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761.
Ekaterina V. Bobkova Michael J. Weber Zangwei Xu Yan-Ling Zhang Joon Jung Peter Blume-Jensen Alan Northrup Priya Kunapuli Jannik N. Andersen Ilona Kariv 《The Journal of biological chemistry》2010,285(24):18838-18846
The phosphoinositide 3-kinase/AKT signaling pathway plays a key role in cancer cell growth, survival, and angiogenesis. Phosphoinositide-dependent protein kinase-1 (PDK1) acts at a focal point in this pathway immediately downstream of phosphoinositide 3-kinase and PTEN, where it phosphorylates numerous AGC kinases. The PDK1 kinase domain has at least three ligand-binding sites: the ATP-binding pocket, the peptide substrate-binding site, and a groove in the N-terminal lobe that binds the C-terminal hydrophobic motif of its kinase substrates. Based on the unique PDK1 substrate recognition system, ultrahigh throughput TR-FRET and Alphascreen® screening assays were developed using a biotinylated version of the PDK1-tide substrate containing the activation loop of AKT fused to a pseudo-activated hydrophobic motif peptide. Using full-length PDK1, Km values were determined as 5.6 μm for ATP and 40 nm for the fusion peptide, revealing 50-fold higher affinity compared with the classical AKT(Thr-308)-tide. Kinetic and biophysical studies confirmed the PDK1 catalytic mechanism as a rapid equilibrium random bireactant reaction. Following an ultrahigh throughput screen of a large library, 2,000 compounds were selected from the reconfirmed hits by computational analysis with a focus on novel scaffolds. ATP-competitive hits were deconvoluted by dose-response studies at 1× and 10× Km concentrations of ATP, and specificity of binding was assessed in thermal shift assay. Inhibition studies using fusion PDK1-tide1 substrate versus AKT(Thr-308)-tide and kinase selectivity profiling revealed a novel selective alkaloid scaffold that evidently binds to the PDK1-interacting fragment pocket. Molecular modeling suggests a structural paradigm for the design of inhibitory versus activating allosteric ligands of PDK1. 相似文献
762.
Hima bindu Reddy Seerapu Geetha Priya Subramaniam Syamantak Majumder Swaraj Sinha Swathi Bisana Sahil Mahajan Gopi Krishna Kolluru Ajit Muley Jamila H. Siamwala Govindan Illavazagan Suvro Chatterjee 《Cell biology international》2010,34(7):755-761
Hypoxia induces barrier dysfunctions in endothelial cells. Nitric oxide is an autacoid signalling molecule that confers protection against hypoxia‐mediated barrier dysfunctions. Dyn‐2 (dynamin‐2), a large GTPase and a positive modulator of eNOS (endothelial nitric oxide synthase), plays an important role in maintaining vascular homeostasis. The present study aims to elucidate the role of dyn‐2 in hypoxia‐mediated leakiness of the endothelial monolayer in relation to redox milieu. Inhibition of dyn‐2 by transfecting the cells with K44A, a dominant negative construct of dyn‐2, elevated leakiness of the endothelial monolayer under hypoxia. Sodium nitroprusside (nitric oxide donor) and uric acid (peroxynitrite quencher) were used to evaluate the role of nitric oxide and peroxynitrite in maintaining endothelial barrier functions under hypoxia. Administration of nitric oxide and uric acid recovered hypoxia‐mediated leakiness of K44A‐overexpressed endothelial monolayer. Our study confirms that inhibition of dyn‐2 induces leakiness in the endothelial monolayer by increasing the load of peroxynitrite under hypoxia. 相似文献
763.
Ozlem Equils Priya Nambiar Calvin J. Hobel Roger Smith Charles F. Simmons Shireen Vali 《PloS one》2010,5(1)
Background
Sufficient information from in vitro and in vivo studies has become available to permit computer modeling of the processes that occur in the myometrium during labor. This development allows the in silico investigation of pathological mechanisms and the trialing of potential treatments.Methods/Results
Based on the human literature, we developed a computer model of the immune-endocrine environment of the myometrial cell. The interactions between molecules are represented by differential equations. The model is designed to simulate the estrogen and progesterone receptor changes during pregnancy and particularly the changes in the progesterone receptor (PR) isoforms A and B that are thought to mediate functional progesterone withdrawal in the human at labor. Parturition is represented by an increase in the PRA to PRB ratio to levels seen in women in labor. Infection is shown by inducing inflammation in the system by increasing phospho-IkB kinase concentration (IKK) levels; which lead to increased NF-κB activation, causing an increase in the PRA/PRB ratio. We examined the effects of progesterone or cyclo-oxygenase 2 (Cox2) inhibitor treatments on the PRA/PRB ratio in silico. The model predicted that high doses of progesterone and Cox2 inhibition would be effective in preventing an NF-κB-induced PRA/PRB ratio increase to the levels found during labor.Conclusions
Our data illustrate the use of dynamic biological computer simulations to test the effectiveness of therapeutic interventions. This may allow the early rejection of ineffective therapies prior to expensive field trials. 相似文献764.
