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  • 1
    Language: English
    In: Science (New York, N.Y.), 25 January 2013, Vol.339(6118), pp.446-8
    Description: The human genome contains ~50 genes that were derived from transposable elements or transposons, and many are now integral components of cellular gene expression programs. The human THAP9 gene is related to the Drosophila P-element transposase. Here, we show that human THAP9 can mobilize Drosophila P-elements in both Drosophila and human cells. Chimeric proteins formed between the Drosophila P-element transposase N-terminal THAP DNA binding domain and the C-terminal regions of human THAP9 can also mobilize Drosophila P elements. Our results indicate that human THAP9 is an active DNA transposase that, although "domesticated," still retains the catalytic activity to mobilize P transposable elements across species.
    Keywords: DNA Transposable Elements ; Transposases -- Genetics
    ISSN: 00368075
    E-ISSN: 1095-9203
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  • 2
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 28 May 2013, Vol.110(22), pp.8960-5
    Description: Establishment and maintenance of apico-basolateral trafficking pathways are critical to epithelial homeostasis. Loss of polarity and trafficking fidelity are thought to occur as a consequence of transformation; however, here we report that selective mistrafficking of the epidermal growth factor receptor (EGFR) ligand epiregulin (EREG) from the basolateral to the apical cell surface drives transformation. Normally, EREG is preferentially delivered to the basolateral surface of polarized Madin-Darby canine kidney cells. EREG basolateral trafficking is regulated by a conserved tyrosine-based basolateral sorting motif in its cytoplasmic domain (YXXΦ: Y(156)ERV). Both Y156 and V159 are required for basolateral sorting of EREG, because Y156A and V159G substitutions redirect EREG to the apical cell surface. We also show that basolateral sorting of EREG is adaptor protein 1B-independent. Apical mistrafficking of EREG has a distinctive phenotype. In contrast to transient EGFR tyrosine phosphorylation after basolateral EREG stimulation, apical EREG leads to prolonged EGFR tyrosine phosphorylation, which may be related, at least in part, to a lack of negative regulatory Y1045 phosphorylation and subsequent ubiquitylation. Notably, Madin-Darby canine kidney cells stably expressing apically mistrafficked EREG form significantly larger, hyperproliferative, poorly differentiated, and locally invasive tumors in nude mice compared with WT EREG-expressing cells.
    Keywords: Epithelial Transformation ; Growth Factor Trafficking ; Protein Sorting ; Receptor Tyrosine Kinase ; Cell Polarity -- Physiology ; Cell Transformation, Neoplastic -- Metabolism ; Epidermal Growth Factor -- Metabolism ; Epithelial Cells -- Physiology ; Signal Transduction -- Physiology
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 3
    Language: English
    In: Proceedings of the National Academy of Sciences of the United States of America, 04 March 2014, Vol.111(9), pp.3389-94
    Description: Proline utilization A (PutA) proteins are bifunctional peripheral membrane flavoenzymes that catalyze the oxidation of L-proline to L-glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. Located at the inner membrane of Gram-negative bacteria, PutAs play a major role in energy metabolism by coupling the oxidation of proline imported from the environment to the reduction of membrane-associated quinones. Here, we report seven crystal structures of the 1,004-residue PutA from Geobacter sulfurreducens, along with determination of the protein oligomeric state by small-angle X-ray scattering and kinetic characterization of substrate channeling and quinone reduction. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å-long tunnel that links the two active sites and six smaller tunnels that connect the main tunnel to the bulk medium. The locations of these tunnels and their responses to ligand binding and flavin reduction suggest hypotheses about how proline, water, and quinones enter the tunnel system and where L-glutamate exits. Kinetic measurements show that glutamate production from proline occurs without a lag phase, consistent with substrate channeling and implying that the observed tunnel is functionally relevant. Furthermore, the structure of reduced PutA complexed with menadione bisulfite reveals the elusive quinone-binding site. The benzoquinone binds within 4.0 Å of the flavin si face, consistent with direct electron transfer. The location of the quinone site implies that the concave surface of the PutA dimer approaches the membrane. Altogether, these results provide insight into how PutAs couple proline oxidation to quinone reduction.
