Relevant bibliographies by topics / Complex substrates

Academic literature on the topic 'Complex substrates'

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Published: 11 March 2023

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Journal articles on the topic "Complex substrates"

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Kim, Ikjin, Jungmi Ahn, Chang Liu, Kaori Tanabe, Jennifer Apodaca, Tadashi Suzuki, and Hai Rao. "The Png1–Rad23 complex regulates glycoprotein turnover." Journal of Cell Biology 172, no. 2 (January 9, 2006): 211–19. http://dx.doi.org/10.1083/jcb.200507149.

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Misfolded proteins in the endoplasmic reticulum (ER) are destroyed by a pathway termed ER-associated protein degradation (ERAD). Glycans are often removed from glycosylated ERAD substrates in the cytosol before substrate degradation, which maintains the efficiency of the proteasome. Png1, a deglycosylating enzyme, has long been suspected, but not proven, to be crucial in this process. We demonstrate that the efficient degradation of glycosylated ricin A chain requires the Png1–Rad23 complex, suggesting that this complex couples protein deglycosylation and degradation. Rad23 is a ubiquitin (Ub) binding protein involved in the transfer of ubiquitylated substrates to the proteasome. How Rad23 achieves its substrate specificity is unknown. We show that Rad23 binds various regulators of proteolysis to facilitate the degradation of distinct substrates. We propose that the substrate specificity of Rad23 and other Ub binding proteins is determined by their interactions with various cofactors involved in specific degradation pathways.
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Wu, Xi, Lanlan Li, and Hui Jiang. "Doa1 targets ubiquitinated substrates for mitochondria-associated degradation." Journal of Cell Biology 213, no. 1 (April 4, 2016): 49–63. http://dx.doi.org/10.1083/jcb.201510098.

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Mitochondria-associated degradation (MAD) mediated by the Cdc48 complex and proteasome degrades ubiquitinated mitochondrial outer-membrane proteins. MAD is critical for mitochondrial proteostasis, but it remains poorly characterized. We identified several mitochondrial Cdc48 substrates and developed a genetic screen assay to uncover regulators of the Cdc48-dependent MAD pathway. Surprisingly, we identified Doa1, a substrate-processing factor of Cdc48 that inhibits the degradation of some Cdc48 substrates, as a critical mediator of the turnover of mitochondrial Cdc48 substrates. Deletion of DOA1 causes the accumulation and mislocalization of substrates on mitochondria. Profiling of Cdc48 cofactors shows that Doa1 and Cdc48-Ufd1-Npl4 form a functional complex mediating MAD. Biochemically, Doa1 interacts with ubiquitinated substrates and facilitates substrate recruitment to the Cdc48-Ufd1-Npl4 complex. Functionally, Doa1 is critical for cell survival under mitochondrial oxidative stress, but not ER stress, conditions. Collectively, our results demonstrate the essential role of the Doa1–Cdc48-Ufd1-Npl4 complex in mitochondrial proteostasis and suggest that Doa1 plays dual roles on the Cdc48 complex.
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Dayan, Peter. "Simple substrates for complex cognition." frontiers in Neuroscience 2, no. 2 (December 15, 2008): 255–63. http://dx.doi.org/10.3389/neuro.01.031.2008.

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Kanehara, Kazue, Wei Xie, and Davis T. W. Ng. "Modularity of the Hrd1 ERAD complex underlies its diverse client range." Journal of Cell Biology 188, no. 5 (March 8, 2010): 707–16. http://dx.doi.org/10.1083/jcb.200907055.

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Secretory protein folding is monitored by endoplasmic reticulum (ER) quality control mechanisms. Misfolded proteins are retained and targeted to ER-associated degradation (ERAD) pathways. At their core are E3 ubiquitin ligases, which organize factors that recognize, ubiquitinate, and translocate substrates. Of these, we report that the Hrd1 complex manages three distinct substrate classes. A core complex is required for all classes and is sufficient for some membrane proteins. The accessory factors Usa1p and Der1p adapt the complex to process luminal substrates. Their integration is sufficient to process molecules bearing glycan-independent degradation signals. The presence of Yos9p extends the substrate range by mediating the recognition of glycan-based degradation signals. This modular organization enables the Hrd1 complex to recognize topologically diverse substrates. The Hrd1 system does not directly evaluate the folding state of polypeptides. Instead, it does so indirectly, by recognizing specific embedded signals displayed upon misfolding.
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Min, Mingwei, Ugo Mayor, and Catherine Lindon. "Ubiquitination site preferences in anaphase promoting complex/cyclosome (APC/C) substrates." Open Biology 3, no. 9 (September 2013): 130097. http://dx.doi.org/10.1098/rsob.130097.

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Ordered progression of mitosis requires precise control in abundance of mitotic regulators. The anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase plays a key role by directing ubiquitin-mediated destruction of targets in a temporally and spatially defined manner. Specificity in APC/C targeting is conferred through recognition of substrate D-box and KEN degrons, while the specificity of ubiquitination sites, as another possible regulated dimension, has not yet been explored. Here, we present the first analysis of ubiquitination sites in the APC/C substrate ubiquitome. We show that KEN is a preferred ubiquitin acceptor in APC/C substrates and that acceptor sites are enriched in predicted disordered regions and flanked by serine residues. Our experimental data confirm a role for the KEN lysine as an ubiquitin acceptor contributing to substrate destruction during mitotic progression. Using Aurora A and Nek2 kinases as examples, we show that phosphorylation on the flanking serine residue could directly regulate ubiquitination and subsequent degradation of substrates. We propose a novel layer of regulation in substrate ubiquitination, via phosphorylation adjacent to the KEN motif, in APC/C-mediated targeting.
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Knape, Matthias J., Maximilian Wallbott, Nicole C. G. Burghardt, Daniela Bertinetti, Jan Hornung, Sven H. Schmidt, Robin Lorenz, and Friedrich W. Herberg. "Molecular Basis for Ser/Thr Specificity in PKA Signaling." Cells 9, no. 6 (June 25, 2020): 1548. http://dx.doi.org/10.3390/cells9061548.

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cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.
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Bourreau, D., P. Guillon, and M. Chatard-Moulin. "Complex permittivity measurement of optoelectronic substrates." Electronics Letters 22, no. 7 (1986): 399. http://dx.doi.org/10.1049/el:19860271.

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Neal, Sonya, Raymond Mak, Eric J. Bennett, and Randolph Hampton. "A Cdc48 “Retrochaperone” Function Is Required for the Solubility of Retrotranslocated, Integral Membrane Endoplasmic Reticulum-associated Degradation (ERAD-M) Substrates." Journal of Biological Chemistry 292, no. 8 (January 11, 2017): 3112–28. http://dx.doi.org/10.1074/jbc.m116.770610.

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A surprising feature of endoplasmic reticulum (ER)-associated degradation (ERAD) is the movement, or retrotranslocation, of ubiquitinated substrates from the ER lumen or membrane to the cytosol where they are degraded by the 26S proteasome. Multispanning ER membrane proteins, called ERAD-M substrates, are retrotranslocated to the cytosol as full-length intermediates during ERAD, and we have investigated how they maintain substrate solubility. Using an in vivo assay, we show that retrotranslocated ERAD-M substrates are moved to the cytoplasm as part of the normal ERAD pathway, where they are part of a solely proteinaceous complex. Using proteomics and direct biochemical confirmation, we found that Cdc48 serves as a critical “retrochaperone” for these ERAD-M substrates. Cdc48 binding to retrotranslocated, ubiquitinated ERAD-M substrates is required for their solubility; removal of the polyubiquitin chains or competition for binding by addition of free polyubiquitin liberated Cdc48 from retrotranslocated proteins and rendered them insoluble. All components of the canonical Cdc48 complex Cdc48-Npl4-Ufd1 were present in solubilized ERAD-M substrates. This function of the complex was observed for both HRD and DOA pathway substrates. Thus, in addition to the long known ATP-dependent extraction of ERAD substrates during retrotranslocation, the Cdc48 complex is generally and critically needed for the solubility of retrotranslocated ERAD-M intermediates.
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Saunders, Reuben A., Benjamin M. Stinson, Tania A. Baker, and Robert T. Sauer. "Multistep substrate binding and engagement by the AAA+ ClpXP protease." Proceedings of the National Academy of Sciences 117, no. 45 (October 26, 2020): 28005–13. http://dx.doi.org/10.1073/pnas.2010804117.

