Probing the role of aromatic residues at the secondary saccharide-binding sites of human salivary alpha-amylase in substrate hydrolysis and bacterial binding

Human salivary α-amylase (HSAmy) has three distinct functions relevant to oral health: (1) hydrolysis of starch, (2) binding to hydroxyapatite (HA), and (3) binding to bacteria (e.g., viridans streptococci). Although the active site of HSAmy for starch hydrolysis is well-characterized, the regions responsible for bacterial binding are yet to be defined. Since HSAmy possesses several secondary saccharide-binding sites in which aromatic residues are prominently located, we hypothesized that one or more of the secondary saccharide-binding sites harboring the aromatic residues may play an important role in bacterial binding. To test this hypothesis, the aromatic residues at five secondary binding sites were mutated to alanine to generate six mutants representing either single (W203A, Y276A, and W284A), double (Y276A/W284A and W316A/W388A), or multiple [W134A/W203A/Y276A/W284A/W316A/W388A; human salivary α-amylase aromatic residue multiple mutant (HSAmy-ar)] mutations. The crystal structure of HSAmy-ar as an acarbose complex was determined at a resolution of 1.5 Å and compared with the existing wild-type acarbose complex. The wild-type and the mutant enzymes were characterized for their abilities to exhibit enzyme activity, starch-binding activity, HA-binding activity, and bacterial binding activity. Our results clearly showed that (1) mutation of aromatic residues does not alter the overall conformation of the molecule; (2) single or double mutants showed either moderate or minimal changes in both starch-binding activity and bacterial binding activity, whereas HSAmy-ar showed significant reduction in these activities; (3) starch-hydrolytic activity was reduced by 10-fold in HSAmy-ar; (4) oligosaccharide-hydrolytic activity was reduced in all mutants, but the action pattern was similar to that of the wild-type enzyme; and (5) HA binding was unaffected in HSAmy-ar. These results clearly show that the aromatic residues at the secondary saccharide-binding sites in HSAmy play a critical role in bacterial binding and in starch-hydrolytic functions of HSAmy.     

Predominance of terrestrial organic matter in sediments from a cyanobacteria- blooming hypereutrophic lake

Although an understanding of the quantity and quality of sedimentary organic matter (SOM) pools is necessary to design sound environmental management strategies for lacustrine systems, the characterization of organic matter sources and the assessment of their relative contributions to eutrophic and inland lake sediments remain insufficient. In this study, the contribution of potential organic matter sources to sediments in shallow and hypereutrophic lake Taihu, China was assessed on the molecular level using source-specific fatty acid biomarkers. The results indicated that SOM was composed mainly of terrestrial plants with a maximal contribution of 45.3 ± 2.4% to the total organic carbon, which accounted for approximately 66% among the determined organic matter sources. Evidence suggests the terrestrial plants remained in a fresh state in surface sediments: the correlation (R² = 0.62, p < 0.05) between bacterial and terrestrial plant carbon was strong. On the other hand, aquatic plant and bacterial carbon contributed 5–15% to the total organic carbon, which was followed by the faint contribution (<5% of total organic carbon) of algae-derived organic carbon including cyanobacteria, diatoms, and dinoflagellates. The results provided details of the contributions of SOM sources, illustrating the usefulness of fatty acid biomarkers in discriminating organic matter sources within lake environments. Although organic matter sources of sediments varied in spatial and temporal patterns, the strong correlation between terrestrial plant and total organic carbon (R² = 0.60, p < 0.05) indicates that terrestrial plants were the dominant source in lake sediments.     

Dipole source localization by the mottled sculpin II. The role of lateral line excitation patterns

Extracellular, single unit recording techniques were used to measure the responses of posterior lateral line nerve fibers to a 50-Hz dipole source that slowly changed its location along the length of the fish. The flow-field equations for a dipole source were used to model the pressure gradient pattern and thus, the expected excitation pattern along a linear array of lateral line receptor organs for different source locations. Finally, excitation patterns were similarly modeled along the left and right side of the fish's head for actual steps taken by sculpin in approach pathways to the 50-Hz dipole source. Spatial histograms of posterior lateral line nerve fiber responses to different locations of the dipole source could be predicted from pressure gradient patterns modeled from the flow-field equations, confirming that the modeling approach applied to behavioral results was a good predictor of excitation patterns likely to be encoded by the lateral line periphery. An examination of how modeled excitation patterns changed from one position to the next in typical approach pathways and how patterns differed between positions from which successful and unsuccessful strikes were launched suggests that approach and strike strategies can indeed be explained by the information available in excitation patterns. In particular, changes in the spatial distribution of pressure gradient directions (polarities), available only when the source is lateral (as opposed to directly in front of the fish), appear to enhance the ability of sculpin to determine source distance. Without such information, misses are more likely to occur and successful strikes are more likely to be launched from short distances only. Accepted: 23 October 1996     

Polycyclic aromatic hydrocarbons (PAHs) in the environment of Beijing,China: Levels,distribution, trends and sources

