Готові списки джерел за темами / Molecular Modifications

Добірка наукової літератури з теми "Molecular Modifications"

Автор: Grafiati

Опубліковано: 6 вересня 2023

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Статті в журналах з теми "Molecular Modifications"

Rehpenn, Andreas, Alexandra Walter, and Golo Storch. "Molecular Editing of Flavins for Catalysis." Synthesis 53, no. 15 (March 22, 2021): 2583–93. http://dx.doi.org/10.1055/a-1458-2419.

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AbstractThe diverse activity of flavoenzymes in organic transformations has fascinated researchers for a long time. However, when applied outside an enzyme environment, the isolated flavin cofactor only shows largely reduced activity. This highlights the importance of embedding the reactive isoalloxazine core of flavins in defined surroundings. The latter include crucial non-covalent interactions with amino acid side chains or backbone as well as controlled access to reactants such as molecular oxygen. Nevertheless, molecular flavins are increasingly applied in the organic laboratory as valuable organocatalysts. Chemical modification of the parent isoalloxazine structure is of particular interest in this context in order to achieve reactivity and selectivity in transformations, which are so far only known with flavoenzymes or even unprecedented. This review aims to give a systematic overview of the reported designed flavin catalysts and highlights the impact of each structural alteration. It is intended to serve as a source of information when comparing the performance of known catalysts, but also when designing new flavins. Over the last few decades, molecular flavin catalysis has emerged from proof-of-concept reactions to increasingly sophisticated transformations. This stimulates anticipating new flavin catalyst designs for solving contemporary challenges in organic synthesis.1 Introduction2 N1-Modification3 N3-Modification4 N5-Modification5 C6–C9-Modification6 N10-Modification7 Conclusion

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Li, Yinglu, Zhiming Li, and Wei-Guo Zhu. "Molecular Mechanisms of Epigenetic Regulators as Activatable Targets in Cancer Theranostics." Current Medicinal Chemistry 26, no. 8 (May 16, 2019): 1328–50. http://dx.doi.org/10.2174/0929867324666170921101947.

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Epigenetics is defined as somatically inheritable changes that are not accompanied by alterations in DNA sequence. Epigenetics encompasses DNA methylation, covalent histone modifications, non-coding RNA as well as nucleosome remodeling. Notably, abnormal epigenetic changes play a critical role in cancer development including malignant transformation, metastasis, prognosis, drug resistance and tumor recurrence, which can provide effective targets for cancer prognosis, diagnosis and therapy. Understanding these changes provide effective means for cancer diagnosis and druggable targets for better clinical applications. Histone modifications and related enzymes have been found to correlate well with cancer incidence and prognosis in recent years. Dysregulated expression or mutation of histone modification enzymes and histone modification status abnormalities have been considered to play essential roles in tumorigenesis and clinical outcomes of cancer treatment. Some of the histone modification inhibitors have been extensively employed in clinical practice and many others are still under laboratory research or pre-clinical assessment. Here we summarize the important roles of epigenetics, especially histone modifications in cancer diagnostics and therapeutics, and also discuss the developmental implications of activatable epigenetic targets in cancer theranostics.

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Eichler, Jerry, and Michael W. W. Adams. "Posttranslational Protein Modification in Archaea." Microbiology and Molecular Biology Reviews 69, no. 3 (September 2005): 393–425. http://dx.doi.org/10.1128/mmbr.69.3.393-425.2005.

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SUMMARY One of the first hurdles to be negotiated in the postgenomic era involves the description of the entire protein content of the cell, the proteome. Such efforts are presently complicated by the various posttranslational modifications that proteins can experience, including glycosylation, lipid attachment, phosphorylation, methylation, disulfide bond formation, and proteolytic cleavage. Whereas these and other posttranslational protein modifications have been well characterized in Eucarya and Bacteria, posttranslational modification in Archaea has received far less attention. Although archaeal proteins can undergo posttranslational modifications reminiscent of what their eucaryal and bacterial counterparts experience, examination of archaeal posttranslational modification often reveals aspects not previously observed in the other two domains of life. In some cases, posttranslational modification allows a protein to survive the extreme conditions often encountered by Archaea. The various posttranslational modifications experienced by archaeal proteins, the molecular steps leading to these modifications, and the role played by posttranslational modification in Archaea form the focus of this review.

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Winter, Stefan, and Wolfgang Fischle. "Epigenetic markers and their cross-talk." Essays in Biochemistry 48 (September 20, 2010): 45–61. http://dx.doi.org/10.1042/bse0480045.

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Post-translational modifications of histone proteins in conjunction with DNA methylation represent important events in the regulation of local and global genome functions. Advances in the study of these chromatin modifications established temporal and spatial co-localization of several distinct ‘marks’ on the same histone and/or the same nucleosome. Such complex modification patterns suggest the possibility of combinatorial effects. This idea was originally proposed to establish a code of histone modifications that regulates the interpretation of the genetic code of DNA. Indeed, interdependency of different modifications is now well documented in the literature. Our current understanding is that the function of a given histone modification is influenced by neighbouring or additional modifications. Such context sensitivity of the readout of a modification provides more flexible translation than would be possible if distinct modifications function as isolated units. The mechanistic principles for modification cross-talk can originate in the modulation of the activity of histone-modifying enzymes or may be due to selective recognition of these marks via modification of specific binding proteins. In the present chapter, we discuss fundamental biochemical principles of modification cross-talk and reflect on the interplay of chromatin marks in cellular signalling, cell-cycle progression and cell-fate determination.

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Chukwuma Sr, Chrysanthus. "Characterization of the Clinical and Molecular Perspectives of Epigenetics." Archives of Clinical Investigation 1, no. 1 (October 17, 2022): 01–07. http://dx.doi.org/10.31579/2834-8087/003.

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This review provides the latitude to examine the extant information in the univeral characterizations of epigenetic formulations. Epigenetics encompasses the interaction of behaviours and environment culminating in changes which influence gene activity. In contrast to genetic modifications, epigenetic modifications are reversible; and do not alter the DNA sequence but are capable of interferring in the way the DNA sequence is read. Epigenetic alterations involve genetic changes which effect gene functionality without modifying the underlying DNA sequence. DNA methylation depicts the covalent superimposed methyl group to cytosine in CpG dinucleotides. DNA methylation presents as a veritable epigenetic modification; and it governs gene expression by changing chromosome structure, DNA conformation and stability as well as the function trajectory between DNA and protein. DNA methylation regulates gene expression via the conscription of proteins associated with gene expression or by the inhibition of the binding of transcription factor(s) to DNA. Whereas genetic alterations do modify protein formation, it is clear that epigenetic alterations impact on gene expression to put genes ''on'' and ''off'', as appropriate. The resultant impact of environmental and anthropogenic idiosyncracies, such as diet and physical activity are liable to induce epigenetic modifications in behaviours and gene-environment interactions. Genes are not always in functional mode. DNA methylation constitutes a select epigenetic process applied by cells for the control of gene expression. within the genome. Alterations in gene activity and epigenetic errors can result in varying genetic, metabolic and degenerative disorders which may disparately or in comorbid presentaions influence inter alia health, gene activity or expression, protein production and functionality. This entry exemplifies the reading and understanding of epigenetics in relation to inter alia beneficial developmental theories within the human race.

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Miki, Keishu, Takeshi Watanabe, and Shinji Koh. "Electrochemical Characterization of CVD-Grown Graphene for Designing Electrode/Biomolecule Interfaces." Crystals 10, no. 4 (March 26, 2020): 241. http://dx.doi.org/10.3390/cryst10040241.