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Escherichia coliClpXP is one of the most thoroughly studied AAA+ proteases, but relatively little is known about the reactions that allow it to bind and then engage specific protein substrates before the adenosine triphosphate (ATP)-fueled mechanical unfolding and translocation steps that lead to processive degradation. Here, we employ a fluorescence-quenching assay to study the binding of ssrA-tagged substrates to ClpXP. Polyphasic stopped-flow association and dissociation kinetics support the existence of at least three distinct substrate-bound complexes. These kinetic data fit well to a model in which ClpXP and substrate form an initial recognition complex followed by an intermediate complex and then, an engaged complex that is competent for substrate unfolding. The initial association and dissociation steps do not require ATP hydrolysis, but subsequent forward and reverse kinetic steps are accelerated by faster ATP hydrolysis. Our results, together with recent cryo-EM structures of ClpXP bound to substrates, support a model in which the ssrA degron initially binds in the top portion of the axial channel of the ClpX hexamer and then is translocated deeper into the channel in steps that eventually pull the native portion of the substrate against the channel opening. Reversible initial substrate binding allows ClpXP to check potential substrates for degrons, potentially increasing specificity. Subsequent substrate engagement steps allow ClpXP to grip a wide variety of sequences to ensure efficient unfolding and translocation of almost any native substrate.
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Twomey, Edward C., Zhejian Ji, Thomas E. Wales, Nicholas O. Bodnar, Scott B. Ficarro, Jarrod A. Marto, John R. Engen, and Tom A. Rapoport. "Substrate processing by the Cdc48 ATPase complex is initiated by ubiquitin unfolding." Science 365, no. 6452 (June 27, 2019): eaax1033. http://dx.doi.org/10.1126/science.aax1033.

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The Cdc48 adenosine triphosphatase (ATPase) (p97 or valosin-containing protein in mammals) and its cofactor Ufd1/Npl4 extract polyubiquitinated proteins from membranes or macromolecular complexes for subsequent degradation by the proteasome. How Cdc48 processes its diverse and often well-folded substrates is unclear. Here, we report cryo–electron microscopy structures of the Cdc48 ATPase in complex with Ufd1/Npl4 and polyubiquitinated substrate. The structures show that the Cdc48 complex initiates substrate processing by unfolding a ubiquitin molecule. The unfolded ubiquitin molecule binds to Npl4 and projects its N-terminal segment through both hexameric ATPase rings. Pore loops of the second ring form a staircase that acts as a conveyer belt to move the polypeptide through the central pore. Inducing the unfolding of ubiquitin allows the Cdc48 ATPase complex to process a broad range of substrates.
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Dissertations / Theses on the topic "Complex substrates"

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Alhijjaji, Fariha. "Studies on the microbial degradation of complex substrates." Thesis, University of Sheffield, 2015. http://etheses.whiterose.ac.uk/10040/.

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Microorganisms, mainly bacteria and fungi, are key agents involved in the breakdown and decomposition of plant and animal polymers in ecosystems. The aim of this research project was to study the mechanisms of degradation of four complex substrates; keratin, pectin, alginate and chitin. In this study, keratinophylic fungi were isolated from agricultural soil via a hair-baiting technique (HBT) using wool and hair as baits; additionally, keratinophylic species were isolated from contaminated feathers. The isolates from hair, wool and feathers were grown on solid media supported by keratin azure as a source of carbon and nitrogen. Keratinolytic activities were observed by the formation of a clearing zone in the medium. A study of keratinolytic assay in shaking culture was made by measuring the activity of keratinase (release keratin azure). In addition, scanning electron microscopy (SEM) studies were included in this study. Qualitative assays of pectin degradation, using apple pectin as a carbon source are reported. Pectin degradation in plates was detected using a solution of iodine-potassium iodide. Pectinase activity was determined in the supernatants by release of reducing sugars (galacturonic acid) using dinitrosalicylate reagent (DNS). Antimicrobial activities of pectin esterified potassium salt against some pathogens partically the bacteria which cause infection in wounds was determined by measuring inhibition zones around the wells. Alginolytic microorganisms were isolated from two fresh seaweeds, namely Fucus and Laminaria. The enzymatic activities were quantified by the formation of new unsaturated non-reducing ends and as reducing sugar (RS). The amount of reducing sugar formed was determined using 3, 5-dinitrosalicylic acid (DNS) methods. Crab shell chitin was hydrolysed by acid to produce colloidal chitin. Fungal and bacterial isolates were tested to determine chitinolytic properties in plates by measuring purple zones against yellow background. The supernatants derived from selected isolates were then used to determine chitinase activity by measuring reducing sugars (RS). RS calculated as glucose using Nelson and DNS methods. The fertilizer-potential of the substrates was determined by measuring nitrification and the oxidation of sulphur in soil amendment with the individual complex substrates. A variety of bacteria and fungal isolates were identified using molecular identification techniques. Finally, four enzymes were isolated and partially purified using ammonium sulphate in order to determine their molecular weight using SDS polyacrylamide gel electrophoresis (SDS-PAGE). In addition, liquid chromatography mass spectrometry (LC-MS/MS) has been used to identify three enzymes namely; keratinase, pectinases and chitinases.
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Selander, Nicklas. "Catalytic Functionalization of Allylic Substrates by Palladium Pincer Complexes." Doctoral thesis, Stockholms universitet, Institutionen för organisk kemi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-39065.

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This thesis is based on the development of novel catalytic reactions for the synthesis and application of organometallic reagents. The main focus is directed towards organoboronate derivatives. We developed an efficient procedure for converting allylic alcohols to the corresponding allylboronates using palladium pincer complexes as catalysts. The reactions were performed under mild conditions with high selectivity, allowing further one-pot transformations. Using this approach, a variety of stereodefined homoallylic alcohols and amino acid derivatives were synthesized via trapping of the in situ generated allylboronate derivatives with an appropriate electrophile. The synthetic scope of these types of multi-component reactions is broad as many different substrate allylic alcohols may be used together with various electrophiles. Several aspects of these reactions were studied, including different reagents, catalysts and electrophiles. Furthermore, we studied the possibility to use oxidizing reagents as an essential component in the functionalization of olefins. Two main strategies were utilized for these catalytic methods using palladium pincer complexes. The functional group was either transferred from the oxidizing reagent, or introduced via an oxidation-transmetallation route. We propose that both methods involve palladium(IV) intermediates thus expanding both the coordination sphere of palladium and the synthetic scope of pincer complex catalysis.
At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 11: In press.
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Thoresen, Mariska. "An investigation into the synergistic action of cellulose-degrading enzymes on complex substrates." Thesis, Rhodes University, 2015. http://hdl.handle.net/10962/d1017915.

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Gössl, Illdiko Maria. "Supramolecular structures of dendronized polymers and DNA on solid substrates." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2003. http://dx.doi.org/10.18452/14893.

