somato-dendritic interactions. (1) This results in a burst signal that is robust in the context of noise in bottom-up signals. (2) Investigating the influence of additional top-down information, priming-like effects on the processing of bottom-up input can be demonstrated. (3) In accordance with recent physiological findings, interareal coupling in low-frequency ranges is characteristically enhanced by top-down mechanisms. The proposed scheme combines a qualitative influence of top-down directed signals on the temporal dynamics of neuronal activity with a limited effect on the mean firing rate of the targeted neurons. As it gives an account of the system properties on the cellular level, it is possible to derive several experimentally testable predictions.     

Bottom-Up Effects on Biomass Versus Top-Down Effects on Identity: A Multiple-Lake Fish Community Manipulation Experiment

The extent to which ecosystems are regulated by top-down relative to bottom-up control has been a dominant paradigm in ecology for many decades. For lakes, it has been shown that predation by fish is an important determinant of variation in zooplankton and phytoplankton community characteristics. Effects of fish are expected to not only be a function of total fish biomass, but also of functional composition of the fish community. Previous research on the importance of trophic cascades in lakes has largely focused on the role of zooplanktivorous and piscivorous fish. We conducted a large-scale multiple-lake fish community manipulation experiment to test for the effect of differences in fish functional community composition on the trophic structure of lakes. We examine the effect of top-down and bottom-up factors on phytoplankton and zooplankton biomass as well as on their community composition. We put our data in a broader perspective by comparing our results to data of a survey that also included ponds with low fish densities as well as ponds with very high densities of fish. Our results indicate that the overall food web structure under relative high fish densities is primarily structured by bottom-up factors, whereas community characteristics seem to be primarily regulated by top-down factors. Our results suggest a subtle interplay between bottom-up and top-down factors, in which bottom-up factors dominate in determining quantities while top-down effects are important in determining identities of the communities.     

Relative importance of top-down and bottom-up forces in food webs of Sarracenia pitcher communities at a northern and a southern site

The relative importance of resources (bottom-up forces) and natural enemies (top-down forces) for regulating food web dynamics has been debated, and both forces have been found to be critical for determining food web structure. How the relative importance of top-down and bottom-up forces varies between sites with different abiotic conditions is not well understood. Using the pitcher plant inquiline community as a model system, I examine how the relative importance of top-down and bottom-up effects differs between two disparate sites. Resources (ant carcasses) and top predators (mosquito larvae) were manipulated in two identical 4 × 4 factorial press experiments, conducted at two geographically distant sites (Michigan and Florida) within the range of the purple pitcher plant, Sarracenia purpurea, and the aquatic community that resides in its leaves. Overall, top predators reduced the density of prey populations while additional resources bolstered them, and the relative importance of top-down and bottom-up forces varied between sites and for different trophic levels. Specifically, top-down effects on protozoa were stronger in Florida than in Michigan, while the opposite pattern was found for rotifers. These findings experimentally demonstrate that the strength of predator–prey interactions, even those involving the same species, vary across space. While only two sites are compared in this study, I hypothesize that site differences in temperature, which influences metabolic rate, may be responsible for variation in consumer–resource interactions. These findings warrant further investigation into the specific factors that modify the relative importance of top-down and bottom-up effects.     

Top-down and bottom-up control on cougar and its prey in a central Mexican natural reserve

Top-down and bottom-up controls are hypothesized to regulate population structures in many ecosystems. However, few studies have had the opportunity to analyze both processes in the natural environment, especially on large carnivores like the cougar (Puma concolor). Previously, studies show that cougar diet in the Sierra Nanchititla Natural Reserve (SNNR), central Mexico, is mainly armadillo, coati, and white-tailed deer. We assess whether top-down and/or bottom-up control regulate this endangered food web: (a) we predicted that seasonal per capita changes in abundance (pca) of cougar will be positively affected by the abundance of their main prey; (b) primary productivity in SNNR will affect the pca of prey species, driving bottom-up control; and (c) armadillo, coati, and white-tailed deer pca will be affected by the abundance of cougar, generating top-down control. Using 15 camera traps for 6 years in the SNNR, we calculated a relative abundance index (RAI) and pca for cougar and each of the focal prey, and we used the normalized difference vegetation index (NDVI) as a proxy of primary productivity. We constructed multiple regression models and selected the best linear models based on ranking the AIC_c values. Our analysis suggests that P. concolor pca is best explained by bottom-up control and intraspecific feedback. White-tailed deer and armadillo pca were both significantly affected by cougar abundance, indicating top-down control for these prey species, but NDVI was not retained in any of the models selected for prey pca. Our results indicate that both bottom-up and top-down control are involved in regulating this endangered food web in the SNNR, Mexico.     