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In research on enzyme-based biofuel cells, covalent or noncovalent molecular modifications of carbon-based electrode materials are generally used as a method for immobilizing enzymes and/or mediators. However, the influence of these molecular modifications on the electrochemical properties of electrode materials has not been clarified. In this study, we present the electrochemical properties of chemical vapor deposition (CVD)-grown monolayer graphene electrodes before and after molecular modification. The electrochemical properties of graphene electrodes were evaluated by cyclic voltammetry and electrochemical impedance measurements. A covalently modified graphene electrode showed an approximately 25-fold higher charge transfer resistance than before modification. In comparison, the electrochemical properties of a noncovalently modified graphene electrode were not degraded by the modification.

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Wang, Ya-Nan, Chen-Yang Yu, and Hong-Zhong Jin. "RNA N6-Methyladenosine Modifications and the Immune Response." Journal of Immunology Research 2020 (January 21, 2020): 1–6. http://dx.doi.org/10.1155/2020/6327614.

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N6-methyladenosine (m6A) is the most important modification of messenger RNAs (mRNAs) and long noncoding RNAs (lncRNAs) in higher eukaryotes. Modulation of m6A modifications relies on methyltransferases and demethylases. The discovery of binding proteins confirms that the m6A modification has a wide range of biological effects and significance at the molecular, cellular, and physiological levels. In recent years, techniques for investigating m6A modifications of RNA have developed rapidly. This article reviews the biological significance of RNA m6A modifications in the innate immune response, adaptive immune response, and viral infection.

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Han, Dali, and Meng Michelle Xu. "RNA Modification in the Immune System." Annual Review of Immunology 41, no. 1 (April 26, 2023): 73–98. http://dx.doi.org/10.1146/annurev-immunol-101921-045401.

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Characterization of RNA modifications has identified their distribution features and molecular functions. Dynamic changes in RNA modification on various forms of RNA are essential for the development and function of the immune system. In this review, we discuss the value of innovative RNA modification profiling technologies to uncover the function of these diverse, dynamic RNA modifications in various immune cells within healthy and diseased contexts. Further, we explore our current understanding of the mechanisms whereby aberrant RNA modifications modulate the immune milieu of the tumor microenvironment and point out outstanding research questions.

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Wölk, Michele, Theres Schröter, Ralf Hoffmann, and Sanja Milkovska-Stamenova. "Profiling of Low-Molecular-Weight Carbonyls and Protein Modifications in Flavored Milk." Antioxidants 9, no. 11 (November 23, 2020): 1169. http://dx.doi.org/10.3390/antiox9111169.

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Thermal treatments of dairy products favor oxidations, Maillard reactions, and the formation of sugar or lipid oxidation products. Additives including flavorings might enhance these reactions or even induce further reactions. Here we aimed to characterize protein modifications in four flavored milk drinks using samples along the production chain—raw milk, pasteurization, mixing with flavorings, heat treatment, and the commercial product. Therefore, milk samples were analyzed using a bottom up proteomics approach and a combination of data-independent (MSE) and data-dependent acquisition methods (DDA). Twenty-one small carbonylated lipids were identified by shotgun lipidomics triggering 13 protein modifications. Additionally, two Amadori products, 12 advanced glycation end products (AGEs), and 12 oxidation-related modifications were targeted at the protein level. The most common modifications were lactosylation, formylation, and carboxymethylation. The numbers and distribution of modification sites present in raw milk remained stable after pasteurization and mixing with flavorings, while the final heat treatment significantly increased lactosylation and hexosylation in qualitative and quantitative terms. The processing steps did not significantly affect the numbers of AGE-modified, oxidized/carbonylated, and lipid-carbonylated sites in proteins.

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Ogihara, Takuo, Kenta Mizoi, and Akiko Ishii-Watabe. "Pharmacokinetics of Biopharmaceuticals: Their Critical Role in Molecular Design." Biomedicines 11, no. 5 (May 16, 2023): 1456. http://dx.doi.org/10.3390/biomedicines11051456.

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Biopharmaceuticals have developed rapidly in recent years due to the remarkable progress in gene recombination and cell culture technologies. Since the basic structure of biopharmaceuticals can be designed and modified, it is possible to control the duration of action and target specific tissues and cells by kinetic modification. Amino acid sequence modifications, albumin fusion proteins, polyethylene glycol (PEG) modifications, and fatty acid modifications have been utilized to modify the duration of action control and targeting. This review first describes the position of biopharmaceuticals, and then the kinetics (absorption, distribution, metabolism, elimination, and pharmacokinetics) of classical biopharmaceuticals and methods of drug quantification. The kinetic innovations of biopharmaceuticals are outlined, including insulin analog, antibody-related drugs (monoclonal antibodies, Fab analogs, Fc analogs, Fab-PEG conjugated proteins, antibody-drug conjugates, etc.), blood coagulation factors, interferons, and other related drugs. We hope that this review will be of use to many researchers interested in pharmaceuticals derived from biological components, and that it aids in their knowledge of the latest developments in this field.

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Більше джерел

Дисертації з теми "Molecular Modifications"

Pattarini, Lucia. "Post-translational modifications and molecular interactions regulating VEGFR2 activity." Doctoral thesis, Scuola Normale Superiore, 2009. http://hdl.handle.net/11384/85997.

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The work described in this thesis has been mainly focused on the study of a key molecule involved in blood vessel formation, the tyrosine kinase receptor VEGFR2. Considering that VEGFR2 biology should be tighly regulated to allow proper blood vessel formation and maintenance, we investigated two different mechanims influencing VEGFR2 activity: post translational modification and receptor complex formation. Since VEGFR2 biology is governed through protein modication, mainly phosphorylation, we decided to investigate the possible role of acetylation in VEGFR2 activity. Combining biochemical and proteomic studies, we showed that VEGFR2 is modified by acetylation. Starting from this observation, we further investigated the impact of VEGFR2 acetylation on protein stability and phosphorylation in response to ligand. These findings are of particular interest, since, to our knowledge, this is the first report that a tyrosine kinase receptor might be regulated by acetylation. Additionally, we decided to elucidate the interaction of VEGFR2 with its coreceptor Neuropilin1, with particular attention to the Neuropilin1 molecule, by taking advantage of the FRET imaging technique. Collectively, our work characterizes VEGFR2-Neuropilin1 and Neuropilin1-Neuropilin1 complex formation in response to VEGFs and SEMA3A. Altough we do not provide direct evidence for Neuropilin1 direct signalling, our data suggest that Neuropilin1 oligomer formation might be a key step in Neuropilin1 biology.

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Motleleng, Liabo Lilian. "Histone modifications and the Arabidopsis thaliana circadian clock." Master's thesis, University of Cape Town, 2010. http://hdl.handle.net/11427/14719.

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Includes bibliographical references (leaves 61-84).
The circadian system has a regulatory role in almost all aspects of a plant's life. In Arabidopsis thaliana, almost 36% of the genome has been shown to be circadianly regulated and many genes that are circadianly regulated have been shown to be light responsive or involved in light responses. Rhythmic histone acetylation has been demonstrated in the promoter of TIMING OF CAB EXPRESSION1 (TOC1). Here, I used semi-quantitative Reverse Transcriptase Polymerase Chain Reaction (semi-quantitative RT -PCR) to investigate which enzymes are involved in the rhythmic expression of TOC1. I also determined whether loss-of-function histone acetylation and methylation mutants could affect the overall functioning of the circadian oscillator by measuring their circadian leaf movement and delayed fluorescence (DF) rhythms. GCN5/ HAG1 mutant plants (gcn5) exhibited erratic TOC1 expression in both constant dark (DD) and constant light (LL) conditions. Although TOC1 expression appeared to be rhythmic in both DD and LL conditions, the waveform of the rhythm was altered in TATA-binding protein associated factor 1 (taf1) mutants. This suggested that TAF1 and GCN5 might play different roles in the rhythmic histone acetylation affecting TOC1 expression. DF data and leaf movement data indicated that both TAF1 and GCN5 might play a role in the overall functioning of the A. thaliana circadian clock. Arrhythmic TOC1 expression and DF was observed in histone deacetylase 1 (hd1) mutants, suggesting that HD1 is not only involved in the rhythmic histone deacetylation affecting TOC1 expression but in the overall functioning of the circadian clock. Semi-quantitative RTPCR, DF and leaf movement studies demonstrated that CURLY LEAF (CLF), a histone methylase is involved in both the histone methylation affecting TOC1 expression and in the overall functioning of the A. thaliana circadian clock.

