A covalently bound photoisomerizable agonist. Comparison with reversibly bound agonists at electrophorus electroplaques

After disulphide bonds are reduced with dithiothreitol, trans-3- (α-bromomethyl)-3’-[α- (trimethylammonium)methyl]azobenzene (trans-QBr) alkylates a sulfhydryl group on receptors. The membrane conductance induced by this “tethered agonist” shares many properties with that induced by reversible agonists. Equilibrium conductance increases as the membrane potential is made more negative; the voltage sensitivity resembles that seen with 50 [mu]M carbachol. Voltage- jump relaxations follow an exponential time-course; the rate constants are about twice as large as those seen with 50 μM carbachol and have the same voltage and temperature sensitivity. With reversible agonists, the rate of channel opening increases with the frequency of agonist-receptor collisions: with tethered trans-Qbr, this rate depends only on intramolecular events. In comparison to the conductance induced by reversible agonists, the QBr-induced conductance is at least 10-fold less sensitive to competitive blockade by tubocurarine and roughly as sensitive to “open-channel blockade” bu QX-222. Light-flash experiments with tethered QBr resemble those with the reversible photoisomerizable agonist, 3,3’,bis-[α-(trimethylammonium)methyl]azobenzene (Bis-Q): the conductance is increased by cis {arrow} trans photoisomerizations and decreased by trans {arrow} cis photoisomerizations. As with Bis-Q, ligh-flash relaxations have the same rate constant as voltage-jump relaxations. Receptors with tethered trans isomer. By comparing the agonist-induced conductance with the cis/tans ratio, we conclude that each channel’s activation is determined by the configuration of a single tethered QBr molecule. The QBr-induced conductance shows slow decreases (time constant, several hundred milliseconds), which can be partially reversed by flashes. The similarities suggest that the same rate-limiting step governs the opening and closing of channels for both reversible and tethered agonists. Therefore, this step is probably not the initial encounter between agonist and receptor molecules.     

Structural characterization of β-ketoacyl ACP synthase I bound to platencin and fragment screening molecules at two substrate binding sites

The bacterial fatty acid pathway is essential for membrane synthesis and a range of other metabolic and cellular functions. The β-ketoacyl-ACP synthases carry out the initial elongation reaction of this pathway, utilizing acetyl-CoA as a primer to elongate malonyl-ACP by two carbons, and subsequent elongation of the fatty acyl-ACP substrate by two carbons. Here we describe the structures of the β-ketoacyl-ACP synthase I from Brucella melitensis in complex with platencin, 7-hydroxycoumarin, and (5-thiophen-2-ylisoxazol-3-yl)methanol. The enzyme is a dimer and based on structural and sequence conservation, harbors the same active site configuration as other β-ketoacyl-ACP synthases. The platencin binding site overlaps with the fatty acyl compound supplied by ACP, while 7-hydroxyl-coumarin and (5-thiophen-2-ylisoxazol-3-yl)methanol bind at the secondary fatty acyl binding site. These high-resolution structures, ranging between 1.25 and 1.70 å resolution, provide a basis for in silico inhibitor screening and optimization, and can aid in rational drug design by revealing the high-resolution binding interfaces of molecules at the malonyl-ACP and acyl-ACP active sites.     

Palatability and feeding preferences of Uresiphita maorialis (Lepidoptera: Crambidae) for three Sophora species

In a three-hour bioassay, we tested the palatability and feeding preferences of Uresiphita maorialis (kōwhai moth) for Sophora tetraptera, Sophora microphylla and Sophora prostrata. Palatability tests showed no differences among the Sophora species. Feeding preferences, on the other hand, showed that S. tetraptera and S. microphylla leaves are preferred over S. prostrata leaves. Our results support our field observations in Wellington city parks and gardens showing that S. tetraptera and S. microphylla plants frequently have higher densities of larvae than S. prostrata.     