Background
Henrich Klebahn was a German linguist, mycologist and phytopathologist, who was known as Dr. Dr. h. c. Henrich Klebahn, Hauptcustos a. D., Honorarprofessor an der Hanischen Universität. He was born February 20, 1859 in Bremen, and died October 5, 1942 in Hamburg. He taught linguistics from 1885-1899, studied Natural Science at the Universities of Jena and Berlin (1881) and received his PhD from the University of Jena. In 1899, he was appointed scientific assistant at the Hamburg botanical garden, where he worked until 1905. From 1905 to 1930, he was at the agricultural institute of Bromberg. In 1921, he was named honorary professor and lecturer in cryptogams and soil biology at the Institut für Allgemeine Botanik where he taught until 1934. He is well known for his work on gas vesicles and halophiles, among other topics. This re-print of 'Die Schädlinge des Klippfisches. Ein Beitrag zur Kenntnis der salzliebenden Organismen. Von H. Klebahn. Mit zwei Tafeln und vier Abbildungen im Text.' was originally published in 1919 in the Jahrbuch der Hamb. Wissensch. Anstaltes. XXXVI. Beiheft pages 11-69, by Latcke & Waltt, E. H. Buchdrucker. The translators have tried to remain faithful to the contents and to the original sense of the article by minimizing modifications.Results
The original paper reported the conclusions of a 3 year long study of the microbes causing damage to the fish industry as well as a summation of work on the subject up until 1919. The findings were that the causative agents were fungi and other microbes, the chief of which was a red, Gram-negative rod-shaped bacillus, Bacillus halobius ruber, that formed pale reddish colonies and was found to oscillate, but after extensive testing, was found not possess flagella. The initial appearance of "a shiny corpuscle" at the ends of cells was determined not to be spores; rather that it was the "result of the coherence of the light beams due to a total reflection of the light in the optically denser little rods". The cells were osmotically sensitive to the addition of water. In addition, a Gram-negative, red Sarcina morrhuae that appeared pinker in color, was less salt-sensitive than the red bacillus, in fact surviving the transfer to water. These were "round individual cells or groups of only two or four cells, usually; however, there are eight or more round cells that are arranged like cube corners to great cube-like or irregular packages lying together, just in the same manner as with the familiar Sarcina ventriculi." This organism was also identified from the walls of a fish storage room. Finally, a third, red microorganism was isolated: a Gram-negative micrococcus, Micrococcus (Diplococcus) morrhuae, which was "spherically rounded" and barely sensitive to water: "If one distributes a sample of a colony in water, the cells partly separate, to a great degree; however, they stay together in groups of two or four cells."Conclusions
This article provides evidence for identification of halophilic microbes as the major cause of fish spoilage, and is one of the earliest publications in the field of halophile microbiology. 相似文献765.
766.
Wang C Meek DJ Panchal P Boruvka N Archibald FS Driscoll BT Charles TC 《Applied and environmental microbiology》2006,72(1):384-391
The goal of this study was to initiate investigation of the genetics of bacterial poly-3-hydroxybutyrate (PHB) metabolism at the community level. We constructed metagenome libraries from activated sludge and soil microbial communities in the broad-host-range IncP cosmid pRK7813. Several unique clones were isolated from these libraries by functional heterologous complementation of a Sinorhizobium meliloti bdhA mutant, which is unable to grow on the PHB cycle intermediate D-3-hydroxybutyrate due to absence of the enzyme D-3-hydroxybutyrate dehydrogenase activity. Clones that conferred D-3-hydroxybutyrate utilization on Escherichia coli were also isolated. Although many of the S. meliloti bdhA mutant complementing clones restored D-3-hydroxybutyrate dehydrogenase activity to the mutant host, for some of the clones this activity was not detectable. This was also the case for almost all of the clones isolated in the E. coli selection. Further analysis was carried out on clones isolated in the S. meliloti complementation. Transposon mutagenesis to locate the complementing genes, followed by DNA sequence analysis of three of the genes, revealed coding sequences that were broadly divergent but lay within the diversity of known short-chain dehydrogenase/reductase encoding genes. In some cases, the amino acid sequence identity between pairs of deduced BdhA proteins was <35%, a level at which detection by nucleic acid hybridization based methods would probably not be successful. 相似文献
767.
