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排序方式: 共有466条查询结果,搜索用时 15 毫秒
121.
Re-establishment of mouse strains used for mutagenesis and transgenesis has been hindered by difficulties in freezing sperm. The use of intracytoplasmic sperm injection (ICSI) enables the production of embryos for the restoration of mouse lines using sperm with reduced quality. By using ICSI, simplified sperm-freezing methods such as snap freezing can be explored. We examined the capacity of embryos from the inbred C57Bl/6J and 129Sv/ImJ mouse strains, commonly used for transgenic and N-ethyl-N-nitrosourea mutagenesis purposes to develop to blastocysts in vitro and to term following ICSI with sperm frozen without cryoprotectant. The results were compared to F1 (C57BlxCBA) hybrid embryos. Following freezing, sperm were immotile but could fertilize oocytes at similar rates to fresh sperm. However, embryo development in vitro to the blastocyst stage was reduced in all three strains. No pups were born from C57Bl/6J or 129Sv/ImJ embryos obtained from frozen sperm following transfer to foster females, and only a limited number of F1 embryos developed to term. Activation of oocytes injected with frozen sperm with 1.7 mM Sr2+ (SrCl2) did result in the birth of pups in all three strains. We conclude that the inability of sperm frozen without cryoprotectants to effectively activate oocytes may affect embryo development to term and can be overcome by strontium activation. This may become an effective strategy for sperm preservation and the restoration of most popular strains used for genetic modifications. 相似文献
122.
Berberich C Ramírez-Pineda JR Hambrecht C Alber G Skeiky YA Moll H 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(6):3171-3179
Upon loading with microbial Ag and adoptive transfer, dendritic cells (DC) are able to induce immunity to infections. This offers encouragement for the development of DC-based vaccination strategies. However, the mechanisms underlying the adjuvant effect of DC are not fully understood, and there is a need to identify Ag with which to arm DC. In the present study, we analyzed the role of DC-derived IL-12 in the induction of resistance to Leishmania major, and we evaluated the protective efficacy of DC loaded with individual Leishmania Ag. Using Ag-pulsed Langerhans cells (LC) from IL-12-deficient or wild-type mice for immunization of susceptible animals, we showed that the inability to release IL-12 completely abrogated the capacity of LC to mediate protection against leishmaniasis. This suggests that the availability of donor LC-derived IL-12 is a requirement for the development of protective immunity. In addition, we tested the protective effect of LC loaded with Leishmania homolog of receptor for activated C kinase, gp63, promastigote surface Ag, kinetoplastid membrane protein-11, or Leishmania homolog of eukaryotic ribosomal elongation and initiation factor 4a. The results show that mice vaccinated with LC that had been pulsed with selected molecularly defined parasite proteins are capable of controlling infection with L. major. Moreover, the protective potential of DC pulsed with a given Leishmania Ag correlated with the level of their IL-12 expression. Analysis of the cytokine profile of mice after DC-based vaccination revealed that protection was associated with a shift toward a Th1-type response. Together, these findings emphasize the critical role of IL-12 produced by the sensitizing DC and suggest that the development of a DC-based subunit vaccine is feasible. 相似文献
123.
Morse D Otterbein LE Watkins S Alber S Zhou Z Flavell RA Davis RJ Choi AM 《American journal of physiology. Lung cellular and molecular physiology》2003,285(1):L250-L257
Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/- mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1-/- mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1-/- mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1-/- and wild-type mice. Associated with increased mortality, Jnk1-/- mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice. 相似文献
124.
A family of eukaryotic-like Ser/Thr protein kinases occurs in bacteria, but little is known about the structures and functions of these proteins. Here we characterize PknB, a transmembrane signaling kinase from Mycobacterium tuberculosis. The intracellular PknB kinase domain is active autonomously, and the active enzyme is phosphorylated on residues homologous to regulatory phospho-acceptors in eukaryotic Ser/Thr kinases. The crystal structure of the PknB kinase domain in complex with an ATP analog reveals the active conformation. The predicted fold of the PknB extracellular domain matches the proposed targeting domain of penicillin-binding protein 2x. The structural and chemical similarities of PknB to metazoan homologs support a universal activation mechanism of Ser/Thr protein kinases in prokaryotes and eukaryotes. 相似文献
125.
126.