Biodeterioration of Mayan buildings at uxmal and tulum,Mexico

Uxmal and Tulum are two important Mayan sites in the Yucatan peninsula. The buildings are mainly composed of limestone and grey/black discoloration is seen on exposed walls and copious greenish biofilms on inner walls. The principal microorganisms detected on interior walls at both Uxmal and Tulum were cyanobacteria; heterotrophic bacteria and filamentous fungi were also present. A dark‐pigmented mitosporic fungus and Bacillus cereus, both isolated from Uxmal, were shown to be acidogenic in laboratory cultures. Cyanobacteria belonging to rock‐degrading genera Synechocystis and Gloeocapsa were identified at both sites. Surface analysis previously showed that calcium ions were present in the biofilms on buildings at Uxmal and Tulum, suggesting the deposition of biosolubilized stone. Apart from their potential to degrade the substrate, the coccoid cyanobacteria supply organic nutrients for bacteria and fungi, which can produce organic acids, further increasing stone degradation.     

Cytidine derivatives as IspF inhibitors of Burkolderia pseudomallei

Published biological data suggest that the methyl erythritol phosphate (MEP) pathway, a non-mevalonate isoprenoid biosynthetic pathway, is essential for certain bacteria and other infectious disease organisms. One highly conserved enzyme in the MEP pathway is 2C-methyl-d-erythritol 2,4-cyclodiphosphate synthase (IspF). Fragment-bound complexes of IspF from Burkholderia pseudomallei were used to design and synthesize a series of molecules linking the cytidine moiety to different zinc pocket fragment binders. Testing by surface plasmon resonance (SPR) found one molecule in the series to possess binding affinity equal to that of cytidine diphosphate, despite lacking any metal-coordinating phosphate groups. Close inspection of the SPR data suggest different binding stoichiometries between IspF and test compounds. Crystallographic analysis shows important variations between the binding mode of one synthesized compound and the pose of the bound fragment from which it was designed. The binding modes of these molecules add to our structural knowledge base for IspF and suggest future refinements in this compound series.     

毛乌素沙地南缘沙丘生物结皮对凝结水形成和蒸发的影响

在水分极度匮乏的荒漠生态系统,凝结水是除降雨之外最重要的水分来源之一,它对荒漠生态系统结构、功能和过程的维持产生重要的影响。为探明半干旱沙区生物结皮表面的凝结水形成和蒸发特征,采用自制的微型蒸渗计(直径7 cm、高5 cm的PVC管)实验观测了不同类型地表(裸沙、浅灰色藻类结皮、黑褐色藻类结皮和苔藓结皮)对凝结水形成和蒸发的影响。结果表明:(1)观测期间共有20次凝结水形成记录,除降雨天气外,几乎每天都能观测到水分凝结现象;(2)不同类型地表凝结水总量依次为(1.998±0.075),(2.326±0.083),(2.790±0.058)和(3.416±0.068) mm,生物结皮表面的凝结水总量显著大于裸沙(P < 0.05);随生物结皮的发育,不同类型生物结皮表面的凝结水总量呈增加的趋势,凝结水总量之间差异显著(P < 0.05);观测期间不同类型地表日平均凝结水量依次为(0.100±0.003),(0.116±0.004),(0.140±0.002)和(0.171± 0.003) mm,不同类型地表日平均凝结水量之间差异极显著(P < 0.01);(3)凝结水形成过程的观测结果显示,凝结水19:00开始形成,23:00-凌晨1:00形成不明显,1:00-7:00继续形成,除浅灰色藻类结皮外,太阳升出后在黑褐色藻类结皮和苔藓结皮表面继续形成少量的凝结水;凝结水7:30开始蒸发,10:30到11:00之间结束蒸发,凝结水在裸沙和浅灰色藻类结皮中的保持时间显著大于黑褐色藻类结皮和苔藓结皮中的保持时间(P < 0.05);(4)凝结水的形成受大气温度、地表温度、空气相对湿度和大气地表温度差等气象因素的影响,但其形成过程不与某一个气象因素呈简单的线性关系。     

The role of fish-poultry integration on fish growth performance,yields and economic benefits among smallholder farmers in sub-Saharan Africa,Tanzania

Aquaculture practices from sub-Saharan Africa are characterised by low production, owing to improper technology. Production can be increased through integrating fish farming with other existing on-farm activities, particularly livestock husbandry. We assessed the role of fish-poultry integration on all male Nile tilapia, Oreochromis niloticus growth performance, yields and economic benefits among smallholder farmers in sub-Saharan Africa, Tanzania. The study also compared phytoplankton species composition, abundance and biomass between the fish-poultry integration and non-integrated system. After 180 days of the experiment, all male O. niloticus cultured under fish-poultry integration exhibited significantly higher growth rates than those in the non-integrated system (p < 0.05). Gross fish yield (GFY), net fish yield (NFY) and net annual yields (NAY) obtained from fish-poultry integration were significantly higher than those from non-integrated system (p < 0.05). Partial enterprise budget analysis revealed that fish-poultry integration was more profitable than the non-integrated system. Moreover, fish-poultry integrated system produced significantly higher phytoplankton abundance and biomass than those from the non-integrated system. Results demonstrate that rural smallholder farmers can achieve higher growth rate, farm net yields and income by integrating all male O. niloticus with other on-farm activities than practising a stand-alone fish culture system.     