    Keywords: X-Ray Crystallography ; Membrane Association ; Proline Catabolism ; Models, Molecular ; Protein Conformation ; Bacterial Proteins -- Chemistry ; Benzoquinones -- Metabolism ; Geobacter -- Enzymology ; Membrane Proteins -- Chemistry ; Metabolic Networks and Pathways -- Genetics
    ISSN: 00278424
    E-ISSN: 1091-6490
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  • 4
    Language: English
    In: Biophysical Journal, 05 September 2012, Vol.103(5), pp.1087-1096
    Description: Homogeneous cell populations can exhibit considerable cell-to-cell variability in protein levels arising from the stochastic nature of the gene-expression process. In particular, transcriptional bursting of mRNAs from the promoter has been implicated as a major source of stochasticity in the expression of many genes. In eukaryotes, transcribed pre-mRNAs have to be exported outside the nucleus and in many cases, export rates can be slow and comparable to mRNA turnover rates. We investigate whether such export processes can be effective mechanisms in buffering protein levels from transcriptional bursting of pre-mRNAs in the nucleus. For a stochastic gene-expression model with both transcriptional bursting and export, we derive an exact solution of the steady-state probability-generating function for both the nuclear and the cytoplasmic mRNA levels. These formulas reveal that decreasing export rates can dramatically reduce variability in cytoplasmic mRNA levels. However, our results also show that decreasing export rates enhance mRNA autocorrelation times, which function to increase heterogeneity in protein levels. Our overall analysis concludes that under physiologically relevant parameter regimes, a pre-mRNA export step can decrease steady-state variability at the mRNA level but not at the protein level. Finally, we reinforce previous observations that saturation in the pre-mRNA transport machinery can be an important mechanism in suppressing protein variability from underlying transcriptional bursts.
    Keywords: Biology
    ISSN: 0006-3495
    E-ISSN: 1542-0086
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  • 5
    In: Journal of Applied Microbiology, March 2014, Vol.116(3), pp.654-666
    Description: Byline: B.N. Singh, A. Singh, B.R. Singh, H.B. Singh Keywords: defence responses; lignin; phenolics; Rhizoctonia ; sunflower; Trichoderma harzianum NBRI-1055 Abstract Aims To investigate the efficacy of Trichoderma harzianum NBRI-1055 (denoted as 'T-1055') in suppression of seedling blight of sunflower caused by Rhizoctonia solani Kuhn and their impact on host defence responses. Methods and Results T-1055 was applied as seed treatment, soil application and combined application (seed treatment + soil application). Higher protection afforded by combined application of T-1055 was associated with the marked induction of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO), peroxidase (PO) and cinnamyl alcohol dehydrogenase (CAD) activities. The activities of PAL and PPO reached maximum at 10 days after sowing (DAS), while PO and CAD levels reached maximum at 12 DAS. This was further supported by the accumulation of total phenolic content that showed an increase up to threefold at 14 DAS. In addition, HPLC analysis revealed that the contents of ferulic and p-coumaric acids increased by 6ae3 and 4ae6 times, respectively, at 14 DAS. Amount of gallic acid was also little more than double. Lignin deposition in sunflower root increased by 2ae7, 3ae4 and 3ae7 times through combined application of T-1055 at 16, 18 and 20 DAS, respectively. Combined application also increased the accumulation of PR-2 and PR-3 proteins by 3ae3 and 3ae9 times, respectively, at 12 DAS in followed by seed treatment alone. Conclusions The combined application of T-1055 triggered defence responses in an enhanced level in sunflower than the soil and seed alone and provided better protection against Rhizoctonia seedling blight. Significance and Impact of the Study Rhizospheric fungal bioagent 'T-1055' can enhance protection in sunflower against the R. solani pathogen through augmented elicitation of host defence responses.