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Zheng, Gang Gang Zheng. "A molecular 'switchboard' - lysine modifications and their impact on transcription." Connect to text online, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1131636831.

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Zheng, Gang. "A MOLECULAR ‘SWITCHBOARD’-LYSINE MODIFICATIONS AND THEIR IMPACT ON TRANSCRIPTION." Case Western Reserve University School of Graduate Studies / OhioLINK, 2006. http://rave.ohiolink.edu/etdc/view?acc_num=case1131636831.

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Baker, Daniel Lindley. "Relaxation dynamics in molecular glass-formers with systematic structure modifications." Thesis, University of Leeds, 2014. http://etheses.whiterose.ac.uk/8734/.

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Glasses are amorphous materials which do not exhibit the long-range order or periodicity found in crystalline solids. A glass is formed by cooling a liquid at a sufficient rate such that crystallisation can be avoided. The structural disorder of glasses give them unique properties which make them suitable for a wide range of industrial, pharmaceutical or biological applications. Glass-forming materials generally exhibit several characteristic mechanisms of molecular motion. The physical origins and interrelation between these mechanisms are not well understood. In order to address this, detailed investigations of how glass-transition related dynamics are affected by systematic modification of the molecular structure are needed. This thesis concerns the measurement of the glass-forming properties of three series of molecular glass-formers. These series are comprised of samples which vary systematically in their structure: an alkylbenzene series involving the systematic variation of the length of an alkyl-tail attached to a phenyl-ring and two series involving the successive oligomerisation of styrene and alpha-methylstyrene. The glass forming properties of these series were analysed using Broadband Dielectric Spectroscopy (BDS) and Differential Scanning Calorimetry (DSC). Thermogravimetric Analysis was also employed in order to optimise the sample preparation procedure. The work in this thesis identifies and characterises the detailed molecular weight dependent behaviour of several key relaxation mechanisms in the glassy and supercooled state of three different glass-forming systems. Strong similarities between the relaxation behaviour of the two polymeric and the alkyl chain modified benzene series were found. This demonstrates that much of the observed phenomenology is remarkably general and the work forms a basis for developments of models to address the glass-transition and glassy behaviour. Moreover, it is demonstrated that the glass transition in the three different series of samples behave in a highly similar manner with regards to the system molecular weight and strong support is found for a link between the primary structural relaxation that exists in the supercooled state and the secondary relaxation mechanisms that persist within the glassy state.

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Chen, Peng. "Function of wobble nucleoside modifications in tRNAs of Salmonella enterica Serovar Typhimurium." Doctoral thesis, Umeå universitet, Molekylärbiologi (Teknat- och Medfak), 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-328.

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Transfer RNA from all organisms has modified nucleosides and position 34 (the wobble position) is one of the most extensively modified positions. Some wobble nucleoside modifications restrict codon choice (e.g. 5-methylaminomethyl-2-thiouridine, mnm5s2U) while some extend the decoding capacity (e.g. uridine-5-oxyacetic acid, cmo5U). In this thesis the influence of wobble nucleoside modification on cell physiology and translation efficiency and accuracy is described. A mutant proL tRNA (proL207) was isolated that had an unmodified adenosine in the wobble position. Surprisingly, the proL207 mutant grows normally and is efficiently selected at the non-complementary CCC codon. The explanation of how an A34 containing tRNA can read CCC codon could be that a protonated A can form a base pair with C. cmo5U (uridine-5-oxyacetic acid) is present in the wobble position of five tRNA species in S.enterica. Two genes (cmoA and cmoB) have been identified that are involved in the synthetic pathway of cmo5U. Mutants were constructed in alanine, valine, proline, and threonine codon boxes which left only a cmo5U containing tRNA present in the cell. The influence of cmo5U on growth or on A site selection rates of the ternary complex was found to be tRNA dependent. During the study of the frameshift suppressor sufY of the hisC3737 frameshift mutation, a dominant mutation was found in YbbB protein, a selenouridine synthetase. The frameshifting occurs at CCC-CAA codon contexts and is specific for CAA codons, which are read by tRNAGlncmnm5s2UUG . The sufY204 mutation is a dominant mutation resulting in a change from Gly67 to Glu67 in the YbbB protein, and mediates the synthesis of several novel modified nucleosides/nucleotides (UKs) with unknown structure. The synthesis of these UKs is connected to the synthesis of cmnm5s2U34. The presence of UK on tRNAGlnU*UG reduced aminoacylation and therefore might account for the slow entry at CAA codons which could result in +1 frameshifting by P site tRNA. The selenourdine synthetase activity is not required for the synthesis of UKs. We hypothesize that an intrinsic activity that is low in the wild type protein has been elevated by the single amino acid substitution and results in the synthesis of UKs.

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Coll, San Martín Laia. "Genetic disruption of transfer RNA modifications in human cancer." Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/672905.

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Cancer was reported as the second leading cause of death in 2018 by the World Health Organisation. Cancer is defined as the set of diseases that proceeds in multiple phases generating a transformation lead by an accumulation of genetic (mutations, copy number), epigenetic (CpG methylation, histone modifications), and epitranscriptomic alterations. Lung cancer is the primary cause of cancer-related deaths. It is commonly diagnosed at advanced stages, leading to a 5-year survival of about 21%. Lung cancer is historically subdivided according to resection specimens in small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC is an epithelial tumour with neuroendocrine features representing 10-15% of all lung tumours, and the most lethal and aggressive subtype of lung cancer. Epitranscriptomics is the field of study of RNA modifications. RNA modifications are inserted in transcripts derived from both coding and non-coding genes. These modifications are added to ribonucleotide residues on the purine/pyrimidine ring or ribose, and a significant number of enzymes regulates their dynamic. The role of these modifications ranges from provide stability, to be involved in export, maturation, splicing, folding and function of the RNA. Transfer RNAs (tRNAs) tRNA play a fundamental role in protein biosynthesis as an adaptor molecule acting as a biological link between mRNA and protein sequences on the ribosome during the translation process. Transfer RNAs show the highest density of modifications, specifically the anticodon‐loop region is a hotspot of highly diverse modifications. TRIT1 gene encodes the enzyme tRNA-isopentenyltransferase-1 that transfers an isopentenyl group to form the N6‐isopentenyladenosine (i6A) at position 37 of the cytoplasmic tRNA[Ser]Sec and tRNASer(HGA), and mitochondrial (mt) tRNACys(GCA), tRNATyr(GUA), tRNATrp, tRNASer(UGA), and tRNAPhe(GAA). tRNA[Ser]Sec carries the selenocysteine amino acid that is incorporated in 25 proteins in humans called selenoproteins. We investigated genetic and epigenetic alterations that would affect transfer RNAs modifications in cancer. After data-mining the copy number and DNA methylation data for tRNA modifier enzymes in about 1000 cell lines, we identified an outstanding TRIT1 copy number amplification restricted only to small cell lung cancer (SCLC) cell lines (11 of 60). Thus, this was the starting point of an exhaustive study. We focused on evaluating the implication of this event in tumorigenesis, as well as its potential as a drug target candidate. The TRIT1 gene amplification in cancer cell lines was associated to a significant increase at transcriptional and protein levels. To elucidate the functional role of TRIT1 amplification in SCLC, we assessed the consequences of decrease the expression of TRIT1 using the shRNA approach in the TRIT1-amplified DMS-273 SCLC cell line. The level of i6A decreased upon decreasing TRIT1 expression. In vitro assays did not provide keys to elucidate the role of TRIT1 in cancer, and we decided to perform in vivo assays in mice. We observed a significant reduction in tumour growth upon TRIT1 knockdown, providing robust evidence of the impact of TRIT1 amplification in cancer. Moreover, we carried out massive RNA sequencing of the loss-of-function model in the DMS-273 cell line. After computational analysis, 4510 differential expressed mRNA were identified. Notably, 3409 (75.9%) of these mRNAs were found downregulated upon TRIT1 depletion reflecting the significant impact of i6A hypomodification. Gene set enrichment analysis from the downregulated genes showed “regulation of cell differentiation” as the most enriched process upon TRIT1 depletion. Considering that therapeutic options in SCLC are limited, we performed an exhaustive revision of the literature and a computational approach to identify drugs affecting TRIT1-amplified SCLC. The gene amplification-associated overexpression of TRIT1 confers sensitivity to arsenic trioxide and dimethyloxalylglycine (DMOG), as shown in vitro cellular assays and in vivo experiments in mice. Finally, TRIT1 gene amplification was identified in about 10-15% of SCLC primary samples.
Las modificaciones del ARN son claves en multitud de procesos celulares que al verse alteradas pueden participar en el proceso de tumorogénesis. En la presente tesis se aborda el efecto de estas alteraciones implicadas en la transformación celular en a las moléculas de los ARN de transferencia (ARNt). Se identificó la amplificación génica de TRIT1 exclusivamente en líneas celulares de cáncer de pulmón microcítico (SCLC). La amplificación de TRIT1 también fue detectada en pacientes de SCLC. La enzima TRIT1 que se encarga de introducir la modificación i6A la posición A37 de ciertos ARNt. La amplificación de TRIT1 lleva a un aumento de su expresión a nivel transcripcional y traduccional. Se generó un modelo celular de pérdida de expresión empleando una línea celular de SCLC con amplificación de TRIT1, y se realizaron ensayos in vitro. También se evaluaron las consecuencias funcionales in vivo mediante la inyección de las células en ratón, se observó una reducción del crecimiento tumoral en los tumores derivados de las células con pérdida de expresión de TRIT1. El análisis transcriptómico del modelo celular, mediante secuenciación masiva de ARN (RNA-seq), mostro una desregulación de procesos biológicos relacionados con la diferenciación celular debido a la amplificación de TRIT1. Este hallazgo condujo a la identificación del trióxido de arsénico como un posible candidato como tratamiento para SCLC por su acción en la diferenciación celular. Ensayos in vitro e in vivo indican que la amplificación de TRIT1 confiere sensibilidad al trióxido de arsénico, proponiendo un nuevo candidato terapéutico para el tratamiento de una enfermedad con tan pocas opciones terapéuticas como es SCLC.