Orly Ardon Raphael Nudelman Catherine Caris Jacqueline Libman Abraham Shanzer Yona Chen Yitzhak Hadar 《Journal of bacteriology》1998,180(8):2021-2026
In this study, we monitored and compared the uptake of iron in the fungus Ustilago maydis by using biomimetic siderophore analogs of ferrichrome, the fungal native siderophore, and ferrioxamine B (FOB), a xenosiderophore. Ferrichrome-iron was taken up at a higher rate than FOB-iron. Unlike ferrichrome-mediated uptake, FOB-mediated iron transport involved an extracellular reduction mechanism. By using fluorescently labeled siderophore analogs, we monitored the time course, as well as the localization, of iron uptake processes within the fungal cells. A fluorescently labeled ferrichrome analog, B9-lissamine rhodamine B, which does not exhibit fluorescence quenching upon iron binding, was used to monitor the entry of the compounds into the fungal cells. The fluorescence was found intracellularly 4 h after the application and later was found concentrated in two to three vesicles within each cell. The fluorescence of the fluorescently labeled FOB analog CAT18, which is quenched by iron, was visualized around the cell membrane after 4 h of incubation with the ferrated (nonfluorescent) compounds. This fluorescence intensity increased with time, demonstrating fungal iron uptake from the siderophores, which remained extracellular. We here introduce the use of fluorescent biomimetic siderophores as tools to directly track and discriminate between different pathways of iron uptake in cells. 相似文献
127.
B D Pilger R Perozzo F Alber C Wurth G Folkers L Scapozza 《The Journal of biological chemistry》1999,274(45):31967-31973
Herpes simplex virus type 1 (HSV 1) thymidine kinase (TK) exhibits an extensive substrate diversity for nucleobases and sugar moieties, in contrast to other TKs. This substrate diversity is the crucial molecular basis of selective antiviral and suicide gene therapy. The mechanisms of substrate binding of HSV 1 TK were studied by means of site-directed mutagenesis combined with isothermal calorimetric measurements and guided by theoretical calculations and sequence comparison. The results show the link between the exceptionally broad substrate diversity of HSV 1 TK and the presence of structural features such as the residue triad His-58/Met-128/Tyr-172. The mutation of Met-128 into a Phe and the double mutant M128F/Y172F result in mutants that have lost their activity. However, by exchanging His to form the triple mutant H58L/M128F/Y172F, the enzyme regains activity. Strikingly, this triple mutant becomes resistant toward acyclovir. Furthermore, we give evidence for the importance of Glu-225 of the flexible LID region for the catalytic reaction. The data presented give new insights to understand mechanisms ruling substrate diversity and thus are crucial for both the development of new antiviral drugs and engineering of mutant TKs apt to accept novel substrate analogs for gene therapeutic approaches. 相似文献
128.
Ella DoronMandel Indrek Koppel Ofri Abraham Ida Rishal Terika P Smith Courtney N Buchanan Pabitra K Sahoo Jan Kadlec Juan A OsesPrieto Riki Kawaguchi Stefanie Alber Eitan Erez Zahavi Pierluigi Di Matteo Agostina Di Pizio DidiAndreas Song Nataliya Okladnikov Dalia Gordon Shifra BenDor Rebecca HaffnerKrausz Giovanni Coppola Alma L Burlingame Pavel Jungwirth Jeffery L Twiss Mike Fainzilber 《The EMBO journal》2021,40(20)
Nucleolin is a multifunctional RNA Binding Protein (RBP) with diverse subcellular localizations, including the nucleolus in all eukaryotic cells, the plasma membrane in tumor cells, and the axon in neurons. Here we show that the glycine arginine rich (GAR) domain of nucleolin drives subcellular localization via protein‐protein interactions with a kinesin light chain. In addition, GAR sequences mediate plasma membrane interactions of nucleolin. Both these modalities are in addition to the already reported involvement of the GAR domain in liquid‐liquid phase separation in the nucleolus. Nucleolin transport to axons requires the GAR domain, and heterozygous GAR deletion mice reveal reduced axonal localization of nucleolin cargo mRNAs and enhanced sensory neuron growth. Thus, the GAR domain governs axonal transport of a growth controlling RNA‐RBP complex in neurons, and is a versatile localization determinant for different subcellular compartments. Localization determination by GAR domains may explain why GAR mutants in diverse RBPs are associated with neurodegenerative disease. 相似文献
129.
Pullen KE Ng HL Sung PY Good MC Smith SM Alber T 《Structure (London, England : 1993)》2004,12(11):1947-1954
Serine/threonine protein phosphatases are central mediators of phosphorylation-dependent signals in eukaryotes and a variety of pathogenic bacteria. Here, we report the crystal structure of the intracellular catalytic domain of Mycobacterium tuberculosis PstPpp, a membrane-anchored phosphatase in the PP2C family. Despite sharing the fold and two-metal center of human PP2Calpha, the PstPpp catalytic domain binds a third Mn(2+) in a site created by a large shift in a previously unrecognized flap subdomain adjacent to the active site. Mutations in this site selectively increased the Michaelis constant for Mn(2+) in the reaction of a noncognate, small-molecule substrate, p-nitrophenyl phosphate. The PstP/Ppp structure reveals core functional motifs that advance the framework for understanding the mechanisms of substrate recognition, catalysis, and regulation of PP2C phosphatases. 相似文献
130.
A structural perspective on protein-protein interactions 总被引:1,自引:0,他引:1
Russell RB Alber F Aloy P Davis FP Korkin D Pichaud M Topf M Sali A 《Current opinion in structural biology》2004,14(3):313-324