Cascade blue as a donor for resonance energy transfer studies of heme-containing proteins

Cascade Blue acetyl azide is an amine reactive compound with spectral properties ideally suited for fluorescence resonance energy transfer (FRET) studies in which heme prosthetic groups serve as acceptors. To demonstrate utility of the Cascade Blue-heme spectroscopic ruler, cytochrome c was employed as a test case to calibrate distance measurements obtained from FRET analysis. Following modification, stoichiometrically labeled cytochrome c was digested with trypsin and derivatized fragments were analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry to identify Lys25 as the predominant site of covalent modification. FRET analysis on derivatized protein demonstrated nearly complete quenching of Cascade Blue fluorescence, indicating the labeled lysine residue to reside within 30 A of the heme prosthetic group. Sodium dodecyl sulfate (SDS) denaturation resulted in an approximately 28% recovery of fluorescence, demonstrating the utility of this donor-acceptor pair for evaluating distance changes of 30-90 A. Modeling the Cascade Blue donor molecule onto Lys25 of a cytochrome c NMR structure confirmed a distance of < or =30 A from the heme acceptor, as determined by FRET analysis. Further modeling of the SDS-denatured state as an extended chain suggested a maximum separation distance of 45 A, also consistent with results derived from FRET analysis.     

In vitro and in vivo aspects of cascade impactor tests and inhaler performance: A review

The purpose of this review is to discuss the roles of cascade impactor (CI) data in inhaler assessment and to examine the relationship between aerodynamic particle size distribution (APSD) and the clinical response to inhaled drugs. A systematic literature search of studies linking APSD to clinical response was undertaken. Two distinct roles for CI-generated data were identified: (1) the control of inhaler/drug product quality; and (2) the provision of data that may be predictive of particle deposition in the respiratory tract. Method robustness is required for the former application, combined with simplicity in operation, resulting in rudimentary attempts to mimic the anatomy of the respiratory tract. The latter necessitates making the apparatus and its operation more closely resemble patient use of the inhaler. A CI cannot perfectly simulate the respiratory tract, since it operates at constant flow rate, while the respiratory cycle has a varying flow-time profile. On the basis of a review of studies linking APSD to clinical response of inhaled drugs, it is concluded that attempts to use CI-generated data from quality control testing to compare products for bioequivalence are likely to have only limited success, as links between laboratory-measured APSD, particle deposition in the respiratory tract, and clinical response are not straightforward.     

以生物防治为主持续治理中华稻蝗的初步研究

1～3龄蝗蝻主要聚集于田埂、地头取食杂草,随后逐渐扩散到稻田内,4～6龄蝗蝻在稻田内趋于均匀分布。卡死克对中华稻蝗的致死中浓度(LC₅₀)为54.27 μg/mL,田间用５%卡死克^®水剂喷雾防治,以105 mL/hm²为最佳用量。卡死克与蝗虫微孢子虫协调应用于防治高密度稻蝗时,两者以２∶１的比例进行地毯式喷施效果最佳,当年感染微孢子虫的蝗虫达35.6%。这种防治措施费用低,持效期长,在持续性综合治理稻田蝗害中有很好的应用前景。     

Ecosystem services classification: A systems ecology perspective of the cascade framework

Ecosystem services research faces several challenges stemming from the plurality of interpretations of classifications and terminologies. In this paper we identify two main challenges with current ecosystem services classification systems: i) the inconsistency across concepts, terminology and definitions, and; ii) the mix up of processes and end-state benefits, or flows and assets. Although different ecosystem service definitions and interpretations can be valuable for enriching the research landscape, it is necessary to address the existing ambiguity to improve comparability among ecosystem-service-based approaches. Using the cascade framework as a reference, and Systems Ecology as a theoretical underpinning, we aim to address the ambiguity across typologies. The cascade framework links ecological processes with elements of human well-being following a pattern similar to a production chain. Systems Ecology is a long-established discipline which provides insight into complex relationships between people and the environment. We present a refreshed conceptualization of ecosystem services which can support ecosystem service assessment techniques and measurement. We combine the notions of biomass, information and interaction from system ecology, with the ecosystem services conceptualization to improve definitions and clarify terminology. We argue that ecosystem services should be defined as the interactions (i.e. processes) of the ecosystem that produce a change in human well-being, while ecosystem components or goods, i.e. countable as biomass units, are only proxies in the assessment of such changes. Furthermore, Systems Ecology can support a re-interpretation of the ecosystem services conceptualization and related applied research, where more emphasis is needed on the underpinning complexity of the ecological system.     