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Hartsough, Edward J. "Molecular regulation of VEGFR-2 function and expression through post-translational modifications." Thesis, Boston University, 2012. https://hdl.handle.net/2144/12417.

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Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
Vascular endothelial growth factor receptor-2 (VEGFR-2) is an endothelial cell receptor tyrosine kinase (RTK) whose activity is an obligate requirement for both normal development and pathological angiogenesis. A critical post-translational modification (PTM) of RTKs such as VEGFR-2 is tyrosine phosphorylation, which regulates these proteins at multiple levels including, tyrosine kinase activation, signaling, trafficking, and degradation. Similarly, growing evidence now suggests that protein methylation is another important type of PTM that plays a role in protein-protein interaction and signal transduction. In an effort to explore the possibility that methylation plays a role in regulation of VEGFR-2 function, we have employed mass spectrometry analysis coupled with pharmacological inhibitors of the methylation pathway. Our analysis revealed the presence of five methylated residues, three lysine and two arginine. Particularly, methylation of lysine 1041 (K1041), which is proximal to the conserved activation loop of the kinase domain, significantly contributed to VEGFR-2 kinase activation. Mutation of K1041 to multiple different amino acids rendered VEGFR-2 inactive and inhibited the activation of key downstream signaling proteins. Moreover, these mutations reduced VEGFR-2 mediated cell proliferation and capillary tube formation. Single mutations of R817, K856, K861 and R1115 yielded no apparent effect on tyrosine phosphorylation of VEGFR-2, however compounding the methyl deficiencies with triple and quadruple mutations markedly weakened tyrosine phosphorylation and the ligand-mediated downregulation of VEGFR-2. Furthermore, treatment of endothelial cells with global methylation inhibitors including adenosine dialdehyde (AdOx) and 3-deazaneplanocin A (DZNep) decreased ligand mediated tyrosine phosphorylation of VEGFR-2. The study presented here provides evidence that arginine and lysine methylation of VEGFR-2 through both combinatorial and non-combinatorial mechanisms regulate VEGFR-2 phosphorylation and function. This study also demonstrates that RNF121, an endoplasmic reticulum (ER) resident ubiquitin E3 ligase, binds to nascent VEGFR-2 protein and controls the abundance of cell surface VEGFR-2. Taken together, our data describes a novel role for arginine and lysine methylation in the regulation of VEGFR-2 functions and identifies a link between RNF121 ubiquitin E3 ligase and cell surface expression of VEGFR-2.

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Di, Antonio Marco. "New Molecular Devices for Selective Structural Modifications of G-Quadruplex Folded Oligonucleotides." Doctoral thesis, Università degli studi di Padova, 2011. http://hdl.handle.net/11577/3427493.