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Komplexe aus entgegengesetzt geladenen Polyelektrolyten haben sowohl in der Biologie als auch in den Materialwissenschaften eine große Bedeutung. Im Mittelpunkt des Interesses stehen besonders die Kondensation der DNA in vitro, die Struktur des Nukleosoms im Zellkern, nicht-virale Systeme zur Transfektion von DNA in Zellen oder der Vorgang der layer-by-layer Adsorption. Verschiedene Theorien befassen sich mit den treibenden Kräften solcher Komplexbildungen. Allerdings standen experimentelle Untersuchungen auf diesem Gebiet bisher noch aus. Dieser Arbeit liegt die Fragestellung zu Grunde, ob es mit Hilfe der Rasterkraftmikroskopie möglich ist, die Struktur einzelner Polyelektrolytkomplexe, bestehend aus den beiden Polyelektrolyten DNA und dendronisierten Polymer, aufzuklären und ihre Komplexbildung zu untersuchen. Die Komplexe bildeten sich in Lösung und wurden anschließend auf einer unbeschichteten oder mit positiven Polymeren beschichteten Glimmeroberfläche adsorbiert. Auf der positiv beschichteten Glimmeroberfläche hafteten DNA-dendronisierte Polymer Komplexe mit einem Ladungsverhältnis von 1:1 bis 1:0.7 (DNA:dendronisiertes Polymer). Anhand der hochaufgelösten rasterkraftmikroskopischen Aufnahmen wurde ein Modell entwickelt, das die Umwicklung der DNA um das dendronisierte Polymer beschreibt. Der DNA-DNA Abstand ergab sich zu (2.30 ± 0.27) nm für den Komplex mit DNA und zweiter Generation dendronisierter Polymere und zu (2.16 ± 0.27) nm mit vierter Generation. Die theoretische Vorhersage der Überladung der Komplexe konnte experimentell bestätigt werden. Mit Hilfe der Rasterkraftmikroskopie konnte überdies der Einfluss des Salzgehaltes der Lösung auf die Bildung der Komplexe mit DNA und zweiter Generation dendronisierter Polymere untersucht werden. Wie man anhand des Zusammenwirkens von elektrostatischen Kräften und entropischen Wechselwirkungen bei der Adsorption von Polyelektrolyten vorhersagen kann, durchlief der DNA-DNA Abstand ein Minimum bei ansteigendem Salzgehalt. Bei sehr hohem Salzgehalt (2.4 M NaCl) konnte das Ablösen der DNA von dem Komplex beobachtet werden. Die untersuchten DNA/dendroniserten Polymer Komplexe bilden ein neues Modellsystem, mit dem einzelne Polyelektrolyt-Wechselwirkungen direkt untersucht werden können. Ein Vergleich der experimentellen Daten mit den vorhandenen Theorien zeigte, dass der Prozess des Überladens weitgehend durch elektrostatische Wechselwirkung zwischen den beiden Polyelektrolyten beschrieben werden kann. Sowohl entropische Beiträge als auch die Biegeenergie der umwickelnden DNA sind vernachlässigbar. Basierend auf diesen Ergebnissen können neue Trägerstrukturen für effizientere nicht-virale DNA-Transfektionssysteme entwickelt werden.
Complexes of oppositely charged polyelectrolytes play an important role in both biology and material science, for instance DNA condensation in vitro, nucleosomal structure, non-viral gene transfection systems as well as layer-by-layer adsorption. Although there are theories predicting overcharging of polyelectrolyte complexes, the driving forces are still under debate and systematic experimental studies on single polyelectrolytes remain challenging. Therefore the question arose if it is possible to analyze single polyelectrolyte complexes, using DNA and dendronized polymers, with the scanning force microscope in order to investigate the complexation in detail. For the complex analysis, the polyelectrolytes were allowed to interact in solution and then to adsorb on negatively charged mica or on mica coated with a positively charged polymer. Scanning force microscopy was used to investigate the adsorbed species. DNA/dendronized polymer complexes of charge ratio of 1/1 through 1/0.7 adsorbed on mica coated with a positively charged polymer. The analysis of high resolution molecular images indicated that DNA wraps around the dendronized polymer with an estimated pitch of (2.30 ± 0.27) nm and (2.16 ± 0.27) nm for dendronized polymers of generation two and four, respectively. In the proposed model the polyelectrolyte with the smaller linear charge density is wrapped around the more highly charged dendronized polymer, resulting in a negatively overcharged complex. This overcharging is consistent within recent theories of spontaneous overcharging of complexes of one polyelectrolyte wrapping around the other. Using the complex of DNA and dendronized polymers of second generation, the influence of monovalent salt concentration on the molecular structure was studied. By increasing the salt concentration the pitch showed a minimum as predicted by the interplay of electrostatic forces and entropic interactions of polyelectrolyte adsorption. At high salt concentration (2.4 M NaCl) the release of DNA from the complex can be observed. The results showed that the DNA/dendronized polymer system can be used as a new, high potential model system to investigate single polyelectrolyte interactions. With regard to recent theories, the experimental results indicate that the overcharging of the complex is mainly driven by electrostatic forces whereas contributions of counterion entropy and bending energy seem to be negligible. This understanding may be useful for the design of single polyelectrolyte complexes for non-viral gene delivery systems and might help to optimize the transfection efficiency based on the structure of the vector system.
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Zich, Judith. "Analysis of Mph1 kinase and its substrates in spindle checkpoint signalling." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/8253.

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Accurate chromosome segregation is crucial as mis-segregation results in aneuploidy, which can lead to severe diseases such as cancer. The spindle checkpoint monitors sister-chromatid attachment and inhibits the onset of anaphase until all chromosomes are correctly bi-oriented on the mitotic spindle. The spindle checkpoint machinery of S.pombe is composed of many proteins, one of which is the kinase Mph1 (Mps1p-like pombe homolog). It previously has been shown that Mph1 is essential for the spindle checkpoint but not whether this is due to its kinase activity. In this study we determined the role of Mph1 kinase activity in the spindle checkpoint. To do so a kinase-dead version of Mph1, which had no detectable kinase activity, was analysed. Using this kinase-dead allele we showed that lack of Mph1 kinase activity abolished the spindle checkpoint and led to chromosome missegregation. As a result of these two defects cell viability of cells lacking Mph1 kinase activity was severely impaired. These results led to the question of how Mph1 kinase activity regulates the spindle checkpoint. Spindle checkpoint signalling is thought to mainly take place at two sites, at the kinetochore and at the anaphase promoting complex (APC). The APC is an E3 ubiquitin ligase that drives cells into anaphase by targeting the separase inhibitor securin and cyclin B for degradation by the 26 S proteasome. Upon activation of the spindle checkpoint the APC is inhibited by the mitotic checkpoint complex (MCC) composed of Slp1, Mad2 and Mad3. In this study we wanted to test whether the regulatory role of Mph1 kinase in the spindle checkpoint is via MCC binding to the APC. Using the kinase-dead version of Mph1 we showed that Mad2 and Mad3 binding to the APC is severely impaired in the absence of Mph1 kinase activity. This result led to the hypothesis that Mph1 might regulate Mad2 and Mad3 binding Using kinase assays Mad2 and Mad3 were identified as in vitro substrates of Mph1 and phosphorylation sites in Mad2 and Mad3 were determined by mass spectrometry. Phosphorylation mutants of Mad2 and Mad3 showed spindle checkpoint defects, indicating that they are important Mph1 substrates.
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Kothe, Thomas. "Reductive Binding of C‒O and Nitro Substrates at a Pyrazolate-Bridged Preorganized Dinickel Scaffold." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-1524-B.

