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361.
Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.  相似文献   
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PTPμ is expressed in the developing nervous system and promotes growth and guidance of chick retinal ganglion cells. Using a newly developed growth cone rearrangement assay, we examined whether the small G‐proteins were involved in PTPμ‐dependent signaling. The stimulation of retinal cultures with purified PTPμ resulted in a striking morphological change in the growth cone, which becomes dominated by filopodia within 5 min of addition. This rearrangement in response to PTPμ stimulation was mediated by homophilic binding. We perturbed GTPase signaling using Toxin B, which inhibits Cdc42, Rac, and Rho, as well as the toxin Exoenzyme C3 that inhibits Rho. The PTPμ‐induced growth cone rearrangement was blocked by Toxin B, but not by Exoenzyme C3. This result suggests that either Cdc42 or Rac are required but not Rho. To determine which GTPase was involved in PTPμ signaling, we utilized dominant‐negative mutants of Cdc42 and Rac. Dominant‐negative Cdc42 blocked PTPμ‐induced rearrangement, while wild‐type Cdc42 and dominant‐negative Rac did not. Together, these results suggest a molecular signaling cascade beginning with PTPμ homophilic binding at the plasma membrane and the activation of Cdc42, which acts on the actin cytoskeleton to result in rearrangement of the growth cone. © 2003 Wiley Periodicals, Inc. J Neurobiol 56:199–208, 2003  相似文献   
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The lifestyle of parasitic plants is associated with peculiar morphological, genetic, and physiological adaptations that existing online plant-specific resources fail to adequately represent. Here, we introduce the Web Application for the Research of Parasitic Plants (WARPP) as an online resource dedicated to advancing research and development of parasitic plant biology. WARPP is a framework to facilitate international efforts by providing a central hub of curated evolutionary, ecological, and genetic data. The first version of WARPP provides a community hub for researchers to test this web application, for which curated data revolving around the economically important Broomrape family (Orobanchaceae) is readily accessible. The initial set of WARPP online tools includes a genome browser that centralizes genomic information for sequenced parasitic plant genomes, an orthogroup summary detailing the presence and absence of orthologous genes in parasites compared with nonparasitic plants, and an ancestral trait explorer showing the evolution of life-history preferences along phylogenies. WARPP represents a project under active development and relies on the scientific community to populate the web app’s database and further the development of new analysis tools. The first version of WARPP can be securely accessed at https://parasiticplants.app. The source code is licensed under GNU GPLv2 and is available at https://github.com/wickeLab/WARPP.

The WARPP online resource is a new, expandable, and interactive parasitic plant-specific data hub that provides online tools tailored to the peculiarities of parasitic angiosperms.  相似文献   
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Elevated dissolved iron concentrations in the methanic zone are typical geochemical signatures of rapidly accumulating marine sediments. These sediments are often characterized by co-burial of iron oxides with recalcitrant aromatic organic matter of terrigenous origin. Thus far, iron oxides are predicted to either impede organic matter degradation, aiding its preservation, or identified to enhance organic carbon oxidation via direct electron transfer. Here, we investigated the effect of various iron oxide phases with differing crystallinity (magnetite, hematite, and lepidocrocite) during microbial degradation of the aromatic model compound benzoate in methanic sediments. In slurry incubations with magnetite or hematite, concurrent iron reduction, and methanogenesis were stimulated during accelerated benzoate degradation with methanogenesis as the dominant electron sink. In contrast, with lepidocrocite, benzoate degradation, and methanogenesis were inhibited. These observations were reproducible in sediment-free enrichments, even after five successive transfers. Genes involved in the complete degradation of benzoate were identified in multiple metagenome assembled genomes. Four previously unknown benzoate degraders of the genera Thermincola (Peptococcaceae, Firmicutes), Dethiobacter (Syntrophomonadaceae, Firmicutes), Deltaproteobacteria bacteria SG8_13 (Desulfosarcinaceae, Deltaproteobacteria), and Melioribacter (Melioribacteraceae, Chlorobi) were identified from the marine sediment-derived enrichments. Scanning electron microscopy (SEM) and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH) images showed the ability of microorganisms to colonize and concurrently reduce magnetite likely stimulated by the observed methanogenic benzoate degradation. These findings explain the possible contribution of organoclastic reduction of iron oxides to the elevated dissolved Fe2+ pool typically observed in methanic zones of rapidly accumulating coastal and continental margin sediments.Subject terms: Biogeochemistry, Microbial ecology  相似文献   
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The following study presents a statistical analysis of 595 relevés gathered during a survey of isolated grasslands and dwarf shrub heaths in the region of Halle, eastern Germany. Relevés were classified with UPGMA clustering based on a Bray-Curtis distance matrix; the resulting classification was compared with a previously published phytosociological classification. Most of the 14 target communities were supported by both approaches, but in the case of communities on heavily disturbed sites cluster analysis corresponded to units on higher phytosociological levels (alliances). DCA ordinations clearly revealed that nutrient and moisture availability controlled the community differentiation. Both factors were, however, highly correlated with the distance to the outcrop edge. Thus the outcrop size also influenced differentiation of communities, making larger outcrops richer in xerophytic and endangered species. This appears to be largely due to edge effects since the relationships diminished sharply when we controlled for progressively increasing distances from the edge in partial ordinations. Thus, microtopography of outcrops was the most important factor for community differentiation. It is therefore suggested that future research and conservation management should concentrate on the consequences of nutrient influx and changing land use on larger outcrops.  相似文献   
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