    Keywords: Defence Responses ; Lignin ; Phenolics ; Rhizoctonia ; Sunflower ; ‐1055
    ISSN: 1364-5072
    E-ISSN: 1365-2672
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  • 6
    Language: English
    In: Journal of the American Chemical Society, 12 February 2014, Vol.136(6), pp.2575-82
    Description: Enzyme catalysis has been studied extensively, but the role of enzyme dynamics in the catalyzed chemical conversion is still an enigma. The enzyme dihydrofolate reductase (DHFR) is often used as a model system to assess a network of coupled motions across the protein that may affect the catalyzed chemical transformation. Molecular dynamics simulations, quantum mechanical/molecular mechanical studies, and bioinformatics studies have suggested the presence of a "global dynamic network" of residues in DHFR. Earlier studies of two DHFR distal mutants, G121V and M42W, indicated that these residues affect the chemical step synergistically. While this finding was in accordance with the concept of a network of functional motions across the protein, two residues do not constitute a network. To better define the extent and limits of the proposed network, the current work studied two remote residues predicted to be part of the same network: W133 and F125. The effect of mutations in these residues on the nature of the chemical step was examined via measurements of the temperature-dependence of the intrinsic kinetic isotope effects (KIEs) and other kinetic parameters, and double mutants were used to tie the findings to G121 and M42. The findings indicate that residue F125, which was implicated by both calculations and bioinformatic methods, is a part of the same global dynamic network as G121 and M42, while W133, implicated only by bioinformatics, is not. These findings extend our understanding of the proposed network and the relations between functional and genomic couplings. Delineating that network illuminates the need to consider remote residues and protein structural dynamics in the rational design of drugs and of biomimetic catalysts.
    Keywords: Tetrahydrofolate Dehydrogenase -- Chemistry
    ISSN: 00027863
    E-ISSN: 1520-5126
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  • 7
    Language: English
    In: Journal of the American Chemical Society, 24 April 2013, Vol.135(16), pp.6184-91
    Description: Genetically encoded methods for protein conjugation are of high importance as biological tools. Here we describe the development of a new class of dyes for genetically encoded tagging that add new capabilities for protein reporting and detection via HaloTag methodology. Oligodeoxyfluorosides (ODFs) are short DNA-like oligomers in which the natural nucleic acid bases are replaced by interacting fluorescent chromophores, yielding a broad range of emission colors using a single excitation wavelength. We describe the development of an alkyl halide dehalogenase-compatible chloroalkane linker phosphoramidite derivative that enables the rapid automated synthesis of many possible dyes for protein conjugation. Experiments to test the enzymatic self-conjugation of nine different DNA-like dyes to proteins with HaloTag domains in vitro were performed, and the data confirmed the rapid and efficient covalent labeling of the proteins. Notably, a number of the ODF dyes were found to increase in brightness or change color upon protein conjugation. Tests in mammalian cellular settings revealed that the dyes are functional in multiple cellular contexts, both on the cell surface and within the cytoplasm, allowing protein localization to be imaged in live cells by epifluorescence and laser confocal microscopy.
    Keywords: DNA -- Chemistry ; Fluorescent Dyes -- Chemistry ; Hydrolases -- Chemistry ; Proteins -- Chemistry
    ISSN: 00027863
    E-ISSN: 1520-5126
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  • 8
    Language: English
    In: Biophysical Journal, 04 November 2014, Vol.107(9), pp.2214-2220
    Description: Protein levels differ considerably between otherwise identical cells, and these differences significantly affect biological function and phenotype. Previous work implicated various noise mechanisms that drive variability in protein copy numbers across an isogenic cell population. For example, transcriptional bursting of mRNAs has been shown to be a major source of noise in the expression of many genes. Additional expression variability, referred to as extrinsic noise, arises from intercellular variations in mRNA transcription and protein translation rates attributed to cell-to-cell differences in cell size, abundance of ribosomes, etc. We propose a method to determine the magnitude of different noise sources in a given gene of interest. The method relies on blocking transcription and measuring changes in protein copy number variability over time. Our results show that this signal has sufficient information to quantify both the extent of extrinsic noise and transcription bursting in gene expression. Moreover, if the mean mRNA count is known, then the relative contributions of transcription versus translation rate fluctuations to extrinsic noise can also be determined. In summary, our study provides an easy-to-implement method for characterizing noisy protein expression that complements existing techniques for studying stochastic dynamics of genetic circuits.