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Duan, Peng-Cheng. "A Dinuclear Dihydride Complex for Bimetallic Reductive Activation and Transformation of a Range of Inert Substrates." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://hdl.handle.net/11858/00-1735-0000-002E-E38C-2.

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Bakir, Ilyas. "Molecular studies of the γ-secretase complex activity and selectivity towards the two substrates APP and Notch." Thesis, Mälardalen University, School of Sustainable Development of Society and Technology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:mdh:diva-9622.

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Alzheimer Disease (AD) is the most common neurodegenerative disorder in the world. One of the neuropathological hallmarks of AD is the senile plaques in the brain. The plaques are mainly composed of the amyloid β (Aβ) peptide. Aβ is generated from the amyloid precursor protein, APP, when it is first cleaved by the β-secretase and subsequently the γ-secretase complex. The γ-secretase complex cleaves at different sites, called γ and ε, where the γ-cleavage site generates Aβ peptides of different lengths and ε-cleavage generates the APP intracellular domain (AICD). The two major forms of Aβ is 40 and 42 amino acids long peptides, where the latter is more prone to aggregate and is the main component in senile plaques. The γ-secretase complex is composed of four proteins; Pen-2, Aph-1, nicastrin and presenilin (PS). The PS protein harbours the catalytic site of the complex, where two aspartate residues in position 257 and 385 (Presenilin 1 numbering) are situated. Most Familial AD (FAD) mutations in the PS gene cause a change in the γ-cleavage site, leading to a shift from producing Aβ40 to the longer more toxic variant Aβ42. Frequently, this often leads to impairments of the AICD production. Another substrate for the γ-secretase complex is Notch. It is important to maintain the Notch signaling since an intracellular domain (NICD) is formed after cleavage by the γ-secretase complex in the membrane (S3-site) and this domain is involved in transcription of genes important for cell fate decisions.

It has been reported that certain APP luminal juxtamembrane mutations could drastically alter Aβ secretion, however their effect on AICD production remains unknown. In this study we want to analyse wether the juxtamembrane region is important for the AICD production. To gain more insight into the luminal juxtamembrane function for γ-secretase-dependent proteolysis, we have made a juxtamembrane chimeric construct. A four-residue sequence preceding the transmembrane domain (TMD) of APP (GSNK), was replaced by its topological counterpart from the human Notch1 receptor (PPAQ). The resulting chimeric vector C99GVP-PPAQ and the wildtype counterpart were expressed in cells lacking PS1 and PS2 (BD8) together with PS1wt. We observed that the chimeric construct did not alter production of AICD when using a cell based luciferase reporter gene assay monitoring AICD production. We also introduced a PS1 variant lacking a big portion of the large hydrophilic loop, PS1∆exon10, since our group has previously observed that this region affect Aβ production143. We found that the absence of the large hydrophilic loop in PS1 gave a 2-fold decrease in AICD-GVP formation from C99GVPwt compared to PS1wt.  The activity of PS1wt and PS1Δexon10 using C99GVP-PPAQ as a substrate gave similar result as the C99GVPwt substrate, i.e. a 2-fold decrease in AICD-GVP formation when comparing PS1Δexon10 with PS1wt. From this data we therefore suggest that the four residues in the juxtramembrane domain (JMD) (GSNK) is not altering ε-cleavage of APP when changed to Notch1 counterpart, PPAQ. Furthermore, we also show that the 2-fold decrease in AICD-production by the PS1Δexon10 molecule is not changed between the two substrates C99GVPwt and C99GVP-PPAQ. This indicates that the luminal region of APP is not directly involved in the ε-site processing. If the luminal region is affecting processing in the γ-cleavage sites, remains however to be investigated.

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Cowan, James. "The development and study of chelating substrates for the separation of metal ions in complex sample matrices." Thesis, University of Plymouth, 2002. http://hdl.handle.net/10026.1/1881.

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Stefani, Nicola. "Energy from crops: experimental study and dynamic simulation of biogas production by anaerobic digestion of complex substrates." Doctoral thesis, Università degli studi di Trieste, 2011. http://hdl.handle.net/10077/4506.

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2009/2010
Anaerobic digestion (AD) is a biological process which allows the removal of high organic-loading and potentially polluting substances by their transformation into biogas, a mixture of methane and carbon dioxide, prevalently. AD presents many other advantages: it has a low energy consumption and low construction costs with a relatively simple plant technology. Actually, since anaerobic bacteria work more efficiently at room temperature or higher, AD can be profitably applied in developing countries. Biogas production is a foundamental parameter of AD because it is the main index to be considered in a process economic evaluation and also because it gives a measure of its efficiency as well. Moreover, biogas production, and more frequently methane production, is often used as an index set to control the process. With the increase of energy price, the specific biogas production (SGP) of primary and residuals crops has become a goal for economic energy supply and, as a consequence, a rapid and effective method for measuring the gas produced has to be put forward, because there is not an accepted international standard yet. The effective knowledge of biogas production rate allows study of the biological process through macroscopic indicators, easily usable in industrial field, as well. The present study concerns the development and the validation of a technique for biogas production measurement and kinetic determination which adopts bench-mark laboratory-scale experiments with complex solid substrates, i.e. primary and residual energy crops. A laboratory-scale plant was designed and put up to perform this task. The equipment permits to carry out 4 contemporary tests because it is composed of 4 independent gas-lines, each of which connecting an anaerobic reactor to a gas-meter. Data from the experiments were continuously recorded by a data logger. The equipment was tested with synthetic substrate feeds of ethanol and sodium acetate. By a comparison between experimental gas production data and the theoretical ones, stoichiometrically calculated, the range of the error on methane productions resulted within ± 5%. In addition, the presence of oxygen amounts in the mixture, revealed the inconsistency of a test. These positive results allowed the implementation of different experiments to measure biogas produced from natural substrates. Apple, onion, corn straw, potato and winery wastes mixture were therefore tested in various experiments in order to calculate the SGP and SMA of the different crops. A new mathematical model for the description of complex substrate degradation was developed as well. The model was calibrated on the different biological systems and then applyed on real substrates to carry out their COD fractionation, to analyse the biological variable trends and to test the reliability of results. Finally, the reliability of a procedure for the evaluation of a two-step AD as compared to the one-step AD was tested by using apple and potato substrates.
La digestione anaerobica è un processo biologico che permette la rimozione di sostanze con alto carico organico, potenzialmente inquinanti, e la trasformazione di queste in biogas, costituito prevalentemente da metano e anidride carbonica. La digestione anaerobica ha anche ulteriori vantaggi: ha un basso consumo energetico, bassi costi di costruzione degli impianti, uniti ad una tecnologia impiantistica relativamente semplice. Inoltre, poiché i batteri anaerobici lavorano meglio a temperatura ambiente o superiore, si può applicare con profitto nei paesi in via di sviluppo. La produzione di biogas è un parametro fondamentale della digestione anaerobica perché è il principale indicatore cui fare riferimento nella valutazione economica del processo e perché allo stesso tempo fornisce anche una stima della sua efficienza. Inoltre, la produzione di biogas, o ancor più frequentemente quella di metano, è spesso usata come indice cui fare riferimento per un controllo di processo. Con l'aumento del costo energetico, risulta necessario definire correttamente ed efficacemente un metodo di misura del biogas prodotto, in particolare la produzione specifica (SGP) di biogas da biomasse primarie e residuali. Ad es, il test di attività metanogenica specifica (SMA), non ha ancora uno standard internazionale riconosciuto. La conoscenza effettiva della velocità di produzione di metano, infatti, apre la strada alla possibilità di studiare il processo biologico attraverso indicatori macroscopici, facili da applicare anche in un contesto industriale. Il presente lavoro riguarda lo sviluppo e la validazione di un metodo per effettuare la misurazione del biogas e la determinazione delle cinetiche di processo con esperimenti in scala di laboratorio effettuati su substrati complessi, ovvero biomasse primarie e residuali. Per fare ciò, è stato progettato e realizzato un impianto in scala di laboratorio. L'apparecchiatura permette di effettuare 4 prove contemporanee perché è provvista di 4 linee gas indipendenti, ciascuna delle quali connette un reattore anaerobico ad un gasometro. All'impianto è stato affiancato un sistema automatico di acquisizione dati, che permette la registrazione in continuo dei dati di produzione. L'impianto è stato verificato utilizzando alimentazioni di substrati sintetici quali etanolo e acetato di sodio. A seguito del confronto tra i dati di produzione di gas sperimentale e quelli di produzione teorica, calcolata stechiometricamente, l'errore nella risposta è risultato essere contenuto tra i valori di ± 5%. In aggiunta, la verifica del contenuto in ossigeno della miscela ha permesso di scartare le prove non conformi. Questi risultati positivi hanno consentito di passare ad esperimenti condotti su substrati naturali. Sono stati così testati, con i successivi esperimenti, mela, cipolla, patata, paglia di mais e residui solidi della lavorazione del vino, al fine di calcolarne l'SGP e l'SMA. E' anche stato sviluppato un nuovo modello matematico per simulare la degradazione di un substrato complesso. Tale modello è stato dapprima calibrato sui diversi sistemi biologici e in seguito applicato su alcuni substrati reali al fine di operare un frazionamento del COD, analizzare l'andamento delle variabili biologiche e verificare la compatibilità con i risultati sperimentali. Da ultimo, è stata verificata l'affidabilità di una procedura per la valutazione della digestione anaerobica a due fasi e il confronto con quella a fase singola, condotta con campioni di mele e di patate.
XXIII Ciclo
1979
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Books on the topic "Complex substrates"