Polycyclic aromatic hydrocarbons (PAHs) have become a major type of persistent organic pollutant (POPs) in the world. To well understand the occurrence, spatial distribution and source apportionment of 16 PAHs in different media in Beijing, a comprehensive study was conducted based on 337 obtained and subsequently selected samples in four specific media, including soil, road dust, surface water and atmosphere covering sampling time spanning from 2005 to 2014. Mean concentrations of Σ16 PAHs in soils (878.5 μg/kg) decreased from inner city to exterior areas with different land uses decreased in the order of industrial estate (2510 μg/kg)>educational area (1331 μg/kg)>Park (783 μg/kg)>cultivated (650 μg/kg)>roadside (583 μg/kg)>residential (568 μg/kg)>green area (478 μg/kg)>forest area (125 μg/kg). Meanwhile, PAHs concentrations in branch roads were slightly higher than trunk roads and mainly concentrated in urban areas (1136 μg/kg). Affected by industrial and residential areas, PAHs concentrations in downstream surface rivers were much higher than upstream surface rivers (475 ng/L). Due to strong mobility, PAHs concentrations in atmosphere were not equally distributed around the study area (157.59 ng/m³). The result of source apportionment determined by diagnostic ratios method indicated PAHs in multiple media in Beijing are mainly from coal and biomass combustion.     

Fish recruitment, dispersal, and trophic interactions in a heterogeneous lotic environment

I examine how dispersal of juvenile creek chubs (Semotilusatromaculatus) from beaver ponds into adjacent stream environments interacts with temporal abiotic variability to influence fish foraging, growth, and long-term persistence in the lotic ecosystem. Minnow trapping in upstream and downstream beaver ponds, along with weir traps used to monitor directional movement, indicated that most chubs colonized the stream from the downstream beaver pond. Large annual fluctuations in density of age 0 creek chubs occurred in the stream over a 10-year sampling period. Multiple regression analysis indicated that stream temperature, precipitation, and the density of reproductive creek chubs were not correlated with summer density of age 0 chubs in the stream. The factor most strongly associated with increased density of age 0 creek chubs was creation of the downstream beaver pond during the 6th–7th years of the study, suggesting dispersal from the pond was the primary factor determining age 0 fish density in the stream. Most individuals in the strong year classes neither persisted in the stream through their first winter nor resulted in an increased abundance of older age classes in later years. Comparison of age 0 fish density in summer to the proportion of fish surviving to age 1 in spring suggested that overwinter mortality was density dependent. Furthermore, a comparison of the size structure for age 0 individuals in summer to age 1 individuals the following spring indicated that winter mortality was size dependent. Experiments in an artificial stream adjacent to the natural channel revealed that fish growth was strongly density dependent, decreasing as fish density increased across both spring and summer, and elevated and low discharge. The decline in invertebrate prey captured by the fish and the subsequent decline in fish growth appeared to be particularly pronounced under low discharge in summer. Changes in juvenile creek chub density had no significant effect on benthic insect or crustacean abundance, suggesting that exploitative competition for limited invertebrate drift resources was a more important cause of density- dependent growth than depressed local benthic invertebrate abundance. These results suggest that lotic regions adjacent to beaver ponds act as potential reproductive “sinks” for dispersing juveniles confronting seasonal and flow-mediated restrictions on resource acquisition and growth, and the occurrence of seasonal bottlenecks to their survival, especially harsh winter conditions. Received: 9 September 1996 / Accepted: 8 August 1997     

固体介质中的重金属源解析及其在中国近海沉积物中的应用

沉积物中重金属源解析对甄别人为活动与自然变化对近海生态系统演化的影响有重要作用.本文总结了近年来污染物源解析常用的多元统计分析、地球化学方法和地质统计分析3种主要研究方法,剖析了不同方法的优劣及适用性,提出正定矩阵因子分析、Pb同位素示踪在重金属来源定量化研究中具有良好应用前景.梳理了中国近海沉积物中重金属来源的主要研究结果,发现河口和海湾是沉积物重金属受人为来源影响剧烈的典型近海区域,不同定量解析方法(多元统计分析、背景值估算、Pb同位素分析)均表明中国近海沉积物重金属的人为来源贡献率接近或超过50%.当前中国近海沉积物中重金属源解析研究还存在源识别端元模糊、解析结果缺乏相应的可靠性评价等问题.据此提出近海沉积物重金属源解析研究应使用多种解析技术手段综合、集成与优化,提高源解析的准确性;建立完善指标体系,筛选代表特定人为活动和自然过程的指标;甄别人为源重金属入海方式及过程,为沉积物数据信息的解译提供理论基础.     

