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91.
A family of modular type mannuronan C-5-epimerase genes controls alginate structure in Azotobacter vinelandii 总被引:2,自引:1,他引:2
Helga Ertesvåg Hilde Kristin Høidal Ingrid Kathrin Hals Anne Rian Berit Doseth Svein Valla 《Molecular microbiology》1995,16(4):719-731
The ToxR protein is a transmembrane protein that regulates the expression of several virulence factors of Vibrio cholerae. Previous analysis of fusion proteins between ToxR and alkaline phosphatase (ToxR-PhoA) suggested that ToxR was active as a dimer. In order to determine whether dimerization of the ToxR periplasmic domain was essential for activity, this domain was replaced by monomeric and dimeric protein domains. Surprisingly, PhoA (dimeric), β-lactamase (monomeric, ToxR–Bla), or the leucine zipper of GCN4 (dimeric, ToxR-GCN4-M) could substitute functionally for the ToxR periplasmic domain. ToxR-GCN4 fusion proteins, in which the ToxR trans-membrane domain was eliminated (ToxR-GCN4-C), were inactive, but an additional fusion protein that contained a heterologous membrane-spanning domain retained activity. Strains containing each of these ToxR fusion proteins were analysed for in vivo colonization properties and response to in vitro growth conditions that are known to affect expression of the ToxR regulon. Strains containing ToxR-GCN4-M and ToxR-Bla responded like wild-type strains to in vitro growth conditions. In the infant-mouse colonization model, strains containing ToxR fusion proteins were all deficient in colonization relative to strains containing wild-type ToxR, and strains containing monomeric ToxR-Bla were most severely outcompeted. These results suggest that, under in vitro conditions, ToxR does not require a dimerized periplasmic domain, but that, under in vivo conditions, the correct conformation of the ToxR periplasmic domain may be more important for function. 相似文献
92.
Elisabeth J. Leehr Kathrin Schag Amelie Brinkmann Ann-Christine Ehlis Andreas J. Fallgatter Stephan Zipfel Katrin E. Giel Thomas Dresler 《PloS one》2016,11(4)
ObjectiveFood stimuli are omnipresent and naturally primary reinforcing stimuli. One explanation for the intake of high amounts of food in binge eating disorder (BED) is a deviant valuation process. Valuation of food stimuli is supposed to influence approach or avoidance behaviour towards food. Focusing on self-reported and indirect (facial electromyography) valuation process, motivational aspects in the processing of food stimuli were investigated.MethodsWe compared an overweight sample with BED (BED+) with an overweight sample without BED (BED-) and with normal weight controls (NWC) regarding their self-reported and indirect (via facial electromyography) valuation of food versus non-food stimuli.ResultsRegarding the self-reported valuation, the BED+ sample showed a significantly stronger food-bias compared to the BED- sample, as food stimuli were rated as significantly more positive than the non-food stimuli in the BED+ sample. This self-reported valuation pattern could not be displayed in the indirect valuation. Food stimuli evoked negative indirect valuation in all groups. The BED+ sample showed the plainest approach-avoidance conflict marked by a diverging self-reported (positive) and indirect (negative) valuation of food stimuli.ConclusionsBED+ showed a deviant self-reported valuation of food as compared to BED-. The valuation process of the BED+ sample seems to be characterized by a motivational ambivalence. This ambivalence should be subject of further studies and may be of potential use for therapeutic interventions. 相似文献
93.
Astrid?Adrian Kathrin?Schoppmann Juri?Sromicki Sonja?Brungs Melanie?von der Wiesche Bertold?Hock Waldemar?Kolanus Ruth?Hemmersbach Oliver?Ullrich
Gravity has been a constant force throughout the Earth’s evolutionary history. Thus, one of the fundamental biological questions is if and how complex cellular and molecular functions of life on Earth require gravity. In this study, we investigated the influence of gravity on the oxidative burst reaction in macrophages, one of the key elements in innate immune response and cellular signaling. An important step is the production of superoxide by the NADPH oxidase, which is rapidly converted to H2O2 by spontaneous and enzymatic dismutation. The phagozytosis-mediated oxidative burst under altered gravity conditions was studied in NR8383 rat alveolar macrophages by means of a luminol assay. Ground-based experiments in “functional weightlessness” were performed using a 2 D clinostat combined with a photomultiplier (PMT clinostat). The same technical set-up was used during the 13th DLR and 51st ESA parabolic flight campaign. Furthermore, hypergravity conditions were provided by using the Multi-Sample Incubation Centrifuge (MuSIC) and the Short Arm Human Centrifuge (SAHC). The results demonstrate that release of reactive oxygen species (ROS) during the oxidative burst reaction depends greatly on gravity conditions. ROS release is 1.) reduced in microgravity, 2.) enhanced in hypergravity and 3.) responds rapidly and reversible to altered gravity within seconds. We substantiated the effect of altered gravity on oxidative burst reaction in two independent experimental systems, parabolic flights and 2D clinostat / centrifuge experiments. Furthermore, the results obtained in simulated microgravity (2D clinorotation experiments) were proven by experiments in real microgravity as in both cases a pronounced reduction in ROS was observed. Our experiments indicate that gravity-sensitive steps are located both in the initial activation pathways and in the final oxidative burst reaction itself, which could be explained by the role of cytoskeletal dynamics in the assembly and function of the NADPH oxidase complex. 相似文献
94.