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Gasparini, Evel. Il matriarcato slavo. Edited by Marcello Garzaniti and Donatella Possamai. Florence: Firenze University Press, 2010. http://dx.doi.org/10.36253/978-88-8453-999-1.

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This book on Slavic matriarchy is the result of the studies and researches that Evel Gasparini carried out over the span of his lifetime. Intrigued by the possibility of a close link between the collective ownership of the land and the ancient agricultural-matriarchal substrate of Slav culture, Gasparini launched on the titanic enterprise of analysing the archaeological and historical sources of early Slavic civilisation. Basing himself on a concept of culture elaborated in the ethnological field, he brought to light certain contradictions in the application of the Indo-European paradigm to Slavic culture and identified a series of elements illustrating the matriarchal substrate. Exploiting an uncommon knowledge of cultural anthropology and profound linguistic competencies, in this book Gasparini maps out a complex panorama ranging from the economy to the social structure and from the religious traditions to music and dance. Out of print for some time, the book is now proposed in a new, more convenient form, complete with an appendix on Finns and Slavs – which was originally intended as another chapter in the book but was then left out – a detailed preface by Gasparini's disciple Remo Faccani, and a bibliography of the scholar's oeuvre edited by Donatella Possamai.
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Goldstein, Alan S. Catalytic oxidations of organic substrates by transition metal salts. 1991.

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Estimation of complex permittivity of composite multilayer material at microwave frequency using waveguide measurements. Hampton, VA: National Aeronautics and Space Administration, Langley Research Center, 2003.

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Ang, Xiaolu Lulu Lim. Substrates of the SCF-beta-TRCP E3 ubiquitin ligase complex: Mechanisms of recognition and delivery to the proteasome. 2009.

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Garvey, Marjorie A. TMS: neurodevelopment and perinatal insults. Edited by Charles M. Epstein, Eric M. Wassermann, and Ulf Ziemann. Oxford University Press, 2012. http://dx.doi.org/10.1093/oxfordhb/9780198568926.013.0022.

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Neural substrate for changes in neuromotor skills of typically developing children involves the complex and organized maturation of underlying brain structures. This article gives an overview of the changes that occur in motor function, as children get older and those aspects of central nervous development which may form the neural substrates of motor function development. It describes those TMS evoked parameters, related to the motor system, that have been studied in both typically developing children and in those who have suffered perinatal insults to the central nervous system. TMS has its limitations and is especially useful when used in combination with other neurophysiological modalities. The focus for future studies should be on correlating TMS evoked parameters with behavioural measures in typically developing children and explanation of the neural substrates of the motor abnormalities in children with perinatal insults and developmental disabilities.
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Amzica, Florin, and Fernando H. Lopes da Silva. Cellular Substrates of Brain Rhythms. Edited by Donald L. Schomer and Fernando H. Lopes da Silva. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780190228484.003.0002.

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The purpose of this chapter is to familiarize the reader with the basic electrical patterns of the electroencephalogram (EEG). Brain cells (mainly neurons and glia) are organized in multiple levels of intricate networks. The cellular membranes are semipermeable media between extracellular and intracellular solutions, populated by ions and other electrically charged molecules. This represents the basis of electrical currents flowing across cellular membranes, further generating electromagnetic fields that radiate to the scalp electrodes, which record changes in the activity of brain cells. This chapter presents these concepts together with the mechanisms of building up the EEG signal. The chapter discusses the various behavioral conditions and neurophysiological mechanisms that modulate the activity of cells leading to the most common EEG patterns, such as the cellular interactions for alpha, beta, gamma, slow, delta, and theta oscillations, DC shifts, and some particular waveforms such as sleep spindles and K-complexes and nu-complexes.
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Guo, Yong, and Claudia F. Lucchinetti. Taking a Microscopic Look at Multiple Sclerosis. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780199341016.003.0005.

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The pathology of multiple sclerosis is complex, extends beyond the white matter plaque, and is influenced by stage of demyelinating activity, clinical course, disease duration, and treatment. Technological advances in immunology, molecular biology, and “omic” biology have provided novel insights into the mechanisms for development of white matter plaques, axonal damage, cortical demyelination, and disease progression. Detailed, systematic, and statistically rigorous pathological studies on clinically well-characterized MS cohorts have helped define the heterogeneous pathological substrates of MS and unravel the complex molecular pathogenic mechanisms, with the ultimate goal of identifying targets for therapeutic interventions. It is increasingly clear that the use of human tissues is imperative to improve current diagnostic, prognostic, and therapeutic modalities. Preclinical animal models have been invaluable for discovery of key immune processes, basic disease mechanisms, and candidate immune targeting strategies, but the conclusions have yet be reconciled with the essential features of the human disease.
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Guo, Lucie Y. Aph-1 is a substrate-binding site within the γ-secretase complex. 2010.

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Liaw, Ean-Tun. Characterization of substrate-velocity relationships for the cellulase enzyme complex from Trichoderma viride. 1989.

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Vaghi, M. M., and T. W. Robbins. Task-Based Functional Neuroimaging Studies of Obsessive-Compulsive Disorder: A Hypothesis-Driven Review. Edited by Christopher Pittenger. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780190228163.003.0022.

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The neurobiological basis of Obsessive Compulsive Disorder (OCD) has been probed using functional magnetic resonance in hundreds of studies over three decades. This complex literature can be syntheized using a theory-informed approach. At a theoretical level, separable, independent, constructs of relevance to OCD have been identified. At the experimental level, extensive translational evidence has provided an account that relates specific brain systems to these neuropsychological constructs. Parallels between neural substrates implicated in OCD and functional specialization of different brain regions suggest that abnormalities within fronto-striatal circuitry impinge on executive functions, and their subcomponents, and on goal-directed learning and habit formation. In OCD, this is reflected at a functional level in patterns of abnormal activations in particular brain regions during specific cognitive tasks. However, many issues still need to be addressed. The authors suggest that the experimental context might represent a pivotal variable that should be taken into account.
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Book chapters on the topic "Complex substrates"

1

Semenov, S., V. M. Starov, M. G. Velarde, and R. G. Rubio. "Evaporation of Sessile Droplets of Liquid on Solid Substrates." In Understanding Complex Systems, 285–300. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-34070-3_26.