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The attention around selective recognition of G-Quadruplex has steadily grown during the past 10 years. G-4 are non-canonical DNA structures which can be generated from the self-assembling of guanine rich oligonucleotides. Such an interest is justified by the fact that it is widely recognized that the above structures could act as regulators of biological processes in vivo. In fact, there are several examples of G-Quadruplexes stabilization, by small molecules or engineered antibodies, which is translated into a strong influence of the biological process in which the oligonucleotide is involved. The inhibition of telomerase enzyme as well as the gene transcriptional alteration induced by the stabilization of such structures, represent the most important applications, together with the more recently studied effect on translation mediated by G-4 RNA structures. This makes G-Quadruplex a real therapeutic target useful for the developing of new antitumor drugs. The aim of this work is to develop a new generation of G-4 ligands capable to undergo alkylation triggered by biocompatible protocols. The key aspect of this idea is to regulate the alkylation process, exploiting mild chemical or physical modifications, in order to temporally separate it from the reversible recognition. These molecules would therefore act as molecular devices capable of pre-concentration onto the target and stabilizing the complex through non covalent interactions. These ligands after activation of the masked electrophile are capable to generate the alkylating specie and therefore to achieve a strong covalent interaction. These features would induce an irreversible damage which would not be repaired by the common cellular process, enhancing the effectively drug potency in biological response terms. The basic idea is to exploit molecular recognition properties of already described ligands and tethering to them a masked electrophile. The releasing of the alkylating specie will be achieved exploiting physical or chemical triggers. Such molecules would represent a brand new class of irreversible G-4 ligands which has never been conceived before. The common features that such a class of ligands must have are: 1) A wide electron poor and flat surface, which confers molecular recognition properties, exploiting superficial π stacking interactions with the biological target. 2) A precursor of an alkylating specie, triggerable by mild physical and chemical activation protocols, possibly exploitable under physiological conditions 3) A moiety which can be easily modified through substrate interactions or chemical activation. Such moiety will represent the trigger of the alkylating reactivity. In more details, we had focused our attention on the chemical functionalization of Naphthalimide (NI) and Naphthalendiimide (NDI) derivatives, which both are good G-4 binders according to literature. Quinone Methide precurors (QM) electrophiles have been tethered to the above molecules. QM based alkylating species are particularly suitable for this project because they can be generated from very stable precursors through several biocompatible activation protocols. Moreover, their reactivity could be tuned changing the electronic nature of the phenolic precursor. In this PhD thesis we describe the synthesis, the reactivity and the study concerning the interaction of the synthesized molecules with oligonucleotides capable of G-4 folding. Particular emphasis will be given to the consequences induced by the alkylation damage with potentially achievable application both therapeutic and diagnostic. Moreover, we will discuss a parallel project developed during a period spent in the Cambridge University in Prof. Balasubramanian’s reaserch group.
Nell’ultimo decennio un sempre più crescente interesse è stato rivolto nei confronti del riconoscimento selettivo dei quartetti di guanina (G-quadruplex), strutture supramolecolari in grado di auto-assemblarsi in condizioni fisiologiche da oligonucleotidi ricchi di residui guaninici. La ragione di ciò risiede nel fatto che tali strutture sembrano agire come regolatori di processi a livello cellulare. Infatti, esistono svariati esempi in cui, soprattutto in vitro, molecole o anticorpi in grado di riconoscere e stabilizzare quartetti di guanina influenzino drasticamente il processo biologico in cui l’oligonucleotide stesso è implicato. L’inibizione indiretta della telomerasi e gli studi dell’effetto sulla trascrizione di oncogeni ne rappresentano le applicazioni più importati, assieme ai più recenti effetti sulla traduzione di RNA. Ciò rende G-quadruplex un vero e proprio target terapeutico per lo sviluppo di nuove terapie antitumorali. Questo lavoro nasce con lo scopo di creare una nuova generazione di leganti di G-4 che manifestino proprietà alchilanti attivabili mediante protocolli biocompatibili. Proprietà alchilanti non intrinseche, ma attivabili attraverso modifiche chimiche e fisiche, permetterebbero un controllo temporale del processo di alchilazione. Tali molecole agirebbero pertanto da veri e propri dispositivi molecolari preconcentrandosi sul target e stabilizzando il complesso attraverso interazioni non covalenti per poi, mediante attivazione, generare la specie alchilante così da ancorare fortemente la molecola all’oligonucleotide. Queste caratteristiche renderebbero il danno indotto irreversibile o non riparabile dai comuni processi cellulari, aumentando notevolmente l’efficacia di azione in termini di effetti farmaco-biologici. L’idea pertanto è quella di sfruttare le proprietà di riconoscimento di alcuni tra i leganti noti in letteratura equipaggiandoli, però, con una specie alchilante silente, il cui rilascio può essere controllato temporalmente mediante azione fisica o chimica. Questo nuovo tipo di molecole rappresenterebbe pertanto una classe di leganti irreversibili di G-4, mai progettata in precedenza. Per far ciò la molecola da sintetizzare deve possedere: 1) una superficie aromatica estesa ed elettron-povera che conferisca le proprietà di riconoscimento molecolare attraverso interazioni di π stacking con il target biologico. 2) un precursore di una specie alchilante che presenti una scarsa o assente reattività intrinseca modulabile mediante attivazione, possibilmente compatibile con condizioni fisiologiche. 3) una porzione molecolare facilmente modificabile per interazioni con il substrato o per attivazione chimica, che funga da “grilletto” della reattività del precursore alchilante. Nella fattispecie ci siamo concentrati sulla derivatizzazione di strutture, che dalla letteratura risultano dei buoni leganti di G-4, come Naftalendiimidi (NDI) o Naftalimmidi (NI), variamente sostituite con dei precursori di alchilanti tipo chinone metide (QM). Questi ultimi risultano particolarmente adatti a questo scopo in quanto posso essere generati da precursori molto stabili, attraverso dei protocolli di attivazione biocompatibili. Soprattutto risultano elettrofili la cui reattività è modulabile variando la natura elettronica del precursore stesso. In questo lavoro di tesi descriviamo la sintesi, la reattività e gli studi di interazione con oligonucleotidi ripegabili a strutture tipo G-4, delle molecole progettate e preparate nel corso del dottorato di ricerca. Particolare enfasi verrà posta sull’effetto indotto dal danno da alchilazione osservato e sulle potenziali applicazioni sia terapeutiche che diagnostiche. Inoltre descriveremo brevemente un progetto parallelo svolto durante il periodo trascorso all’Università di Cambridge, presso il gruppo di ricerca del Prof. Balasubramanian.

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Ali, Ibraheem Irfan. "Role of Post-translational Protein Modifications in Regulating HIV-1 and Mammalian Transcription." Thesis, University of California, San Francisco, 2019. http://pqdtopen.proquest.com/#viewpdf?dispub=13423596.

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The molecular gatekeepers of nearly all gene expression in living cells are the proteins that function in the process of transcription. Transcription occurs when a cell must respond to a signal. These signals can be in the form of metabolic responses, signals for growth or differentiation, signals to defend against stress or pathogenic invasion, to name a few. The fundamentals of transcription have been extensively studied in bacterial systems and model organisms, but technical limitations have hindered their studies in mammalian and human systems. Recent developments in mass spectrometric methodologies, next-generation sequencing and techniques to study difficult-to-detect post-translational protein modifications are extensively reviewed here to highlight an important regulatory network through which gene expression is regulated. In addition, I present two vignettes: the first, a study of the regulatory mechanisms of monomethylation of the HIV-1 Tat protein in regulating HIV-1 gene expression and latency; the second, a study investigating the role of acetylation in regulating RNA Polymerase II protein modifications and gene expression in mammalian systems. Together, these studies combine new mass spectrometric techniques, modification-specific antibodies, protein purification methods, and next generation sequencing to better understand the role of these modifications in regulating the transcriptional response in mammalian systems. These findings can be applied to better understand mechanisms that regulate HIV-1 viral latency, along with fundamentally shifting the field of mammalian transcription by pinpointing unique modes of regulation only found in higher eukaryotes relevant to HIV-1 infection and cancer.

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Книги з теми "Molecular Modifications"

Geological Survey (U.S.), ed. Modifications of two palynological processing techniques: Ultrasonic processing and early-stage sieving. [Reston, VA]: Dept. of the Interior, U.S. Geological Survey, 1992.

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Overweg, Arian. The preparation, modification and characterization of some molecular sieve materials. Eindhoven: Eindhoven University, 1998.

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Henri, Grosjean, ed. DNA and RNA modification enzymes: Structure, mechanism, function, and evolution. Austin, Tex: Landes Bioscience, 2009.

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Blair, Mary Elizabeth. Habitat modification and gene flow in Saimiri oerstedii: Landscape genetics, intraspecific molecular systematics, and conservation. [New York, N.Y.?]: [publisher not identified], 2011.

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Susanne, Brakmann, and Johnsson Kai, eds. Directed molecular evolution of proteins: Or how to improve enzymes for biocatalysis. Weinheim: Wiley-VCH, 2002.

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Thor, G. Bioprocesses of biopharmaceuticals: The obligatory role of post translational modifications to create functional bioactive molecules. Westborough, MA: D&MD Publications, 2005.

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Zhou, Xiufen. Molecular genetic analysis of an unusual DNA modification and a phage defence system of streptomyces lividans 66. Norwich: University of East Anglia, 1993.

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A 'Toolkit' of Small Molecules for Polymer Assembly and Post-Synthetic Modification Using 'Click' and Photoactive Chemistries. [New York, N.Y.?]: [publisher not identified], 2011.

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1952-, Zouali Moncef, ed. The epigenetics of autoimmune diseases. Chichester: Wiley-Blackwell, 2009.

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Lamond, Angus I. Pre-mRNA processing. New York: Springer-Verlag, 1995.

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Частини книг з теми "Molecular Modifications"

Carlberg, Carsten, Eunike Velleuer, and Ferdinand Molnár. "Histone Modifications." In Molecular Medicine, 101–15. Cham: Springer International Publishing, 2023. http://dx.doi.org/10.1007/978-3-031-27133-5_7.

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Bross, Peter. "Posttranslational Modifications." In SpringerBriefs in Molecular Science, 53. Cham: Springer International Publishing, 2015. http://dx.doi.org/10.1007/978-3-319-26088-4_11.