Schmid M Kindsmüller K Wimmer P Groitl P Gonzalez RA Dobner T 《Journal of virology》2011,85(14):7081-7094
The adenovirus type 5 (Ad5) E1B-55K and E4orf6 (E1B-55K/E4orf6) proteins are multifunctional regulators of Ad5 replication, participating in many processes required for virus growth. A complex containing the two proteins mediates the degradation of cellular proteins through assembly of an E3 ubiquitin ligase and induces shutoff of host cell protein synthesis through selective nucleocytoplasmic viral late mRNA export. Both proteins shuttle between the nuclear and cytoplasmic compartments via leucine-rich nuclear export signals (NES). However, the role of their NES-dependent export in viral replication has not been established. It was initially shown that mutations in the E4orf6 NES negatively affect viral late gene expression in transfection/infection complementation assays, suggesting that E1B-55K/E4orf6-dependent viral late mRNA export involves a CRM1 export pathway. However, a different conclusion was drawn from similar studies showing that E1B-55K/E4orf6 promote late gene expression without active CRM1 or functional NES. To evaluate the role of the E1B-55K/E4orf6 NES in viral replication in the context of Ad-infected cells and in the presence of functional CRM1, we generated virus mutants carrying amino acid exchanges in the NES of either or both proteins. Phenotypic analyses revealed that mutations in the NES of E1B-55K and/or E4orf6 had no or only moderate effects on viral DNA replication, viral late protein synthesis, or viral late mRNA export. Significantly, such mutations also did not interfere with the degradation of cellular substrates, indicating that the NES of E1B-55K or E4orf6 is dispensable both for late gene expression and for the activity associated with the E3 ubiquitin ligase. 相似文献
95.
Michael Marina Kathrin Ulrich Alexander Martin Christoph 《Journal of electromyography and kinesiology》2009,19(5):e353-e361
The aim of this study was to investigate the cervicocephalic kinaesthesia of healthy subjects for gender and age effects and its reliability in a new virtual reality test procedure. 57 healthy subjects (30 male, 27 females; 18-64 years) were immersed into a virtual 3D scene via a headmounted display, which generated specific head movements. The joint repositioning error was determined in a static and dynamic test at the times T0, T1 (T0 + 10 minutes) and T2 (T0 + 24 hours). The intrasession reliability (T0-T1) and the intersession reliability (T0-T2) were analysed. In both tests no gender- or age-specific effects were found. In the overall group the means of the static test were 6.2°-6.9° and of the dynamic test were 4.5°-4.9°. The intratest difference in the static test was -0.16° and the intertest difference was 0.47°. The intratest difference in the dynamic test was 0.42° and the intertest difference was 0.37°. The static and dynamic test was reproducible in healthy subjects, with minor deviations, irrespective of gender and age. The smaller interindividual differences in the dynamic test could be beneficial in the comparison of healthy individuals and individuals with cervical spine disorders. 相似文献
96.
Erben RG Odörfer KI Siebenhütter M Weber K Rohleder S 《Histochemistry and cell biology》2008,130(5):1041-1046
The assessment of cellular half-life is of fundamental importance for cell biology and biomedicine. Here, we show that cellular
half-life in tissues can be histologically measured under steady state conditions in vivo by analyzing the loss of 5-bromo-2′-deoxyuridine
(BrdU)-labeled cells over time after withdrawal of long-term BrdU labeling. To achieve efficient continuous cell labeling,
we implanted BrdU-containing subcutaneous slow-release pellets into 12-month-old male Fischer 344 rats, delivering BrdU at
a dose of 75 mg/kg per day over 1 (n = 20) or 3 weeks (n = 20). Four to five rats each were killed directly after the labeling or 1, 3, and 7 weeks post-labeling. Cellular half-life
after withdrawal of BrdU was analyzed by nonlinear regression analysis of the labeling index, using a model of one-phase exponential
decay. We initially validated our technique in the duodenum, where we determined a half-life of 2.4 days for crypt cells.
Next, we applied this method to other tissues, and found a half-life of 2.2 weeks for cardiac endothelial cells, and of 5–6
days for pancreatic duct cells. In conclusion, we believe that this novel approach is an important step forward in the histological
assessment of cellular half-life. 相似文献
97.
HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials. 相似文献
98.
Li Y Laue K Temtamy S Aglan M Kotan LD Yigit G Canan H Pawlik B Nürnberg G Wakeling EL Quarrell OW Baessmann I Lanktree MB Yilmaz M Hegele RA Amr K May KW Nürnberg P Topaloglu AK Hammerschmidt M Wollnik B 《American journal of human genetics》2010,87(6):757-767
Altered Bone Morphogenetic Protein (BMP) signaling leads to multiple developmental defects, including brachydactyly and deafness. Here we identify chondroitin synthase 1 (CHSY1) as a potential mediator of BMP effects. We show that loss of human CHSY1 function causes autosomal-recessive Temtamy preaxial brachydactyly syndrome (TPBS), mainly characterized by limb malformations, short stature, and hearing loss. After mapping the TPBS locus to chromosome 15q26-qterm, we identified causative mutations in five consanguineous TPBS families. In zebrafish, antisense-mediated chsy1 knockdown causes defects in multiple developmental processes, some of which are likely to also be causative in the etiology of TPBS. In the inner ears of zebrafish larvae, chsy1 is expressed similarly to the BMP inhibitor dan and in a complementary fashion to bmp2b. Furthermore, unrestricted Bmp2b signaling or loss of Dan activity leads to reduced chsy1 expression and, during epithelial morphogenesis, defects similar to those that occur upon Chsy1 inactivation, indicating that Bmp signaling affects inner-ear development by repressing chsy1. In addition, we obtained strikingly similar zebrafish phenotypes after chsy1 overexpression, which might explain why, in humans, brachydactyly can be caused by mutations leading either to loss or to gain of BMP signaling. 相似文献
99.
Roberts BR Tainer JA Getzoff ED Malencik DA Anderson SR Bomben VC Meyers KR Karplus PA Beckman JS 《Journal of molecular biology》2007,373(4):877-890
Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations. 相似文献