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61.
Jin Young K. Kininmonth Stuart Lundgren Petra B. van Oppen Madeleine J. H. Willis Bette L. 《Coral reefs (Online)》2020,39(1):147-158
Coral Reefs - In the face of unprecedented rates of environmental alterations, the necessity to predict the capacity of corals to respond adaptively in a complex ecological system is becoming... 相似文献
62.
Zoltán Máté Marietta Zita Poles Gábor Szabó Mária Bagyánszki Petra Talapka Éva Fekete Nikolett Bódi 《Cell and tissue research》2013,352(2):199-206
Cholecystokinin (CCK) is an early marker of both neuronal and endocrine cell lineages in the developing gastrointestinal tract. To determine the quantitative properties and the spatial distribution of the CCK-expressing myenteric neurones in early postnatal life, a transgenic mouse strain with a CCK promoter-driven red fluorescent protein (DsRedT3/CCK) was established. The cell-specific expression of DsRedT3/CCK was validated by in situ hybridization with a CCK antisense riboprobe and by in situ hybridization coupled with immunohistochemistry involving a monoclonal antibody to CCK. A gradual increase in the DsRedT3/CCK-expressing enteric neurones with clear regional differences was documented from birth until the suckling to weaning transition, in parallel with the period of rapid intestinal growth and functional maturation. To evaluate the proportion of myenteric neurones in which DsRedT3/CCK transgene expression was colocalized with the enteric neuronal marker peripherin, immunofluorescence techniques were applied. All DsRedT3/CCK neurones were peripherin-immunoreactive and the proportion of DsRedT3/CCK-expressing myenteric neurones in the duodenum was the highest after the third week of life, when the number of peripherin-immunoreactive myenteric neurones in this region had decreased. Nearly all of the DsRedT3/CCK-expressing neurones also expressed 5-hydroxytryptophan (5-HT). Thus, by utilizing a new transgenic mouse strain, we have demonstrated a small number of CCK-expressing myenteric neurones with a developmentally regulated spatiotemporal distribution. The coexistence of CCK and 5-HT in the majority of these neurones suggests their possible regulatory role in feeding at the suckling to weaning transition. 相似文献
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64.
?sa Segerstolpe Sander Granneman Petra Bj?rk Flavia de Lima Alves Juri Rappsilber Charlotta Andersson Martin H?gbom David Tollervey Lars Wieslander 《Nucleic acids research》2013,41(2):1178-1190
Ribosomal subunit biogenesis in eukaryotes is a complex multistep process. Mrd1 is an essential and conserved small (40S) ribosomal subunit synthesis factor that is required for early cleavages in the 35S pre-ribosomal RNA (rRNA). Yeast Mrd1 contains five RNA-binding domains (RBDs), all of which are necessary for optimal function of the protein. Proteomic data showed that Mrd1 is part of the early pre-ribosomal complexes, and deletion of individual RBDs perturbs the pre-ribosomal structure. In vivo ultraviolet cross-linking showed that Mrd1 binds to the pre-rRNA at two sites within the 18S region, in helix 27 (h27) and helix 28. The major binding site lies in h27, and mutational analyses shows that this interaction requires the RBD1-3 region of Mrd1. RBD2 plays the dominant role in h27 binding, but other RBDs also contribute directly. h27 and helix 28 are located close to the sequences that form the central pseudoknot, a key structural feature of the mature 40S subunit. We speculate that the modular structure of Mrd1 coordinates pseudoknot formation with pre-rRNA processing and subunit assembly. 相似文献
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66.
Petra Mackeldanz Jürgen Alves Elisabeth Möncke-Buchner Karol H. Wyszomirski Detlev H. Krüger Monika Reuter 《Biochimie》2013
For efficient DNA hydrolysis, Type III restriction endonuclease EcoP15I interacts with two inversely oriented recognition sites in an ATP-dependent process. EcoP15I consists of two methylation (Mod) subunits and a single restriction (Res) subunit yielding a multifunctional enzyme complex able to methylate or to hydrolyse DNA. Comprehensive sequence alignments, limited proteolysis and mass spectroscopy suggested that the Res subunit is a fusion of a motor or translocase (Tr) domain of superfamily II helicases and an endonuclease domain with a catalytic PD…EXK motif. In the Tr domain, seven predicted helicase motifs (I, Ia, II–VI), a recently discovered Q-tip motif and three additional regions (IIIa, IVa, Va) conserved among Type III restriction enzymes have been identified that are predicted to be involved in DNA binding and ATP hydrolysis. Because DNA unwinding activity for EcoP15I (as for bona fide helicases) has never been found and EcoP15I ATPase rates are only low, the functional importance of the helicase motifs and regions was questionable and has never been probed systematically. Therefore, we mutated all helicase motifs and conserved regions predicted in Type III restriction enzyme EcoP15I and examined the functional consequences on EcoP15I enzyme activity and the structural integrity of the variants by CD spectroscopy. The resulting eleven enzyme variants all, except variant IVa, are properly folded showing the same secondary structure distribution as the wild-type enzyme. Classical helicase motifs I–VI are important for ATP and DNA cleavage by EcoP15I and mutations therein led to complete loss of ATPase and cleavage activity. Among the catalytically inactive enzyme variants three preserved the ability to bind ATP. In contrast, newly assigned motifs Q-tip, Ia and Va are not essential for EcoP15I activity and the corresponding enzyme variants were still catalytically active. DNA binding was only marginally reduced (2–7 fold) in all enzyme variants tested. 相似文献
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68.
Peter M. Bowers Tamlyn Y. Neben Geoffery L. Tomlinson Jennifer L. Dalton Larry Altobell Xue Zhang John L. Macomber Betty F. Wu Rachelle M. Toobian Audrey D. McConnell Petra Verdino Betty Chau Robert A. Horlick David J. King 《The Journal of biological chemistry》2013,288(11):7688-7696
A method for simultaneous humanization and affinity maturation of monoclonal antibodies has been developed using heavy chain complementarity-determining region (CDR) 3 grafting combined with somatic hypermutation in vitro. To minimize the amount of murine antibody-derived antibody sequence used during humanization, only the CDR3 region from a murine antibody that recognizes the cytokine hβNGF was grafted into a nonhomologous human germ line V region. The resulting CDR3-grafted HC was paired with a CDR-grafted light chain, displayed on the surface of HEK293 cells, and matured using in vitro somatic hypermutation. A high affinity humanized antibody was derived that was considerably more potent than the parental antibody, possessed a low pm dissociation constant, and demonstrated potent inhibition of hβNGF activity in vitro. The resulting antibody contained half the heavy chain murine donor sequence compared with the same antibody humanized using traditional methods. 相似文献
69.
Robert A. Horlick John L. Macomber Peter M. Bowers Tamlyn Y. Neben Geoffery L. Tomlinson Irina P. Krapf Jennifer L. Dalton Petra Verdino David J. King 《The Journal of biological chemistry》2013,288(27):19861-19869
A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation. 相似文献
70.
Petra Bonova Jozef BurdaViera Danielisova Miroslava NemethovaMiroslav Gottlieb 《Neurochemistry international》2013
In the clinic delayed post-conditioning would represent an attractive strategy for the survival of vulnerable neurons after an ischemic event. In this paper we studied the impact of ischemia and delayed post-conditioning on blood and brain tissue concentrations of glutamate and protein synthesis. We designed two groups of animals for analysis of brain tissues and blood after global ischemia and post-conditioning, and one for analysis of blood glutamate after transient focal ischemia. 相似文献