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Gait, Michael J., and Sudhir Agrawal. "Introduction and History of the Chemistry of Nucleic Acids Therapeutics." In Methods in Molecular Biology, 3–31. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_1.

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AbstractThis introduction charts the history of the development of the major chemical modifications that have influenced the development of nucleic acids therapeutics focusing in particular on antisense oligonucleotide analogues carrying modifications in the backbone and sugar. Brief mention is made of siRNA development and other applications that have by and large utilized the same modifications. We also point out the pitfalls of the use of nucleic acids as drugs, such as their unwanted interactions with pattern recognition receptors, which can be mitigated by chemical modification or used as immunotherapeutic agents.

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Cullen, Bryan R., and Kevin Tsai. "Mapping RNA Modifications Using Photo-Crosslinking-Assisted Modification Sequencing." In Methods in Molecular Biology, 123–34. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1374-0_8.

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Yu, Bin, and Xuemei Chen. "Analysis of miRNA Modifications." In Methods in Molecular Biology, 137–48. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-60327-005-2_10.

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Lavitrano, Marialuisa, Laura Farina, Maria Grazia Cerrito, and Roberto Giovannoni. "Sperm-Mediated Genetic Modifications." In Methods in Molecular Biology, 125–32. New York, NY: Springer US, 2019. http://dx.doi.org/10.1007/978-1-4939-9837-1_11.

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Treffon, Patrick, Michael Liebthal, Wilena Telman, and Karl-Josef Dietz. "Probing Posttranslational Redox Modifications." In Methods in Molecular Biology, 195–219. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-7136-7_12.

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Widłak, Wiesława. "Synthesis and Posttranslational Modifications of Proteins." In Molecular Biology, 93–107. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-45361-8_6.

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Roy, Bijoyita. "Effects of mRNA Modifications on Translation: An Overview." In Methods in Molecular Biology, 327–56. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1374-0_20.

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AbstractThe mRNA epitranscriptome imparts diversity to gene expression by installing chemical modifications. Advances in detection methods have identified chemical modifications in eukaryotic, bacterial, and viral messenger RNAs (mRNAs). The biological functions of modifications in mRNAs still remain to be understood. Chemical modifications are introduced in synthetic mRNAs meant for therapeutic applications to maximize expression from the synthetic mRNAs and to evade the host immune response. This overview provides a background of chemical modifications found in mRNAs, with an emphasis on pseudouridine and its known effects on the mRNA life cycle, its potential applications in synthetic mRNA, and the methods used to assess its effects on mRNA translation.

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Karijolich, John, Athena Kantartzis, and Yi-Tao Yu. "Quantitative Analysis of RNA Modifications." In Methods in Molecular Biology, 21–32. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-657-3_2.

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Тези доповідей конференцій з теми "Molecular Modifications"

Chao, Chih-Ming, and Kerwin Wang. "Surface modifications for iron oxide nanoparticle assembly." In 2011 IEEE International Conference on Nano/Micro Engineered and Molecular Systems (NEMS). IEEE, 2011. http://dx.doi.org/10.1109/nems.2011.6017460.

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Hamlow, Lucas, J. Oomens, Giel Berden, M. Rodgers, Bo Yang, Ranran Wu, Lin Fan, and Chenchen He. "STRUCTUAL EFFECTS OF CYTIDINE 2′ RIBOSE MODIFICATIONS AS DETERMINED BY IRMPD ACTION SPECTROSCOPY." In 70th International Symposium on Molecular Spectroscopy. Urbana, Illinois: University of Illinois at Urbana-Champaign, 2015. http://dx.doi.org/10.15278/isms.2015.mi13.

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Shao, Hao-Chiang. "UNVEILING VESTIGES OF MAN-MADE MODIFICATIONS ON MOLECULAR-BIOLOGICAL EXPERIMENT IMAGES." In 2018 IEEE Global Conference on Signal and Information Processing (GlobalSIP). IEEE, 2018. http://dx.doi.org/10.1109/globalsip.2018.8646594.

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Rothman, Laurence S. "The HITRAN Molecular Database: Enhancements for Remote Sensing." In Optical Remote Sensing of the Atmosphere. Washington, D.C.: Optica Publishing Group, 1993. http://dx.doi.org/10.1364/orsa.1993.thc.1.

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Arčon, D., R. Blinc, P. Cevc, A. Omerzu, and D. Mihailovič. "Comparative study of ferromagnetic and non-ferromagnetic modifications of TDAE-." In ELECTRONIC PROPERTIES OF NOVEL MATERIALS--SCIENCE AND TECHNOLOGY OF MOLECULAR NANOSTRUCTURES. ASCE, 1999. http://dx.doi.org/10.1063/1.59814.

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Vernier, P. T. "Molecular modeling of membrane modifications after exposure to nanosecond, pulsed electric fields." In SPIE BiOS, edited by Gerald J. Wilmink and Bennett L. Ibey. SPIE, 2013. http://dx.doi.org/10.1117/12.2005996.

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Angioni, MM, A. Denotti, S. Pinna, C. Sanna, A. Floris, M. Piga, A. Loi, and A. Cauli. "SAT0528 Molecular modifications induced by mud-bath therapy in patients with osteoarthritis." In Annual European Congress of Rheumatology, 14–17 June, 2017. BMJ Publishing Group Ltd and European League Against Rheumatism, 2017. http://dx.doi.org/10.1136/annrheumdis-2017-eular.6874.

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Adamek, Maksimiljan. "Molecular Grammar of RNA-binding Protein Interactions in Formation and Function of Ribonucleoprotein Complexes." In Socratic Lectures 8. University of Lubljana Press, 2023. http://dx.doi.org/10.55295/psl.2023.ii15.

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Ribonucleoproteins (RNPs) are macromolecular assemblies of proteins along RNA molecules to carry out specialized cellular processes. Understanding how RNA binding proteins (RBPs) and RNA sequences determine the interactions to form RNPs and ultimately steer biomolecular processes remains poorly understood. There is a mounting evidence that RNP assembly de-pends on the formation of a network of transient, multivalent RBP RNA and RBP RBP interac-tions, particularly between tyrosine residues from intrinsically disordered domains and argi-nine residues from RNA-binding domains of RBPs. Furthermore, RBPs, especially their intrin-sically disordered regions, are hotspots for posttranslational modification (PTM) sites. Alt-hough PTMs have been well catalogued, little is known about how these modifications regulate RNP assembly and function. Some initial studies introduced the concept of the so-called phos-pho-switch, in which RBPs require phosphorylation for condensation of larger RNP complexes, but it remains unclear how this contributes to the protein function and the pattern of selective protein binding to RNA molecules. This short review will take a look at what is currently known in the field of RNPs, their interactions, and the phase-separated biomolecular conden-sates, which are intimately connected to RNPs and are important for several key cell processes. Keywords: Ribonucleoproteins; RNA binding proteins; Multivalency; Intrinsically disordered proteins; Posttranslational modifications

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Rodgers, M., Jos Oomens, Giel Berden, Chase Leslie, Erik Soley, Harrison Roy, Zachary Devereaux, et al. "INFLUENCE OF NATURALLY-OCCURRING AND SYNTHETIC MODIFICATIONS ON THE STRUCTURES AND GLYCOSIDIC BOND STABILITIES OF DNA AND RNA NUCLEOSIDES." In 2020 International Symposium on Molecular Spectroscopy. Urbana, Illinois: University of Illinois at Urbana-Champaign, 2020. http://dx.doi.org/10.15278/isms.2020.ma02.

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Rodgers, M., Jos Oomens, Giel Berden, Chase Leslie, Erik Soley, Harrison Roy, Zachary Devereaux, et al. "INFLUENCE OF NATURALLY-OCCURRING AND SYNTHETIC MODIFICATIONS ON THE STRUCTURES AND GLYCOSIDIC BOND STABILITIES OF DNA AND RNA NUCLEOSIDES." In 2021 International Symposium on Molecular Spectroscopy. Urbana, Illinois: University of Illinois at Urbana-Champaign, 2021. http://dx.doi.org/10.15278/isms.2021.rg02.

