115 research outputs found

    Mechanistic Insights into the Differential Catalysis by RheB and Its Mutants: Y35A and Y35A-D65A

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    RheB GTPase is a Ras-related molecular switch, which regulates the mTOR signaling pathway by cycling between the active [guanosine triphosphate (GTP)] state and inactive [guanine diphosphate (GDP)] state. Impairment of GTPase activity because of mutations in several small GTPases is known to be associated with several cancers. The conventional GTPase mechanism such as in H-Ras requires a conserved glutamine (Q64) in the switch-II region of RheB to align the catalytic water molecule for efficient GTP hydrolysis. The conformation of this conserved glutamine is different in RheB, resulting in an altered conformation of the entire switch-II region. Studies on the atypical switch-II conformation in RheB revealed a distinct, noncanonical mode of GTP hydrolysis. An RheB mutant Y35A was previously shown to exclusively enhance the intrinsic GTPase activity of RheB, whereas the Y35A-D65A double mutant was shown to reduce the elevated GTPase activity. Here, we have used all-atom molecular dynamics (MD) simulations for comprehensive understanding of the conformational dynamics associated with the fast (Y35A) and slow (Y35A-D65A) hydrolyzing mutants of RheB. Using a combination of starting models from PDB structures and in-silico generated mutant structures, we discuss the observed conformational deviations in wild type (WT) versus mutants. Our results show that a number of interactions of RheB with phosphates of GTP as well as Mg<sup>2+</sup> are destabilized in Y35A mutant in the switch-I region. We report distinct water dynamics at the active site of WT and mutants. Furthermore, principal component analysis showed significant differences in the conformational space sampled by the WT and mutants. Our observations provide improved understanding of the noncanonical GTP hydrolysis mechanism adopted by RheB and its modulation by Y35A and Y35A-D65A mutants

    Uptake of mycobacteria by AEC and PuM.

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    <p>Isolated AEC (A and B) and PuM (C and D) were cultured on cover glass at a concentration of 1×10<sup>5</sup> cells per well as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032125#s4" target="_blank">Material and Methods</a>. After overnight culture in medium without antibiotics, the cells were infected with GFP-BCG at a MOI of 1∶100 (cell∶bacteria) for 4 h at 37°C in RPMI medium without antibiotics. To kill all extracellular remained bacteria, the cells were treated with gentamicin for 1 h, washed 3 times and finally incubated for 72 h in RPMI without antibiotics. After that, the cover glasses with infected cells were fixed, mounted and observed under white light (A and C) and green light (B and D) in a fluorescence microscope. Magnification 1000×.</p

    Antigen presentation by AEC.

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    <p>IFN-γ levels after <i>in vitro</i> restimulation of splenocytes from Ag19 kDa immunized mice. As APC we used AEC (AEC<sub>19 kDa</sub>) and PuM (PuM<sub>19 kDa</sub>) pulsed with Ag19 kDa as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032125#s4" target="_blank">Materials and Methods</a>. One week after the last immunization with Ag19 kDa, mice were sacrificed and splenocytes from immunized and unimmunized mice were co-cultured with AEC<sub>19 kDa</sub> and PuM<sub>19 kDa</sub> for 72 h. Data are expressed as mean ± SD from 3 mice per group. A representative of two independent experiments is shown. * represents levels significantly different from unimmunized control, p<0.05.</p

    MMP-9 production by primary cells.

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    <p>Data are expressed in pg/ml of cytokine produced by primary cells. A representative experiment from five independent experiments is shown. Values are given as mean ± SD n = 3–4. ND: not detected.</p

    Chemokine production by primary AEC and PuM after <i>in vitro</i> stimulation.

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    <p>Data are expressed in pg/ml of cytokine produced. A representative experiment from five independent experiments is shown. Values are given as mean ± SD n = 3–4. ND: not detected.</p

    Gene expression in PPDB-stimulated PBMC from BCG vaccinated and control cattle.

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    <p>A. Protective efficacy after <i>M. bovis</i> challenge determined by pathology scoring. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003077#s2" target="_blank">Results</a> are expressed as total pathology scores <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003077#ppat.1003077-Vordermeier1" target="_blank">[5]</a>. Filled circles, unvaccinated control calves; open triangles, BCG vaccinated calves. (B, C). Transcription of the genes expressing IFN-γ (B) and IL-22 (C) following <i>in vitro</i> stimulation. PBMC were collected from BCG vaccinated (filled symbols) and controls (open symbols) before challenge (week -1) and after challenge with <i>M. bovis</i> at weeks 2, 4 and 8, and stimulated with PPD-B for 24 hours. cDNA was prepared and gene expression determined by RT-qPCR. Data are expressed as log10 relative expression levels compared to non-stimulated cells. Statistical analysis: 2-way ANOVA with Bonferroni post test, * P<0.05.</p

    BCG-vaccinated and control cattle samples mapped to <i>ifn-γ</i> and <i>il-22</i> genes.

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    <p>Visualization by IGB of RNA sequencing reads of representative PPD-B stimulated PBMC from vaccinated-protected, vaccinate-un-protected and non-vaccinated control cattle. Y-axis shows the number of reads covering each base along the transcript in RPKM expression values for each sample. Black track: unvaccinated control cattle; red track: vaccinated/un-protected cattle and blue track: vaccinated/protected cattle. The schematic representation of transcript for (A) <i>ifn-γ</i> and (B) <i>il-22</i> is show in green at the bottom of the each figure; the boxes show the exons of the gene.</p

    Flow cytometry analysis of freshly isolated AEC from mouse lungs.

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    <p>Total lung cells were obtained by using Corti's protocol with some modifications as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032125#s4" target="_blank">Material and Methods</a>. Leukocytes were first depleted with anti-CD45 microbeads and subsequently, the CD45<b><sup>−</sup></b> cells were depleted of contaminating endothelial cells using anti-CD146 microbeads. The remaining CD45<sup>−</sup>CD146<sup>−</sup> cells were considered to be AEC. These cells were fixed and stained intracellularly with antibodies to CD74 (AEC II marker) and podoplanin (T1α) (AEC I marker). A representative dot plot of flow cytometry analysis of CD74 and T1α expression in freshly isolated AEC from three independent experiments is shown. Percentage numbers represent gated cells from total cells.</p

    Results of RNA-Seq analysis.

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    <p>A. Signficantly modulated genes in the three treatment groups. B. Venn diagrams of genes significantly up-regulated and (C) down-regulated genes after vaccination but prior to <i>M. bovis</i> challenge. A. Fold change compared to unstimulated PBMC (medium controls) of PPD-B stimulated PBMC compared to medium controls from unvaccinated, naïve calves (group 1), vaccinated/non-protected (group 2), and vaccinated/protected calves (group 3).</p

    Cytokine/chemokine profiling on supernatants from AEC.

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    <p>AEC (5×10<sup>4</sup> cells per well) were stimulated with the indicated stimuli for 24 h. Supernatants were incubated on membranes from a mouse Cytokine Array Panel A Proteome Profiler kit (R&D systems) according to the manufacturer's instructions. The figure shows a+representing a spot with similar or greater density than control spots, whereas +/− indicates a spot with lower density than control spots.</p
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