Ecosystem alteration modifies the relative strengths of bottom-up and top-down forces in a herbivore population

1. Ecosystem alterations can affect the abundance, distribution and diversity of plants and animals, and thus potentially change the relative strength of bottom-up (the plant resource) and top-down (natural enemies) trophic forces acting on herbivore populations. 2. The hypothesis that alterations of the forest ecosystem associated with precommercial thinning have contributed to the increased severity of outbreaks of Neodiprion abietis (Harris), a sawfly defoliator, through the reduction of trophic forces acting on N. abietis larvae, was tested using exclusion techniques. 3. The relative contributions to N. abietis larval mortality of bottom-up and top-down forces both increased with increasing levels of defoliation and were both reduced by thinning. The reduction of bottom-up and top-down forces caused a 58% mean increase in N. abietis larval survival in thinned compared with untreated stands, which is less than would be expected by the sum of the effects of thinning on each source of mortality. Evidence indicates that the partly compensatory, partly additive nature of the mortality associated with trophic forces in the system under study is responsible for this discrepancy. 4. To our knowledge, this is the first study to show the impact of ecosystem alterations on the balance between bottom-up and top-down forces acting on an eruptive herbivore population along a gradient of host-plant defoliation, and how this can lead to increased outbreak severity. It is stressed that accurate estimates of the relative contributions of bottom-up and top-down forces to mortality cannot be obtained if the additive or compensatory nature of the mortality associated with these trophic forces is overlooked.     

Metrics of predation: perils of predator-prey ratios

We developed an original modeling approach using program Stella® to investigate the usefulness of predator–prey ratios (PPRs) for interpreting top-down and bottom-up forcing on moose Alces alces. We included density-dependent feedbacks for the moose population, allowed K to vary based on amount and quality of available forage for moose, integrated effects of compensatory mortality, and added time lags in wolves Canis lupus tracking the moose population. Modeling scenarios we developed included bottom-up and top-down regulation as predetermined outcomes. We then evaluated whether PPRs would reflect the various combinations of trajectories of predator and prey populations under top-down versus bottom-up regulation. The resulting patterns of PPRs were impossible to disentangle from one another, and did not provide reliable insights into whether top-down or bottom-forcing occurred, especially over short time spans where critical decisions related to management of moose and wolves might be necessary. Only under top-down regulation did PPRs reflect the degree of predation experienced by moose, but in that instance, knowledge of top-down regulation must be known a priori to correctly interpret PPRs. Potential problems with interpreting PPRs include their double-variable nature, which resulted in the failure to reflect patterns of increase and decrease for predators and prey. We suggest that confidence intervals for PPRs be calculated from a binomial, similar to that proposed for sex and age ratios, which should help discourage the inappropriate use of this metric. We caution that the temptation to use PPRs often is irresistible, but their reliability is highly questionable. We provide an alternative method to using PPRs or other predation metrics for determining whether top-down or bottom-up forcing is occurring by adopting an approach based on the physical condition and life-history characteristics of prey.     

视觉注意与神经振荡

人脑每时每刻都要接收大量视觉信息，由于人脑加工信息的能力有限，所以在较大视野内将注意分配给相关信息，同时抑制引起注意分散的不相关信息，对执行目标导向的行为至关重要。这种对视觉信息的选择性和主动性加工以适应当前目标的过程被称作视觉注意（visual attention），且视觉注意可分为自上而下的注意与自下而上的注意两种不同功能。由于来自大脑电信号的神经振荡活动在认知加工中发挥重要作用，已有研究综述了视觉注意与神经振荡（neural oscillation）的密切关系，但并未涉及不同的注意功能与神经振荡的关系。本文系统性调查了不同注意功能与神经振荡的关系，发现额-顶区域的theta频带振荡活动反映了自上而下的认知控制，而后部脑区的theta振荡与自下而上的注意相关。顶-枕区域alpha振荡的偏侧化有助于注意分配，而alpha频带的大规模同步促成了注意对视皮层自上而下的影响。Beta振荡介导了自上而下的信息与自下而上的信息之间的互动，作为信息载体促进了视觉信息处理。Gamma振荡则可能与自上而下和自下而上的注意间整合相关。本文就视觉注意功能与神经振荡关系的研究现状展开综述，旨在揭示不同的神经振荡活动在特定的视觉注意功能中的作用。     

Institutions and Cultural Diversity: Effects of Democratic and Propaganda Processes on Local Convergence and Global Diversity