Sonochemically fabricated microelectrode arrays for biosensors offering widespread applicability: Part I

A novel and patented procedure is described for the sonochemical fabrication of a new class of microelectrode array based sensor with electrode element populations of up to 2 x 10(5) cm(-2). For some years it has been accepted that microelectrode arrays offer an attractive route for lowering minimum limits of detection and imparting stir (convectional mass transport) independence to sensor responses; despite this no commercial biosensors, to date, have employed microelectrode arrays, largely due to the cost of conventional fabrication routes that have not proved commercially viable for disposable devices. Biosensors formed by our sonochemical approach offer unrivalled sensitivity and impart stir independence to sensor responses. This format lends itself for mass fabrication due to the simplicity and inexpensiveness of the approach; in the first instance impedimetric and amperometric sensors are reported for glucose as model systems. Sensors already developed for ethanol, oxalate and a number of pesticide determinations will be reported in subsequent publications.     

Structural characterization of a ribose-5-phosphate isomerase B from the pathogenic fungus Coccidioides immitis

Background

F-spondin is a multi-domain extracellular matrix (ECM) protein and a contact-repellent molecule that directs axon outgrowth and cell migration during development. The reelin_N domain and the F-spondin domain (FS domain) comprise a proteolytic fragment that interacts with the cell membrane and guides the projection of commissural axons to floor plate. The FS domain is found in F-spondins, mindins, M-spondin and amphiF-spondin.

Results

We present the crystal structure of human F-spondin FS domain at 1.95Å resolution. The structure reveals a Ca²⁺-binding C2 domain variant with an 8-stranded antiparallel β-sandwich fold. Though the primary sequences of the FS domains of F-spondin and mindin are less than 36% identical, their overall structures are very similar. The unique feature of F-spondin FS domain is the presence of three disulfide bonds associated with the N- and C-termini of the domain and a highly conserved N-linked glycosylation site. The integrin-binding motif found in mindin is not conserved in the F-spondin FS domain.

Conclusion

The structure of the F-spondin FS domain completes the structural studies of the multiple-domain ECM molecule. The homology of its core structure to a common Ca²⁺- and lipid-binding C2 domain suggests that the F-spondin FS domain may be responsible for part of the membrane targeting of F-spondin in its regulation of axon development. The structural properties of the FS domain revealed in this study pave the way for further exploration into the functions of F-spondin.     

Design and initial characterization of the SC-200 proteomics standard mixture

High-throughput (HTP) proteomics studies generate large amounts of data. Interpretation of these data requires effective approaches to distinguish noise from biological signal, particularly as instrument and computational capacity increase and studies become more complex. Resolving this issue requires validated and reproducible methods and models, which in turn requires complex experimental and computational standards. The absence of appropriate standards and data sets for validating experimental and computational workflows hinders the development of HTP proteomics methods. Most protein standards are simple mixtures of proteins or peptides, or undercharacterized reference standards in which the identity and concentration of the constituent proteins is unknown. The Seattle Children's 200 (SC-200) proposed proteomics standard mixture is the next step toward developing realistic, fully characterized HTP proteomics standards. The SC-200 exhibits a unique modular design to extend its functionality, and consists of 200 proteins of known identities and molar concentrations from 6 microbial genomes, distributed into 10 molar concentration tiers spanning a 1,000-fold range. We describe the SC-200's design, potential uses, and initial characterization. We identified 84% of SC-200 proteins with an LTQ-Orbitrap and 65% with an LTQ-Velos (false discovery rate?=?1% for both). There were obvious trends in success rate, sequence coverage, and spectral counts with protein concentration; however, protein identification, sequence coverage, and spectral counts vary greatly within concentration levels.