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Stymne, Sten, Gareth Griffiths, and Keith Stobart. "Desaturation of Fatty Acids on Complex-lipid Substrates." In The Metabolism, Structure, and Function of Plant Lipids, 405–12. Boston, MA: Springer New York, 1987. http://dx.doi.org/10.1007/978-1-4684-5263-1_74.

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Chen, Z., W. Wang, and B. Cotterell. "The Evaluation of the Fracture Strain of ITO Films on Polymeric Substrates." In Properties of Complex Inorganic Solids 2, 409–16. Boston, MA: Springer US, 2000. http://dx.doi.org/10.1007/978-1-4615-1205-9_30.

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Essaaidi, Mohamed, and Otman Mrabet. "Dielectric Substrates Anisotropy Effects on the Characteristics of Microstrip Structures." In Advances in Electromagnetics of Complex Media and Metamaterials, 449–60. Dordrecht: Springer Netherlands, 2002. http://dx.doi.org/10.1007/978-94-007-1067-2_27.

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Schlage, Pascal, Fabian E. Egli, and Ulrich auf dem Keller. "Time-Resolved Analysis of Matrix Metalloproteinase Substrates in Complex Samples." In Methods in Molecular Biology, 185–98. New York, NY: Springer New York, 2017. http://dx.doi.org/10.1007/978-1-4939-6863-3_9.

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Delan-Forino, Clémentine, and David Tollervey. "Mapping Exosome–Substrate Interactions In Vivo by UV Cross-Linking." In Methods in Molecular Biology, 105–26. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9822-7_6.

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AbstractThe RNA exosome complex functions in both the accurate processing and rapid degradation of many classes of RNA in eukaryotes and Archaea. Functional and structural analyses indicate that RNA can either be threaded through the central channel of the exosome or more directly access the active sites of the ribonucleases Rrp44 and Rrp6, but in most cases, it remains unclear how many substrates follow each pathway in vivo. Here we describe the method for using an UV cross-linking technique termed CRAC to generate stringent, transcriptome-wide mapping of exosome–substrate interaction sites in vivo and at base-pair resolution.
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Beloglazova, Natalia, Sofia Lemak, Robert Flick, and Alexander F. Yakunin. "Analysis of Nuclease Activity of Cas1 Proteins Against Complex DNA Substrates." In Methods in Molecular Biology, 251–64. New York, NY: Springer New York, 2015. http://dx.doi.org/10.1007/978-1-4939-2687-9_16.

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Varfolomeev, Sergey, Bella Grigorenko, Sofya Lushchekina, Patrick Masson, Galina Mahaeva, and Alexander Nemuchin. "Human cholinesterases." In ORGANOPHOSPHORUS NEUROTOXINS, 69–126. ru: Publishing Center RIOR, 2020. http://dx.doi.org/10.29039/21_069-126.

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The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.
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Varfolomeev, Sergey, Bella Grigorenko, Sofya Lushchekina, and Alexander Nemuchin. "Human cholinesterases." In Organophosphorous Neurotoxins, 63–120. ru: Publishing Center RIOR, 2020. http://dx.doi.org/10.29039/chapter_5e4132b5f22366.15634219.

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The work is devoted to modeling the elementary stages of the hydrolysis reaction in the active site of enzymes belonging to the class of cholinesterases — acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The study allowed to describe at the molecular level the effect of the polymorphic modification of BChE, causing serious physiolog ical consequences. Cholinesterase plays a crucial role in the human body. AChE is one of the key enzymes of the central nervous system, and BChE performs protective functions in the body. According to the results of calculations using the combined method of quantum and molecular mechanics (KM/MM), the mechanism of the hydrolysis of the native acetylcholine substrate in the AChE active center was detailed. For a series of ester substrates, a method for estimation of dependence of the enzyme reactivity on the structure of the substrate has been developed. The mechanism of hydrolysis of the muscle relaxant of succininylcholine BChE and the effect of the Asp70Gly polymorph on it were studied. Using various computer simulation methods, the stability of the enzyme-substrate complex of two enzyme variants with succinylcholine was studied.
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Mäkelä, Miia R., and Kristiina Hildén. "Efficient Extraction Method for High Quality Fungal RNA from Complex Lignocellulosic Substrates." In Methods in Molecular Biology, 69–73. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-7804-5_6.

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Conference papers on the topic "Complex substrates"

1

Olson Reichhardt, C. J., D. Ray, and C. Reichhardt. "Active matter transport on complex substrates." In SPIE NanoScience + Engineering, edited by Kishan Dholakia and Gabriel C. Spalding. SPIE, 2014. http://dx.doi.org/10.1117/12.2063481.

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Bie, Youqin. "Replicated mirros with magnetic steel as substrates." In 15th Int'l Optics in Complex Sys. Garmisch, FRG, edited by F. Lanzl, H. J. Preuss, and G. Weigelt. SPIE, 1990. http://dx.doi.org/10.1117/12.34914.

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Nakayama, Hideyuki. "Surface Undulation Appearing by Continuous Temperature Elevation of Supercooled Liquids on Metal Substrates." In SLOW DYNAMICS IN COMPLEX SYSTEMS: 3rd International Symposium on Slow Dynamics in Complex Systems. AIP, 2004. http://dx.doi.org/10.1063/1.1764079.

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Modi, Mitul, Deepak Kulkarni, Andy Bao, Ibrahim Bekar, and Steve Cho. "Analytical Homogenization for Microelectronic Substrates." In ASME 2007 International Mechanical Engineering Congress and Exposition. ASMEDC, 2007. http://dx.doi.org/10.1115/imece2007-43460.

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Organic substrates used in microelectronic packages contain complex micro-via, plated-through-hole via, and copper networks in order to accommodate stringent electrical requirements of power delivery and I/O. These complex layouts can produce significant inhomogeniety in the in-plane and out-of-plane mechanical properties of the substrates. An analytical homogenization methodology has been established that uses substrate design geometries and material properties such as the copper density and via locations with two-phase micromechanics models to homogenize the substrate. In this paper the homogenization methodology is discussed and validated with experimental data. A comparison of the various two-phase micromechanics models commonly found in literature are reviewed and compared for their applicability to accurately homogenize organic substrates. Two case studies are provided to highlight the importance of accurately modeling the anisotropy of the substrates for temperature cycling and bend reliability predictions.
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Kotlikov, E. N., Yu A. Novikova, and Yu N. Tsarev. "DEFINITION OF REFRACTIVE INDICES OF MgBaF4 FILMS ON Si SUBSTRATES." In MODELING AND SITUATIONAL QUALITY MANAGEMENT OF COMPLEX SYSTEMS. St. Petersburg State University of Aerospace Instrumentation, 2020. http://dx.doi.org/10.31799/978-5-8088-1449-3-2020-1-80-85.

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Wanga, Shanshan, Peixiang Ma, Feng Qu, and Yulin Deng. "Application of Biologically Functionalized Chromatography to Simulate the Interaction Between MAO and Substrates." In 2007 IEEE/ICME International Conference on Complex Medical Engineering. IEEE, 2007. http://dx.doi.org/10.1109/iccme.2007.4382057.