Arruda LB Sim D Chikhlikar PR Maciel M Akasaki K August JT Marques ET 《Journal of immunology (Baltimore, Md. : 1950)》2006,177(4):2265-2275
Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes. 相似文献
768.
Petchi Iyappan M.Devi Bala M Sureshkumar Vishnu Priya Veeraraghavan Arulselvan Palanisamy 《Bioinformation》2021,17(1):181
Fucoxanthin (Fx) is an active compound commonly found in the many types of seaweed with numerous biological activities. The main goal of this investigation is to explore the effect of Fx against the cell proliferation, apoptotic induction and oxidative stress in the oral squamous (KB) cell line. Cytotoxicity of Fx was determined by MTT assay. The intracellular ROS production, mitochondrial membrane potential (MMP) and apoptosis induction in KB cells were examined through DCFH-DA, Rhodamine-123 and DAPI, and dual staining techniques. Effect of Fx on the antioxidant enzymes and lipid peroxidation in the KB cells was studied through the standard procedures. Fx treated KB cells showed morphological changes and reduced cell survival, which is exhibited by the cytotoxic activity of 50 µM/ml (IC50) Fx against the KB cells. The Fx treatment considerably induced the apoptotosis cells (EB/AO) and decreased the MMP (Rh-123) in KB cells. Further, it was pointed out that there was an increased lipid peroxidation (LPO) with decreased antioxidants (CAT, SOD and GSH). These results concluded that Fx has the cytotoxic effect against KB cells and has the potential to induce the apoptosis via increased oxidative stress. Hence, the Fx can be a promising agent for the treatment of oral cancer and it may lead to the development of cancer therapeutics. 相似文献
769.
Calcium control of waveform in isolated flagellar axonemes of chlamydomonas 总被引:29,自引:24,他引:5
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The effect of Ca(++) on the waveform of reactivated, isolated axonemes of chlamydomonas flagella was investigated. Flagella were detached and isolated by the dibucaine procedure and demembranated by treatment with the detergent Nonidet; the resulting axomenes lack the flagellar membrane and basal bodies. In Ca(++)-buffered reactivation solutions containing 10(-6) M or less free Ca(++), the axonemes beat with a highly asymmetrical, predominantly planar waveform that closely resembled that of in situ flagella of forward swimming cells. In solutions containing 10(-4) M Ca(++), the axonemes beat with a symmetrical waveform that was very similar to that of in situ flagella during backward swimming. In 10(-5) M Ca(++), the axonemes were predominantly quiescent, a state that appears to be closely associated with changes in axomenal waveform or direction of beat in many organisms. Experiments in which the concentrations of free Ca(++), not CaATP(--) complex were independently varied suggested that free Ca(++), not CaATP(--), was responsible for the observed changes. Analysis of the flagellar ATPases associated with the isolated axonemes and the nonidet- soluble membrane-matrix fraction obtained during preparation of the axonemes showed that the axonemes lacked the 3.0S Ca(++)-activated ATPase, almost all of which was recovered in the membrane-matrix fraction. These results indicate that free Ca(++) binds directly to an axonemal component to alter flagellar waveform, and that neither the 3.0S CaATPase nor the basal bodies are directly involved in this change. 相似文献
770.
Ping Jiang Steven Stone Roger Wagner Susan Wang Priya Dayananth Christine A. Kozak Barbara Wold Alexander Kamb 《Journal of molecular evolution》1995,41(6):795-802
Cyclin-dependent kinase inhibitors are a growing family of molecules that regulate important transitions in the cell cycle. At least one of these molecules, p16, has been implicated in human tumorigenesis while its close homolog, p15, is induced by cell contact and transforming growth factor- (TGF-). To investigate the evolutionary and functional features of p15 and p16, we have isolated mouse (Mus musculus) homologs of each gene. Comparative analysis of these sequences provides evidence that the genes have similar functions in mouse and human. In addition, the comparison suggests that a gene conversion event is part of the evolution of the human p15 and p16 genes.
Correspondence to: A. Kamb 相似文献