FACE-ing the global change: Opportunities for improvement in photosynthetic radiation use efficiency and crop yield

The earth is rapidly changing through processes such as rising [CO₂], [O₃], and increased food demand. By 2050 the projected atmospheric [CO₂] and ground level [O₃] will be 50% and 20% higher than today. To meet future agricultural demand, amplified by an increasing population and economic progress in developing countries, crop yields will have to increase by at least 50% by the middle of the century. FACE (Free Air Concentration Enrichment) experiments have been conducted for more than 20 years in various parts of world to estimate, under the most realistic agricultural conditions possible, the impact of the CO₂ levels projected for the middle of this century on crops. The stimulations of crop seed yields by the projected CO₂ levels across FACE studies are about 18% on average and up to 30% for the hybrid rice varieties and vary among crops, cultivars, nitrogen levels and soil moisture. The observed increase in crop yields under the projected CO₂ levels fall short of what would be required to meet the projected future food demand, even with the most responsive varieties. Crop biomass production and seed yield is the product of photosynthetic solar energy conversion. Improvement in photosynthetic radiation use efficiency stands as the most promising opportunity allowing for major increases in crop yield in a future that portends major changes in climate and crop growing environments. Our advanced understanding of the photosynthetic process along with rapidly advancing capabilities in functional genomics, genetic transformation and synthetic biology promises new opportunities for crop improvement by greater photosynthesis and crop yield. Traits and genes that show promise for improving photosynthesis are briefly reviewed, including enhancing leaf photosynthesis capacity and reducing photorespiration loss, manipulating plant hormones’ responses for better ideotypes, extending duration of photosynthesis, and increasing carbon partitioning to the sink to alleviate feedback inhibition of photosynthesis.     

Ubiquitin is a novel substrate for human insulin-degrading enzyme

Insulin-degrading enzyme (IDE) can degrade insulin and amyloid-β, peptides involved in diabetes and Alzheimer's disease, respectively. IDE selects its substrates based on size, charge, and flexibility. From these criteria, we predict that IDE can cleave and inactivate ubiquitin (Ub). Here, we show that IDE cleaves Ub in a biphasic manner, first, by rapidly removing the two C-terminal glycines (k_cat = 2 s^− 1) followed by a slow cleavage between residues 72 and 73 (k_cat = 0.07 s^−  1), thereby producing the inactive 1-74 fragment of Ub (Ub1-74) and 1-72 fragment of Ub (Ub1-72). IDE is a ubiquitously expressed cytosolic protein, where monomeric Ub is also present. Thus, Ub degradation by IDE should be regulated. IDE is known to bind the cytoplasmic intermediate filament protein nestin with high affinity. We found that nestin potently inhibits the cleavage of Ub by IDE. In addition, Ub1-72 has a markedly increased affinity for IDE (∼ 90-fold). Thus, the association of IDE with cellular regulators and product inhibition by Ub1-72 can prevent inadvertent proteolysis of cellular Ub by IDE. Ub is a highly stable protein. However, IDE instead prefers to degrade peptides with high intrinsic flexibility. Indeed, we demonstrate that IDE is exquisitely sensitive to Ub stability. Mutations that only mildly destabilize Ub (ΔΔG <  0.6 kcal/mol) render IDE hypersensitive to Ub with rate enhancements greater than 12-fold. The Ub-bound IDE structure and IDE mutants reveal that the interaction of the exosite with the N-terminus of Ub guides the unfolding of Ub, allowing its sequential cleavages. Together, our studies link the control of Ub clearance with IDE.     

Structural basis for the allosteric regulation and substrate recognition of human cytosolic 5'-nucleotidase II

Cytosolic 5′-nucleotidase II (cN-II) catalyzes the dephosphorylation of 6-hydroxypurine nucleoside 5′-monophosphates and participates in the regulation of purine nucleotide pools within the cell. It interferes with the phosphorylation-dependent activation of nucleoside analogues used in the treatment of cancer and viral diseases. It is allosterically activated by a number of phosphate-containing cellular metabolites such as ATP, diadenosine polyphosphates, and 2,3-bisphosphoglycerate, which couple its activity with the metabolic state of the cell. We present seven high-resolution structures of human cN-II, including a ligand-free form and complexes with various substrates and effectors. These structures reveal the structural basis for the allosteric activation of cN-II, uncovering a mechanism where an effector-induced disorder-to-order transition generates rearrangements within the catalytic site and the subsequent coordination of the catalytically essential magnesium. Central to the activation is the large transition of the catalytically essential Asp356. This study also provides the structural basis for the substrate specificity of cN-II, where Arg202, Asp206, and Phe157 seem to be important residues for purine/pyrimidine selectivity. These structures provide a comprehensive structural basis for the design of cN-II inhibitors. They also contribute to the understanding of how the nucleotide salvage pathway is regulated at a molecular level.     

Crystal structure of the passenger domain of the Escherichia coli autotransporter EspP

Autotransporters represent a large superfamily of known and putative virulence factors produced by Gram-negative bacteria. They consist of an N-terminal “passenger domain” responsible for the specific effector functions of the molecule and a C-terminal “β-domain” responsible for translocation of the passenger across the bacterial outer membrane. Here, we present the 2.5-Å crystal structure of the passenger domain of the extracellular serine protease EspP, produced by the pathogen Escherichia coli O157:H7 and a member of the serine protease autotransporters of Enterobacteriaceae (SPATEs). Like the previously structurally characterized SPATE passenger domains, the EspP passenger domain contains an extended right-handed parallel β-helix preceded by an N-terminal globular domain housing the catalytic function of the protease. Of note, however, is the absence of a second globular domain protruding from this β-helix. We describe the structure of the EspP passenger domain in the context of previous results and provide an alternative hypothesis for the function of the β-helix within SPATEs.