In a connected world where people influence each other, what can cause a globalized monoculture, and which measures help to preserve the coexistence of cultures? Previous research has shown that factors such as homophily, population size, geography, mass media, and type of social influence play important roles. In the present paper, we investigate for the first time the impact that institutions have on cultural diversity. In our first three studies, we extend existing agent-based models and explore the effects of institutional influence and agent loyalty. We find that higher institutional influence increases cultural diversity, while individuals'' loyalty to their institutions has a small, preserving effect. In three further studies, we test how bottom-up and top-down processes of institutional influence impact our model. We find that bottom-up democratic practices, such as referenda, tend to produce convergence towards homogeneity, while top-down information dissemination practices, such as propaganda, further increase diversity. In our last model—an integration of bottom-up and top-down processes into a feedback loop of information—we find that when democratic processes are rare, the effects of propaganda are amplified, i.e., more diversity emerges; however, when democratic processes are common, they are able to neutralize or reverse this propaganda effect. Importantly, our models allow for control over the full spectrum of diversity, so that a manipulation of our parameters can result in preferred levels of diversity, which will be useful for the study of other factors in the future. We discuss possible mechanisms behind our results, applications, and implications for political and social sciences.     

Relative roles of top-down and bottom-up forces in terrestrial tritrophic plant–insect herbivore–natural enemy systems

Whether resources (bottom-up forces), natural enemies (top-down forces), or both, determine the abundance of insect herbivore populations in plant–insect herbivore–natural enemy systems remains a major issue in population ecology. Unlike recent surveys of the tritrophic literature we do not seek to quantify whether top-down or bottom-up forces predominate in any given set of experimental systems. Acknowledging the dearth of empirical synthesis we employ two contrasting literature surveys to determine whether the plant–insect herbivore–natural enemy literature is currently adequate to form a conceptual synthesis of the relative roles of top-down and bottom-up forces. The emergence of a synthesis of the relative roles of top-down and bottom-up forces in plant–insect herbivore–natural enemy systems appears to have been largely prevented by (1) the absence of appropriate empirical data; (2) failure to appreciate the merits of existing data; (3) a continued desire to emphasise either top-down or bottom-up forces to the exclusion of the other; and (4) confusion regarding which processes regulate and which influence the abundance of insect herbivores.     

100% protein sequence coverage: a modern form of surrealism in proteomics

This review intends not only to discuss the current possibilities to gain 100% sequence coverage for proteins, but also to point out the critical limits to such an attempt. The aim of 100% sequence coverage, as the review title already implies, seems to be rather surreal if the complexity and dynamic range of a proteome is taken into consideration. Nevertheless, established bottom-up shotgun approaches are able to roughly identify a complete proteome as exemplary shown by yeast. However, this proceeding ignores more or less the fact that a protein is present as various protein species. The unambiguous identification of protein species requires 100% sequence coverage. Furthermore, the separation of the proteome must be performed on the protein species and not on the peptide level. Therefore, top-down is a good strategy for protein species analysis. Classical 2D-electrophoresis followed by an enzymatic or chemical cleavage, which is a combination of top-down and bottom-up, is another interesting approach. Moreover, the review summarizes further top-down and bottom-up combinations and to which extent middle-down improves the identification of protein species. The attention is also focused on cleavage strategies other than trypsin, as 100% sequence coverage in bottom-up experiments is only obtainable with a combination of cleavage reagents.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

Top-Down but Not Bottom-Up Visual Scanning is Affected in Hereditary Pure Cerebellar Ataxia

The aim of this study was to clarify the nature of visual processing deficits caused by cerebellar disorders. We studied the performance of two types of visual search (top-down visual scanning and bottom-up visual scanning) in 18 patients with pure cerebellar types of spinocerebellar degeneration (SCA6: 11; SCA31: 7). The gaze fixation position was recorded with an eye-tracking device while the subjects performed two visual search tasks in which they looked for a target Landolt figure among distractors. In the serial search task, the target was similar to the distractors and the subject had to search for the target by processing each item with top-down visual scanning. In the pop-out search task, the target and distractor were clearly discernible and the visual salience of the target allowed the subjects to detect it by bottom-up visual scanning. The saliency maps clearly showed that the serial search task required top-down visual attention and the pop-out search task required bottom-up visual attention. In the serial search task, the search time to detect the target was significantly longer in SCA patients than in normal subjects, whereas the search time in the pop-out search task was comparable between the two groups. These findings suggested that SCA patients cannot efficiently scan a target using a top-down attentional process, whereas scanning with a bottom-up attentional process is not affected. In the serial search task, the amplitude of saccades was significantly smaller in SCA patients than in normal subjects. The variability of saccade amplitude (saccadic dysmetria), number of re-fixations, and unstable fixation (nystagmus) were larger in SCA patients than in normal subjects, accounting for a substantial proportion of scattered fixations around the items. Saccadic dysmetria, re-fixation, and nystagmus may play important roles in the impaired top-down visual scanning in SCA, hampering precise visual processing of individual items.     