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Vermeer, C., BA M. Soute, and MM W. Ulrich. "IN VITRO CARBOXYLATION OF EXOGENOUS PROTEIN SUBSTRATES BY VITAMIN K-DEPENDENT CARBOXYLASE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643994.

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In vivo treatment of experimental animals with vitamin K-antagonists induces the accumulation of non-carboxylated coagulation factor precursors in the liver, where they are tightly bound to vitamin K-dependent carboxylase. If hepatic carboxylase is isolated from warfarin-treated animals, it is obtained therefore almost exclusively in the form of an enzyme/substrate complex. If carboxylase is prepared from non-treated animals, on the other hand, the resulting enzyme is predominantly substrate-free. Small substrates like F L E E L or decarboxylated osteocalcinare carboxylated equally well by both types of carboxylase, but protein substrates(Mr > 30 000) are recognized exclusively by substrate-free carboxylase.Initial attempts to purify carboxylasewere performed with livers from warfarin-treated cows as a starting material. Antibodies against the normal blood coagulation factors crossreact with the hepatic precursor proteins so that the enzyme/substrate complexes could be specifically extracted from detergent-solubilized microsomes by the substrate/antibody interaction. This procedure resulted ina substantial purification of carboxylase, but because its endogenous substrate remained firmly bound, even after it had been carboxylated in vitro, the enzyme system was not suitable for the carboxylation of protein substrates.Therefore a second strategy was developed by which substrate-free carboxylase (from normal livers) was partly purified by sequential extraction of the microsomal membranes with detergents, followed by ammonium sulfate precipitation and size exclusion chromatography.This procedure resulted in a soluble carboxylase complex, still consisting of 7 proteins and phosphatidylcholine. Although further dissociation of the complex resulted in a complete loss of activity, it is not sure if all components play a role in the carboxylation reaction. Exogenous substrates which could be carboxylated by substrate-free carboxylase were: the penta-peptide F L E E L, descarboxyprothrombin from bovine plasma, thermally decarboxylated osteocalcin from bovine bone and non-car-boxy lated coagulaton factor precursors which had been produced by recombinant-DNA techniques in various laboratories. The . efficiency of CO^ incorporation was: 1 mole per 100 moles of F L E E L, 1 mole per 240 moles of descarboxy-prothrombin, 1 mole per mole of decarboxylated osteocalcin and 8 moles per mole of a recombinant factor IX precursor. We assume that the high efficiency with which the recombinant coagulation factor precursors were carboxylated is due to the presence of at least part of their leader sequence. The importance of the aminoacid chain preceding the first carboxylatable Glu residue is demonstrated by the fact that descarboxylated osteocalcin of bovine origin is carboxylated with a relatively high efficiency, whereas descarboxylated osteocalcin from monkey bone is not recognized atal.. Yet the only difference between the two substrates is found in their aminoacids 3 and 4, whereas the first carboxylatable Glu occurs at position 17. It seems, therefore, that the aminoacids 1-16 in bovine osteocalcin mimic to some extent part of the leader sequence in the coagulation factor precursors. Chemical or biochemical modification of decarboxylated osteocalcin might reveal which structural features contribute to its recognition by hepatic carboxylase.The optimal conditions for carboxylation include a high concentration of dithiols (e.g. DTT) and under these conditions disulfide bridges are reduced. Obviously this will lead to a complete destruction of the biological activity of various carboxylated products. Therefore we have searched for a more natural reducing system and it was found that the bacterial thioredoxin/thiore-doxin-reductase system in the presence of 40 uM NADFH was able to replace DTT in the reaction mixtures. Since a comparable system also occurs in calf liver it seems not unlikely that this is the physiological counterpart of the dithiols used in vitro.
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Wiatrowska, Aneta, Karolina Fiaczyk, Piotr Kowalczewski, Mateusz Lysien, Lukasz Witczak, Jolanta Gadzalinska, Iwona Gradzka-Kurzaj, Ludovic Schneider, Lukasz Kosior, and Filip Granek. "Printing of Micrometer-Size Features on Complex Substrates for System Integration." In 2022 IEEE 9th Electronics System-Integration Technology Conference (ESTC). IEEE, 2022. http://dx.doi.org/10.1109/estc55720.2022.9939386.

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Xiuqin Jia, Shengfu Lu, Ning Zhong, Yiyu Yao, Kuncheng Li, and Yanhui Yang. "Common and distinct neural substrates of forward-chaining and backward-chaining syllogistic reasoning." In 2009 ICME International Conference on Complex Medical Engineering - CME 2009. IEEE, 2009. http://dx.doi.org/10.1109/iccme.2009.4906618.

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Soukup, L., M. Šícha, L. Jastrabík, and M. Novák. "Thin film deposition on internal walls of cavities and complex hollow substrates." In The XXII. international conference on phenomena in ionized gases (ICPIG). AIP, 1996. http://dx.doi.org/10.1063/1.50122.

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Reports on the topic "Complex substrates"

1

Rine, Kristin, Roger Christopherson, and Jason Ransom. Harlequin duck (Histrionicus histrionicus) occurrence and habitat selection in North Cascades National Park Service Complex, Washington. National Park Service, April 2022. http://dx.doi.org/10.36967/nrr-2293127.

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Harlequin ducks (Histrionicus histrionicus) are sea ducks that migrate inland each spring to nest along fast-flowing mountain streams. They are considered one of the most imperiled duck species in North America and occur in two distinct populations on the Atlantic and Pacific coasts. The Pacific coast population includes Washington State, where harlequin ducks breed in the Olympic, Cascade, and Selkirk Mountains. This species is designated as a Management Priority Species by the National Park Service within North Cascades National Park Service Complex (NOCA). This report summarizes harlequin duck surveys conducted during 15 years across a 27-year period (1990 and 2017) on major streams within NOCA, and incidental observations collected from 1968–2021. The primary objectives of these surveys were to 1) document the distribution and abundance of harlequin duck observations within NOCA boundaries, 2) describe productivity (number of broods and brood size), 3) describe breeding chronology of harlequin ducks, and 4) describe habitat characteristics of breeding streams. Sixty-eight stream surveys over 15 years resulted in observations of 623 individual harlequin ducks comprising various demographics, including single adults, pairs, and broods. In addition, we collected 184 incidental observations of harlequin ducks from visitors and staff between 1968–2021. Harlequin ducks were observed on 22 separate second- to sixth-order streams throughout NOCA across the entire 53-year span of data, both incidentally and during harlequin duck surveys by Park staff. Harlequin ducks were detected on 8 of the 13 streams that were actively surveyed. Excluding recounts, 88.7% (n = 330) of individual harlequin duck observations during surveys occurred in the Stehekin River drainage. Between all surveys and incidental observations, 135 unpaired females without broods were sighted across all NOCA waterways. Thirty-nine broods were recorded between NOCA surveys and incidental observations, with a mean brood size of 3.61 (± 1.44 SD; range = 2–10). Breeding pairs were recorded as early as April 5 and were seen on streams until June 15, a period of less than seven weeks (median: May 2), but most pairs were observed within a 3-week span, between April 26 and May 17. Single females (unpaired with a male, with (an)other female(s), or with a brood) were observed on streams between April 26 and August 25 (median: July 3), though most observations were made within a 5-week period between June 12 and July 19. Habitat data collected at adult harlequin duck observation sites indicate that the birds often used stream reaches with features that are characteristic of high-energy running water. While adults occupied all instream habitat types identified, non-braided rapids and riffles were used most frequently, followed by pools and backwaters. Larger instream substrate sizes (cobbles and boulders) were present at most observation sites. Adult harlequin ducks were more often found at locations that lacked visible drifting or lodged woody debris, but drift debris was a slightly more abundant debris type. The presence of gravel bars and at least one loafing site was common. Adult harlequin ducks were more often observed in association with vegetation that offered some cover over the channel, but not where banks were undercut. The average channel width at adult observation sites was 34.0 m (range: 6-80 m; n = 114) and 27.6 m (± 15.7 m; range: 10-60 m; n = 12) at brood observation sites. Compared to adult harlequin duck sites, broods were observed more frequently in low velocity habitat (pools, backwaters), but rarely in rapids. Cobble and boulder substrates were still the most dominant substrate type. Contrary to adult ducks, broods were observed most often observed in meandering stream channels, a morphology indicative of low gradient, low velocity stream reaches. Most broods were observed in stream reaches with gravel bars, loafing sites, and...
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2

Henson, B. F., S. J. Buelow, and J. M. Robinson. Modification of heterogeneous chemistry by complex substrate morphology. Office of Scientific and Technical Information (OSTI), December 1998. http://dx.doi.org/10.2172/562542.