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Звіти організацій з теми "Molecular Modifications"

Library, Spring. Where Does Current Quorum Sensing Research Stand. Spring Library, December 2020. http://dx.doi.org/10.47496/sl.blog.16.

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Quorum quenching is achieved by inactivating signalling enzymes, by introducing molecules that mimic signalling molecules and block their receptors, by degrading signalling molecules themselves, or by a modification of the quorum sensing signals due to an enzyme activity.

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Clough, S. B., S. Kumar, X. F. Sun, S. Tripathy, and H. Matsuda. Molecular and Crystal Engineering of Polydiacetylenes: Modification of Optical Properties. Fort Belvoir, VA: Defense Technical Information Center, October 1988. http://dx.doi.org/10.21236/ada200808.

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López-Valverde, Nansi, Javier Aragoneses, Antonio López-Valverde, Cinthia Rodríguez, and Juan Manuel Aragoneses. Role in the osseointegration of titanium dental implants, of bioactive surfaces based on biomolecules: A systematic review and meta-analysis of in vivo studies. INPLASY - International Platform of Registered Systematic Review and Meta-analysis Protocols, June 2022. http://dx.doi.org/10.37766/inplasy2022.6.0076.

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Review question / Objective: Does the bioactive surface of titanium dental implants, based on biomolecules, influence osseointegration?. The aim of our study was to evaluate the role and efficacy of bioactive surfaces in osseointegration. Our review study limited the research interest to titanium dental implants coated with a biomolecule, i.e., an organic molecule produced by a living organism. Condition being studied: In recent years, much attention has been paid to topographical modifications of dental implant surfaces, as well as to their coating with biologically active substances.a bioactive surface is one capable of achieving faster and higher quality osseointegration, shortening waiting times and solving situations of poor bone quality. Molecules that can be applied for bioactive purposes include bioceramics, ions and biomolecules. Collagen and bone morphogenetic protein have been suggested as bone stimulating agents. Biofunctionalization of the implant surface with a biomimetic active peptide has also been shown to result in a significant increase in bone-to-implant ratios and an increase in peri-implant bone density.

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Schaffer, Arthur, Jack Preiss, Marina Petreikov, and Ilan Levin. Increasing Starch Accumulation via Genetic Modification of the ADP-glucose Pyrophosphorylase. United States Department of Agriculture, October 2009. http://dx.doi.org/10.32747/2009.7591740.bard.

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The overall objective of the research project was to utilize biochemical insights together with both classical and molecular genetic strategies to improve tomato starch accumulation. The proposal was based on the observation that the transient starch accumulation in the immature fruit serves as a reservoir for carbohydrate and soluble sugar content in the mature fruit, thereby impacting on fruit quality. The general objectives were to optimize AGPase function and activity in developing fruit in order to increase its transient starch levels. The specific research objectives were to: a) perform directed molecular evolution of the limiting enzyme of starch synthesis, AGPase, focussing on the interaction of its regulatory and catalytic subunits; b) determine the mode of action of the recently identified allelic variant for the regulatory subunit in tomato fruit that leads to increased AGPase activity and hence starch content. During the course of the research project major advances were made in understanding the interaction of the small and large subunits of AGPase, in particular the regulatory roles of the different large subunits, in determining starch synthesis. The research was performed using various experimental systems, including bacteria and Arabidopsis, potato and tomato, allowing for broad and meaningful conclusions to be drawn. A novel discovery was that one of the large subunits of tomato AGPase is functional as a monomer. A dozen publications describing the research were published in leading biochemical and horticultural journals. The research results clearly indicated that increasing AGPase activity temporally in the developing fruit increase the starch reservoir and, subsequently, the fruit sugar content. This was shown by a comparison of the carbohydrate balance in near-isogenic tomato lines differing in a gene encoding for the fruit-specific large subunit (LS1). The research also revealed that the increase in AGPase activity is due to a temporal extension of LS1 gene expression in the developing fruit which in turn stabilizes the limiting heterotetrameric enzyme, leading to sustained starch synthesis. This genetic variation can successfully be utilized in the breeding of high quality tomatoes.

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Porat, Ron, Gregory T. McCollum, Amnon Lers, and Charles L. Guy. Identification and characterization of genes involved in the acquisition of chilling tolerance in citrus fruit. United States Department of Agriculture, December 2007. http://dx.doi.org/10.32747/2007.7587727.bard.

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Citrus, like many other tropical and subtropical fruit are sensitive to chilling temperatures. However, application of a pre-storage temperature conditioning (CD) treatment at 16°C for 7 d or of a hot water brushing (HWB) treatment at 60°C for 20 sec remarkably enhances chilling tolerance and reduces the development of chilling injuries (CI) upon storage at 5°C. In the current research, we proposed to identify and characterize grapefruit genes that are induced by CD, and may contribute to the acquisition of fruit chilling tolerance, by two different molecular approaches: cDNA array analysis and PCR cDNA subtraction. In addition, following the recent development and commercialization of the new Affymetrix Citrus Genome Array, we further performed genome-wide transcript profiling analysis following exposure to CD and chilling treatments. To conduct the cDNA array analysis, we constructed cDNA libraries from the peel tissue of CD- and HWB-treated grapefruit, and performed an EST sequencing project including sequencing of 3,456 cDNAs from each library. Based on the obtained sequence information, we chose 70 stress-responsive and chilling-related genes and spotted them on nylon membranes. Following hybridization the constructed cDNA arrays with RNA probes from control and CD-treated fruit and detailed confirmations by RT-PCR analysis, we found that six genes: lipid-transfer protein, metallothionein-like protein, catalase, GTP-binding protein, Lea5, and stress-responsive zinc finger protein, showed higher transcript levels in flavedo of conditioned than in non-conditioned fruit stored at 5 ᵒC. The transcript levels of another four genes: galactinol synthase, ACC oxidase, temperature-induced lipocalin, and chilling-inducible oxygenase, increased only in control untreated fruit but not in chilling-tolerant CD-treated fruit. By PCR cDNA subtraction analysis we identified 17 new chilling-responsive and HWB- and CD-induced genes. Overall, characterization of the expression patterns of these genes as well as of 11 more stress-related genes by RNA gel blot hybridizations revealed that the HWB treatment activated mainly the expression of stress-related genes(HSP19-I, HSP19-II, dehydrin, universal stress protein, EIN2, 1,3;4-β-D-glucanase, and SOD), whereas the CD treatment activated mainly the expression of lipid modification enzymes, including fatty acid disaturase2 (FAD2) and lipid transfer protein (LTP). Genome wide transcriptional profiling analysis using the newly developed Affymetrix Citrus GeneChip® microarray (including 30,171 citrus probe sets) revealed the identification of three different chilling-related regulons: 1,345 probe sets were significantly affected by chilling in both control and CD-treated fruits (chilling-response regulon), 509 probe sets were unique to the CD-treated fruits (chilling tolerance regulon), and 417 probe sets were unique to the chilling-sensitive control fruits (chilling stress regulon). Overall, exposure to chilling led to expression governed arrest of general cellular metabolic activity, including concretive down-regulation of cell wall, pathogen defense, photosynthesis, respiration, and protein, nucleic acid and secondary metabolism. On the other hand, chilling enhanced various adaptation processes, such as changes in the expression levels of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.

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Anderson, Olin, Gad Galili, and Ann Blechl. Heterologous Expression of Wheat High Molecular Weight Glutenin Subunit Genes: Analysis and Modification of Protein Sequences Affecting Dough Quality. United States Department of Agriculture, January 1993. http://dx.doi.org/10.32747/1993.7603826.bard.

