Allosteric Inhibition as a New Mode of Action for BAY 1213790, a Neutralizing Antibody Targeting the Activated Form of Coagulation Factor XI

Factor XI (FXI), the zymogen of activated FXI (FXIa), is an attractive target for novel anticoagulants because FXI inhibition offers the potential to reduce thrombosis risk while minimizing the risk of bleeding. BAY 1213790, a novel anti-FXIa antibody, was generated using phage display technology. Crystal structure analysis of the FXIa–BAY 1213790 complex demonstrated that the tyrosine-rich complementarity-determining region 3 loop of the heavy chain of BAY 1213790 penetrated deepest into the FXIa binding epitope, forming a network of favorable interactions including a direct hydrogen bond from Tyr102 to the Gln451 sidechain (2.9 Å). The newly discovered binding epitope caused a structural rearrangement of the FXIa active site, revealing a novel allosteric mechanism of FXIa inhibition by BAY 1213790. BAY 1213790 specifically inhibited FXIa with a binding affinity of 2.4 nM, and in human plasma, prolonged activated partial thromboplastin time and inhibited thrombin generation in a concentration-dependent manner.     

Validity of surrogates for determination of 30–1000 Hz magnetic field exposure for video display terminal users in office settings

In studies investigating adverse reproductive outcomes associated with video display terminal (VDT) usage, amounts of time spent in front of a VDT or magnetic field (MF) levels in front of the VDT are used as surrogate measures of subject's MF exposure. However, the relevance of such surrogates to actual exposures has not been demonstrated, and the validity of the use of such measures as a surrogate for the actual MF exposure is only speculative. This study examines 1) measurements of MFs at frequencies of approximately 30–1000 Hz at a fixed distance from the VDTs, 2) reported hours of VDT use, and 3) reported distance between the VDT and the subject's waist as surrogate measures for the average MF exposure level of a VDT user during one 8 h workday. The results showed a weak correlation between the average exposure level of a VDT user and the MF 46 cm from a VDT (R = 0.52, n = 67, P < 0.001). This study showed no association between self-reported hours of VDT usage, or self-reported distance between waist and VDT, and the average MF exposures. Moreover, individuals' average MF exposures did not seem to be affected by other variables, such as position of a VDT on the desk, hours of desk use, and the VDT type (color vs. monochrome). These findings indicate that VDT exposures within office settings are complex and cannot be easily predicted by surrogates. © 1996 Wiley-Liss, Inc.     

乙烯诱导水仙成花相关代谢物和基因的筛选与分析

为了解乙烯诱导水仙(Narcissus tazetta var. chinensis)成花的生理和分子机制,利用代谢组和转录组测序技术,筛选乙烯诱导水仙成花的差异表达代谢物和基因。结果表明,乙烯处理的侧芽检测到12个差异表达代谢物(DEM),包括7个上调,5个下调,其中,(±)7-表茉莉酸、多巴胺、亚精胺可能与乙烯诱导水仙成花正相关,而吲哚及其衍生物呈负相关。转录组共获得1 021个差异表达基因(DEG),包括615个上调,406个下调,在DEG中鉴定筛选了45个与乙烯信号传导和开花相关的差异表达基因。乙烯诱导水仙成花启动可能先激活水仙鳞茎内源植物激素(尤其乙烯)信号通路的变化,与开花促进基因FPF1和MADS15的上调表达密切相关。9个基因的qRT-PCR结果验证了RNA-Seq的正确性。这些差异表达的代谢物和基因在水仙成花启动过程中可能具有重要作用。     

郑州地区夜鹭的求偶行为

2003年及2004年3~6月在河南郑州市区对夜鹭(Nycticorax nycticorax)的求偶行为进行了观察。结果表明,3月中旬至5月底雄性夜鹭表现出占区及固定的仪式化求偶行为,主要包括伸展炫耀、扬举炫耀、炫耀羽毛和配偶形成后的相互爱抚4个方面,其中前两种行为是夜鹭主要的求偶行为。在营巢地,求偶行为从早上日出之前夜鹭觅食归来开始,一直持续至日落前后。夜鹭的配偶选择包括雄性之间对巢区的竞争、雄鹭与雌鹭的相互选择等一系列过程。     

Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins

During ricin intoxication in mammalian cells, ricin''s enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin–ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (V_HHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such V_HHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these V_HHs block RTA–P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as “intrabodies,” these V_HHs rendered cells resistant to ricin intoxication. One V_HH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation.     