Incorporating anthropogenic effects into trophic ecology: predator–prey interactions in a human-dominated landscape

Apex predators perform important functions that regulate ecosystems worldwide. However, little is known about how ecosystem regulation by predators is influenced by human activities. In particular, how important are top-down effects of predators relative to direct and indirect human-mediated bottom-up and top-down processes? Combining data on species'' occurrence from camera traps and hunting records, we aimed to quantify the relative effects of top-down and bottom-up processes in shaping predator and prey distributions in a human-dominated landscape in Transylvania, Romania. By global standards this system is diverse, including apex predators (brown bear and wolf), mesopredators (red fox) and large herbivores (roe and red deer). Humans and free-ranging dogs represent additional predators in the system. Using structural equation modelling, we found that apex predators suppress lower trophic levels, especially herbivores. However, direct and indirect top-down effects of humans affected the ecosystem more strongly, influencing species at all trophic levels. Our study highlights the need to explicitly embed humans and their influences within trophic cascade theory. This will greatly expand our understanding of species interactions in human-modified landscapes, which compose the majority of the Earth''s terrestrial surface.     

Integrating 'top-down" and "bottom-up" mass spectrometric approaches for proteomic analysis of Shewanella oneidensis

Here we present a comprehensive method for proteome analysis that integrates both intact protein measurement ("top-down") and proteolytic fragment characterization ("bottom-up") mass spectrometric approaches, capitalizing on the unique capabilities of each method. This integrated approach was applied in a preliminary proteomic analysis of Shewanella oneidensis, a metal-reducing microbe of potential importance to the field of bioremediation. Cellular lysates were examined directly by the "bottom-up" approach as well as fractionated via anion-exchange liquid chromatography for integrated studies. A portion of each fraction was proteolytically digested, with the resulting peptides characterized by on-line liquid chromatography/tandem mass spectrometry. The remaining portion of each fraction containing the intact proteins was examined by high-resolution Fourier transform mass spectrometry. This "top-down" technique provided direct measurement of the molecular masses for the intact proteins and thereby enabled confirmation of post-translational modifications, signal peptides, and gene start sites of proteins detected in the "bottom-up" experiments. A total of 868 proteins from virtually every functional class, including hypotheticals, were identified from this organism.     

Top-down and bottom-up community regulation in marine rocky intertidal habitats

Strong top-down control by consumers has been demonstrated in rocky intertidal communities around the world. In contrast, the role of bottom-up effects (nutrients and productivity), known to have important influences in terrestrial and particularly freshwater ecosystems, is poorly known in marine hard-bottom communities. Recent studies in South Africa, New England, Oregon and New Zealand suggest that bottom-up processes can have important effects on rocky intertidal community structure. A significant aspect of all of these studies was the incorporation of processes varying on larger spatial scales than previously considered (10’s to 1000’s of km). In all four regions, variation in oceanographic factors (currents, upwelling, nutrients, rates of particle flux) was associated with different magnitudes of algal and/or phytoplankton abundance, availability of particulate food, and rates of recruitment. These processes led to differences in prey abundance and growth, secondary production, consumer growth, and consumer impact on prey resources. Oceanographic conditions therefore may vary on scales that generate ecologically significant variability in populations at the bottom of the food chain, and through upward-flowing food chain effects, lead to variation in top-down trophic effects. I conclude that top-down and bottom-up processes can be important joint determinants of community structure in rocky intertidal habitats, and predict that such effects will occur generally wherever oceanographic ‘discontinuities’ lie adjacent to rocky coastlines. I further argue that increased attention by researchers and of funding agencies to such benthic–pelagic coupling would dramatically enhance our understanding of the dynamics of marine ecosystems.     

When art moves the eyes: a behavioral and eye-tracking study

The aim of this study was to investigate, using eye-tracking technique, the influence of bottom-up and top-down processes on visual behavior while subjects, na?ve to art criticism, were presented with representational paintings. Forty-two subjects viewed color and black and white paintings (Color) categorized as dynamic or static (Dynamism) (bottom-up processes). Half of the images represented natural environments and half human subjects (Content); all stimuli were displayed under aesthetic and movement judgment conditions (Task) (top-down processes). Results on gazing behavior showed that content-related top-down processes prevailed over low-level visually-driven bottom-up processes when a human subject is represented in the painting. On the contrary, bottom-up processes, mediated by low-level visual features, particularly affected gazing behavior when looking at nature-content images. We discuss our results proposing a reconsideration of the definition of content-related top-down processes in accordance with the concept of embodied simulation in art perception.