    Keywords: Biology
    ISSN: 0006-3495
    E-ISSN: 1542-0086
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  • 9
    In: PLoS ONE, 2014, Vol.9(7)
    Description: Salicornia brachiata is an extreme halophyte that grows luxuriantly in coastal marshes. Previously, we have reported isolation and characterization of ESTs from Salicornia with large number of novel/unknown salt-responsive gene sequences. In this study, we have selected a novel salt-inducible gene SbSI-2 ( S alicornia b rachiata s alt- i nducible-2 ) for functional characterization. Bioinformatics analysis revealed that SbSI-2 protein has predicted nuclear localization signals and a strong protein-protein interaction domain. Transient expression of the RFP:SbSI2 fusion protein confirmed that SbSI-2 is a nuclear-localized protein. Genomic organization study showed that SbSI-2 is intronless and has a single copy in Salicornia genome. Quantitative RT-PCR analysis revealed higher SbSI-2 expression under salt stress and desiccation conditions. The SbSI-2 gene was transformed in E. coli and tobacco for functional characterization. pET28a-SbSI-2 recombinant E. coli cells showed higher tolerance to desiccation and salinity compared to vector alone. Transgenic tobacco plants overexpressing SbSI-2 have improved salt- and osmotic tolerance, accompanied by better growth parameters, higher relative water content, elevated accumulation of compatible osmolytes, lower Na + and ROS accumulation and lesser electrolyte leakage than the wild-type. Overexpression of the SbSI-2 also enhanced transcript levels of ROS-scavenging genes and some stress-related transcription factors under salt and osmotic stresses. Taken together, these results demonstrate that SbSI-2 might play an important positive modulation role in abiotic stress tolerance. This identifies SbSI-2 as a novel determinant of salt/osmotic tolerance and suggests that it could be a potential bioresource for engineering abiotic stress tolerance in crop plants.
    Keywords: Research Article ; Biology And Life Sciences
    E-ISSN: 1932-6203
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  • 10
    Language: English
    In: Biomaterials, August 2012, Vol.33(23), pp.5753-5767
    Description: Cadmium sulfide (CdS) quantum dots (QDs) have raised great attention because of their superior optical properties and wide utilization in biological and biomedical studies. However, little is known about the cell death mechanisms of CdS QDs in human cancer cells. This study was designed to investigate the possible mechanisms of apoptosis induced by biosurfactant stabilized CdS QDs (denoted as “bsCdS QDs”) in human prostate cancer LNCaP cells. It was also noteworthy that apoptosis correlated with reactive oxygen species (ROS) production, mitochondrial damage, oxidative stress and chromatin condensation in a dose- and time-dependent manner. Results also showed involvement of caspases, Bcl-2 family proteins, heat shock protein 70, and a cell-cycle checkpoint protein p53 in apoptosis induction by bsCdS QDs in LNCaP cells. Moreover, pro-apoptotic protein Bax was upregulated and the anti-apoptotic proteins, survivin and NF-κB were downregulated in bsCdS QDs exposed cells. Protection of N-acetyl cysteine (NAC) against ROS clearly suggested the implication of ROS in hyper-activation of apoptosis and cell death. It is encouraging to conclude that biologically stabilized CdS QDs bear the potential of its applications in biomedicine, such as tumor therapy specifically by inducing caspase-dependent apoptotic cell death of human prostate cancer LNCaP cells.
    Keywords: Cds Quantum Dots ; Apoptosis ; Ros ; Lncap Cells ; Medicine ; Engineering
    ISSN: 0142-9612
    E-ISSN: 1878-5905
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