Can a stench be beautiful? – Osmophores in stem-succulent stapeliads (Apocynaceae-Asclepiadoideae-Ceropegieae-Stapeliinae)

Carrion flower stapeliads are examples of olfactory mimicry, forming sapromyiophilous flowers, which mimic food sources or oviposition sites to attract fly pollinators. The aim of this work was to investigate the ultrastructure of osmophores involved in the release of the carrion odor of Orbea variegata and Boucerosia indica flowers. In spite of their similar architecture (epidermal epithelium+subepidermal secretory layers), the osmophores of stapeliads feature some differences in morphology and ultrastructure. The epidermal epithelial cells of O. variegata and B. indica differ in shape, but both are extremely rich in endoplasmic reticulum and flocculent material in the vacuole. Unlike the Orbea, Boucerosia has starchless leucoplasts in the epidermal epithelium. Orbea features a cuticle with microchannels, while Boucerosia has a different mechanism for the pathway of scent substances to the cell exterior. They are released by rupturing of the outer layer of cuticle at the apex of the papillae. The epidermal cells of the adaxial corolla differ even between parts of the corolla, the corolla lobes and the annulus in the flower. This diversity may be connected with an odor gradient. The morphological and anatomical features of stapeliad (subtribe Stapeliinae) osmophores are generally similar to osmophores of members of subtribe Ceropegiinae (Ceropegia), thus, we suggest that this model of osmophores evolved before early diversification of Ceropegieae. The ultrastructural features of stapeliad osmophores are generally similar to those of Araceae, Orchidaceae and Passifloraceae.     

Floral scent evolution in the section Pseudophrys: pollinator‐mediated selection or phylogenetic constraints?

• Sexually deceptive orchid species from the Mediterranean genus Ophrys usually interact with one or a few pollinator species by means of specific floral scents. In this study, we investigated the respective role of pollinator‐mediated selection and phylogenetic constraints in the evolution of floral scents in the section Pseudophrys.
• We built a phylogenetic tree of 19 Pseudophrys species based on three nuclear loci; we gathered a dataset on their pollination interactions from the literature and from our own field data; and we extracted and analysed their floral scents using solid phase microextraction and gas chromatography‐mass spectrometry. We then quantified the phylogenetic signal carried by floral scents and investigated the link between plant–pollinator interactions and floral scent composition using phylogenetic comparative methods.
• We confirmed the monophyly of the section Pseudophrys and demonstrated the existence of three main clades within this section. We found that floral scent composition is affected by both phylogenetic relationships among Ophrys species and pollination interactions, with some compounds (especially fatty acid esters) carrying a significant phylogenetic signal and some (especially alkenes and alkadienes) generating dissimilarities between closely related Pseudophrys pollinated by different insects.
• Our results show that in the section Pseudophrys, floral scents are shaped both by pollinator‐mediated selection and by phylogenetic constraints, but that the relative importance of these two evolutionary forces differ among compound classes, probably reflecting distinct selective pressures imposed upon behaviourally active and non‐active compounds.

     

Development of neutralising human recombinant antibodies to pertussis toxin

A phage antibody display library of single chain Fv (scFv) was derived from the peripheral blood of two patients recently recovered from pertussis infection. Ten scFv, differentiated by DNA fingerprinting, were isolated by panning the library against pertussis toxin. One scFv (type 1) accounted for 33% of clones after panning. Six of the panned scFv bound to pertussis toxin. The ability of the scFv to neutralise pertussis toxin was assessed using the Chinese hamster ovary cell assay. The predominant scFv (type 1) and two others (types IV and VIII) were able to neutralise the pertussis toxin.     

High expression level of a gene coding for a chloroplastic amino acid selective channel protein is correlated to cold acclimation in cereals

A cold-regulated gene (cor tmc-ap3) coding for a putative chloroplastic amino acid selective channel protein was isolated from cold-treated barley leaves combining the differential display and the 5-RACE techniques. Cor tmc-ap3 is expressed at low level under normal growing temperature, and its expression is strongly enhanced after cold treatment. A positive correlation between the expression of cor tmc-ap3 and frost tolerance was found both among barley cultivars and among cereal species. The COR TMC-AP3 protein was expressed in vitro, purified and used to raise a polyclonal antibody. Western analysis showed that the cor tmc-ap3 gene product is localized to the chloroplastic outer envelope fraction, supporting its putative function. The frost-resistant winter cultivar Onice accumulated COR TMC-AP3 more rapidly and at a higher level than the frost-susceptible spring cultivar Gitane. After 28 days of cold acclimation the winter cultivar had about 2-fold more protein than the spring genotype. All these results suggest that an increased amount of a chloroplastic amino acid selective channel protein could be required for cold acclimation in cereals. Hypotheses about the role of COR TMC-AP3 during the hardening process are discussed.