Escherichia coli acetyl-coenzyme A carboxylase: characterization and development of a high-throughput assay

Bacterial acetyl-coenzyme A carboxylase (ACCase) is a multicomponent system composed of AccA, AccD, AccC, and AccB (also known as BCCP), which is required for fatty acid biosynthesis. It is essential for cell growth and has been chemically validated as a target for antimicrobial drug discovery. To identify ACCase inhibitors, a simple and robust assay that monitors the overall activity by measuring phosphate production at physiologically relevant concentrations of all protein components was developed. Inorganic phosphate production was demonstrated to directly reflect the coupled activities of AccC and AccA/D with BCCP cycling between the two half-reactions. The K(m) apparent values for ATP, acetyl-coenzyme A, and BCCP were estimated to be 60+/-14 microM, 18+/-4 microM, and 39+/-9 nM, respectively. The stoichiometry between the two half-reactions was measured to be 1:1. Carboxy-biotin produced in the first half-reaction was stable over the time course of the assay. The assay was adapted to a high-throughput screen (HTS) 384-well format using a modified published scintillation proximity method. The optimized HTS assay has acceptable Z' factor values and was validated to report inhibitions of either AccC or AccA/D. The assay is not susceptible to signal quenching due to colored compounds.     

Structure-function analysis of grass clip serine protease involved in Drosophila Toll pathway activation

Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.     

Short-Term Response of Land Birds to Ponderosa Pine Restoration

We monitored the short‐term (>3 years) response of land birds to forest restoration treatments in Ponderosa pine forests located on the east slope of the North Cascade Range. Restoration treatments were designed to create stand structure and composition similar to pre‐settlement forests, which were influenced by a frequent fire regime. Overall, avian community composition was changed as a result of the treatments. Cassin’s Finch, Chipping Sparrow, and Yellow‐rumped Warbler were found at higher densities in treated stands, whereas Mountain Chickadee, Western Tanager, and Red‐breasted Nuthatch had higher densities in untreated stands. White‐headed Woodpecker and Western Bluebird were only detected in the treated stands. Brown‐headed Cowbird showed no response to treatments. We detected changes in the density of four of five foraging guilds in response to restoration treatments. Tree seedeaters, low understory and ground insectivores, and aerial insectivores all increased in density in treated stands. Overall, bark insectivores showed no density response to treatments. Tree foliage insectivore density was lower in treated than in untreated stands. Overall avian density, density of neotropical migrants, and density of some focal species were higher in treated stands. Monitoring should be continued to understand the longer‐term (5–10 year) responses of land birds and to guide future forest restoration efforts.     

The role of Cas8?in type?I CRISPR interference

CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use ‘cascade’ [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5–Cas7–crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA.     

Integrating biocatalysis with chemocatalysis for selective transformations

The integration of biocatalysis with chemocatalysis combines the excellent selectivity of the former with the robust reactivity of the latter and offers many advantages, such as lower cost, higher yield, enhanced selectivity, as well as less waste generation. In spite of the challenge of incompatibilities between different classes of catalysts, recent advances in synthetic chemistry and biology provide ample opportunities for multistep cascade transformations that combine biocatalysis and chemocatalysis. Herein, we review recent progress in merging biocatalysis with chemocatalysis, highlighting selected examples of photo-/electricity-driven biotransformations and recently developed strategies for addressing the catalyst incompatibility issue.     

Soil alters seedling establishment responses to climate

Ecologists expect species and biomes to shift poleward and upward with climate change, but non‐climatic factors complicate these predictions. In mountains, forests are expected to expand upward along climate gradients into subalpine/alpine meadows, while meadows expand upward onto bare ground. However, soils also vary across elevation, with bare soil above the meadows potentially poorer for plant establishment. Poor soil might constrain expansion at meadows' upper edges, while rich meadow soil might facilitate contraction at lower edges by promoting tree establishment. We assessed climate and soil effects on establishment by transplanting soil and seedlings of meadow and tree species across climate gradients on Mount Rainier. There were considerable interspecific differences, but some generalisations emerged. Survival often declined with earlier snow disappearance, with somewhat smaller declines in meadow soil. Size often increased with earlier snow disappearance, with larger increases in meadow soil. Thus, soil patterns may complicate range shifts.