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Brown Horowitz, Sigal, Eric L. Davis, and Axel Elling. Dissecting interactions between root-knot nematode effectors and lipid signaling involved in plant defense. United States Department of Agriculture, January 2014. http://dx.doi.org/10.32747/2014.7598167.bard.

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Root-knot nematodes, Meloidogynespp., are extremely destructive pathogens with a cosmopolitan distribution and a host range that affects most crops. Safety and environmental concerns related to the toxicity of nematicides along with a lack of natural resistance sources threaten most crops in Israel and the U.S. This emphasizes the need to identify genes and signal mechanisms that could provide novel nematode control tactics and resistance breeding targets. The sedentary root-knot nematode (RKN) Meloidogynespp. secrete effectors in a spatial and temporal manner to interfere with and mimic multiple physiological and morphological mechanisms, leading to modifications and reprogramming of the host cells' functions, resulted in construction and maintenance of nematodes' feeding sites. For successful parasitism, many effectors act as immunomodulators, aimed to manipulate and suppress immune defense signaling triggered upon nematode invasion. Plant development and defense rely mainly on hormone regulation. Herein, a metabolomic profiling of oxylipins and hormones composition of tomato roots were performed using LC-MS/MS, indicating a fluctuation in oxylipins profile in a compatible interaction. Moreover, further attention was given to uncover the implication of WRKYs transcription factors in regulating nematode development. In addition, in order to identify genes that might interact with the lipidomic defense pathway induced by oxylipins, a RNAseq was performed by exposing M. javanicasecond-stage juveniles to tomato protoplast, 9-HOT and 13-KOD oxylipins. This transcriptome generated a total of 4682 differentially expressed genes (DEGs). Being interested in effectors, we seek for DEGs carrying a predicted secretion signal peptide. Among the DEGs including signal peptide, several had homology with known effectors in other nematode species, other unknown potentially secreted proteins may have a role as root-knot nematodes' effectors which might interact with lipid signaling. The molecular interaction of LOX proteins with the Cyst nematode effectors illustrate the nematode strategy in manipulating plant lipid signals. The function of several other effectors in manipulating plant defense signals, as well as lipids signals, weakening cell walls, attenuating feeding site function and development are still being studied in depth for several novel effectors. As direct outcome of this project, the accumulating findings will be utilized to improve our understanding of the mechanisms governing critical life-cycle phases of the parasitic M. incognita RKN, thereby facilitating design of effective controls based on perturbation of nematode behavior—without producing harmful side effects. The knowledge from this study will promote genome editing strategies aimed at developing nematode resistance in tomato and other nematode-susceptible crop species in Israel and the United States.

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Weiss, David, and Neil Olszewski. Manipulation of GA Levels and GA Signal Transduction in Anthers to Generate Male Sterility. United States Department of Agriculture, 2000. http://dx.doi.org/10.32747/2000.7580678.bard.

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The original objectives of the research were: i. To study the role of GA in anther development, ii. To manipulate GA and/or GA signal transduction levels in the anthers in order to generate male sterility. iii. To characterize the GA signal transduction repressor, SPY. Previous studies have suggested that gibberellins (GAs) are required for normal anther development. In this work, we studied the role of GA in the regulation of anther development in petunia. When plants were treated with the GA-biosynthesis inhibitor paclobutrazol, anther development was arrested. Microscopic analysis of these anthers revealed that paclobutrazol inhibits post-meiotic developmental processes. The treated anthers contained pollen grains but the connective tissue and tapetum cells were degenerated. The expression of the GA-induced gene, GIP, can be used in petunia as a molecular marker to: study GA responses. Analyses of GIP expression during anther development revealed that the gene is induced only after microsporogenesis. This observation further suggests a role for GA in the regulation of post-meiotic processes during petunia anther development. Spy acts as a negative regulator of gibberellin (GA) action in Arabidopsis. We cloned the petunia Spy homologue, PhSPY, and showed that it can complement the spy-3 mutation in Arabidopsis. Overexpression of Spy in transgenic petunia plants affected various GA-regulated processes, including seed germination, shoot elongation, flower initiation, flower development and the expression of a GA- induced gene, GIP. In addition, anther development was inhibited in the transgenic plants following microsporogenesis. The N-terminus of Spy contains tetratricopeptide repeats (TPR). TPR motifs participate in protein-protein interactions, suggesting that Spy is part of a multiprotein complex. To test this hypothesis, we over-expressed the SPY's TPR region without the catalytic domain in transgenic petunia and generated a dominant- negative Spy mutant. The transgenic seeds were able to germinate on paclobutrazol, suggesting an enhanced GA signal. Overexpression of PhSPY in wild type Arabidopsis did not affect plant stature, morphology or flowering time. Consistent with Spy being an O-GlcNAc transferase (OGT), Spy expressed in insect cells was shown to O-GlcNAc modify itself. Consistent with O-GlcNAc modification playing a role in GA signaling, spy mutants had a reduction in the GlcNAc modification of several proteins. After treatment of the GA deficient, gal mutant, with GA3 the GlcNAc modification of proteins of the same size as those affected in spy mutants exhibited a reduction in GlcNAcylation. GA-induced GlcNAcase may be responsible for this de-GlcNAcylation because, treatment of gal with GA rapidly induced an increase in GlcNAcase activity. Several Arabidopsis proteins that interact with the TPR domain of Spy were identified using yeast two-hybrids screens. One of these proteins was GIGANTEA (GI). Consistent with GI and Spy functioning as a complex in the plant the spy-4 was epistatic to gi. These experiments also demonstrated that, in addition to its role in GA signaling, Spy functions in the light signaling pathways controlling hypocotyl elongation and photoperiodic induction of flowering. A second Arabidopsis OGT, SECRET AGENT (SCA), was discovered. Like SPY, SCA O-GlcNAc modifies itself. Although sca mutants do not exhibit dramatic phenotypes, spy/sca double mutants exhibit male and female gamete and embryo lethality, indicating that Spy and SCA have overlapping functions. These results suggest that O-GlcNAc modification is an essential modification in plants that has a role in multiple signaling pathways.

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Tzfira, Tzvi, Michael Elbaum, and Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, December 2005. http://dx.doi.org/10.32747/2005.7695881.bard.

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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.

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Cohen, Jerry D., and Ephraim Epstein. Metabolism of Auxins during Fruit Development and Ripening. United States Department of Agriculture, August 1995. http://dx.doi.org/10.32747/1995.7573064.bard.

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We had proposed to look at several aspects of auxin metabolism in fruit tissues: 1) IAA biosynthesis from tryptophan and IAA biosynthesis via the non-tryptophan pathway; 2) changes in the capacity to form conjugates and catabolites of auxin at different times during fruit development and; 3) the effects of modifying auxin metabolism in fruit tissues. The latter work focused primarily on the maize iaglu gene, with initial studies also using a bacterial gene for hydrolysis of IAA-aspartate. These metabolic and molecular studies were necessary to define potential benefits of auxin metabolism modification and will direct future efforts for crop improvement by genetic methods. An in vitro system was developed for the production of tomato fruit in culture starting from immature flowers in order to ascertain the effect of auxin modification on fruit ripening. IAA supplied to the fruit culture media prior to breaker stage resulted in an increase in the time period between breaker and red-ripe stages from 7 days without additional IAA to 12 days when 10-5 M IAA was added. These results suggest that significant changes in the ripening period could be obtained by alteration of auxin relationships in tomato fruit. We generated transgenic tomato plants that express either the maize iaglu gene or reduced levels of the gene that encodes the enzyme IAA-glucose synthetase. A modified shuttle vector pBI 121 expressing the maize iaglu gene in both sense and antisense orientations under a 35S promoter was used for the study. The sense plants showed total lack of root initiation and development. The antisense transgenic plants, on the other hand, had unusually well developed root systems at early stages in development. Analysis showed that the amount and activity of the endogenous 75 kDa IAGLU protein was reduced in these plants and consequently these plants had reduced levels of IAA-glucose and lower overall esterified IAA.

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