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Elbaum, Michael, and Peter J. Christie. Type IV Secretion System of Agrobacterium tumefaciens: Components and Structures. United States Department of Agriculture, March 2013. http://dx.doi.org/10.32747/2013.7699848.bard.

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Objectives: The overall goal of the project was to build an ultrastructural model of the Agrobacterium tumefaciens type IV secretion system (T4SS) based on electron microscopy, genetics, and immunolocalization of its components. There were four original aims: Aim 1: Define the contributions of contact-dependent and -independent plant signals to formation of novel morphological changes at the A. tumefaciens polar membrane. Aim 2: Genetic basis for morphological changes at the A. tumefaciens polar membrane. Aim 3: Immuno-localization of VirB proteins Aim 4: Structural definition of the substrate translocation route. There were no major revisions to the aims, and the work focused on the above questions. Background: Agrobacterium presents a unique example of inter-kingdom gene transfer. The process involves cell to cell transfer of both protein and DNA substrates via a contact-dependent mechanism akin to bacterial conjugation. Transfer is mediated by a T4SS. Intensive study of the Agrobacterium T4SS has made it an archetypal model for the genetics and biochemistry. The channel is assembled from eleven protein components encoded on the B operon in the virulence region of the tumor-inducing plasmid, plus an additional coupling protein, VirD4. During the course of our project two structural studies were published presenting X-ray crystallography and three-dimensional reconstruction from electron microscopy of a core complex of the channel assembled in vitro from homologous proteins of E. coli, representing VirB7, VirB9, and VirB10. Another study was published claiming that the secretion channels in Agrobacterium appear on helical arrays around the membrane perimeter and along the entire length of the bacterium. Helical arrangements in bacterial membranes have since fallen from favor however, and that finding was partially retracted in a second publication. Overall, the localization of the T4SS within the bacterial membranes remains enigmatic in the literature, and we believe that our results from this project make a significant advance. Summary of achievements : We found that polar inflations and other membrane disturbances relate to the activation conditions rather than to virulence protein expression. Activation requires low pH and nutrient-poor medium. These stress conditions are also reflected in DNA condensation to varying degrees. Nonetheless, they must be considered in modeling the T4SS as they represent the relevant conditions for its expression and activity. We identified the T4SS core component VirB7 at native expression levels using state of the art super-resolution light microscopy. This marker of the secretion system was found almost exclusively at the cell poles, and typically one pole. Immuno-electron microscopy identified the protein at the inner membrane, rather than at bridges across the inner and outer membranes. This suggests a rare or transient assembly of the secretion-competent channel, or alternatively a two-step secretion involving an intermediate step in the periplasmic space. We followed the expression of the major secreted effector, VirE2. This is a single-stranded DNA binding protein that forms a capsid around the transferred oligonucleotide, adapting the bacterial conjugation to the eukaryotic host. We found that over-expressed VirE2 forms filamentous complexes in the bacterial cytoplasm that could be observed both by conventional fluorescence microscopy and by correlative electron cryo-tomography. Using a non-retentive mutant we observed secretion of VirE2 from bacterial poles. We labeled the secreted substrates in vivo in order detect their secretion and appearance in the plant cells. However the low transfer efficiency and significant background signal have so far hampered this approach.
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Choudhary, Ruplal, Victor Rodov, Punit Kohli, Elena Poverenov, John Haddock, and Moshe Shemesh. Antimicrobial functionalized nanoparticles for enhancing food safety and quality. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598156.bard.

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Original objectives The general goal of the project was to utilize the bactericidal potential of curcumin- functionalizednanostructures (CFN) for reinforcement of food safety by developing active antimicrobial food-contact surfaces. In order to reach the goal, the following secondary tasks were pursued: (a) further enhancement of the CFN activity based on understanding their mode of action; (b) preparing efficient antimicrobial surfaces, investigating and optimizing their performance; (c) testing the efficacy of the antimicrobial surfaces in real food trials. Background to the topic The project dealt with reducing microbial food spoilage and safety hazards. Cross-contamination through food-contact surfaces is one of the major safety concerns, aggravated by bacterial biofilm formation. The project implemented nanotech methods to develop novel antimicrobial food-contact materials based on natural compounds. Food-grade phenylpropanoidcurcumin was chosen as the most promising active principle for this research. Major conclusions, solutions, achievements In agreement with the original plan, the following research tasks were performed. Optimization of particles structure and composition. Three types of curcumin-functionalizednanostructures were developed and tested: liposome-type polydiacetylenenanovesicles, surface- stabilized nanoparticles and methyl-β-cyclodextrin inclusion complexes (MBCD). The three types had similar minimal inhibitory concentration but different mode of action. Nanovesicles and inclusion complexes were bactericidal while the nanoparticlesbacteriostatic. The difference might be due to different paths of curcumin penetration into bacterial cell. Enhancing the antimicrobial efficacy of CFN by photosensitization. Light exposure strengthened the bactericidal efficacy of curcumin-MBCD inclusion complexes approximately three-fold and enhanced the bacterial death on curcumin-coated plastic surfaces. Investigating the mode of action of CFN. Toxicoproteomic study revealed oxidative stress in curcumin-treated cells of E. coli. In the dark, this effect was alleviated by cellular adaptive responses. Under light, the enhanced ROS burst overrode the cellular adaptive mechanisms, disrupted the iron metabolism and synthesis of Fe-S clusters, eventually leading to cell death. Developing industrially-feasible methods of binding CFN to food-contact surfaces. CFN binding methods were developed for various substrates: covalent binding (binding nanovesicles to glass, plastic and metal), sonochemical impregnation (binding nanoparticles to plastics) and electrostatic layer-by-layer coating (binding inclusion complexes to glass and plastics). Investigating the performance of CFN-coated surfaces. Flexible and rigid plastic materials and glass coated with CFN demonstrated bactericidal activity towards Gram-negative (E. coli) and Gram-positive (Bac. cereus) bacteria. In addition, CFN-impregnated plastic material inhibited bacterial attachment and biofilm development. Testing the efficacy of CFN in food preservation trials. Efficient cold pasteurization of tender coconut water inoculated with E. coli and Listeriamonocytogeneswas performed by circulation through a column filled with CFN-coated glass beads. Combination of curcumin coating with blue light prevented bacterial cross contamination of fresh-cut melons through plastic surfaces contaminated with E. coli or Bac. licheniformis. Furthermore, coating of strawberries with CFN reduced fruit spoilage during simulated transportation extending the shelf life by 2-3 days. Implications, both scientific and agricultural BARD Report - Project4680 Page 2 of 17 Antimicrobial food-contact nanomaterials based on natural active principles will preserve food quality and ensure safety. Understanding mode of antimicrobial action of curcumin will allow enhancing its dark efficacy, e.g. by targeting the microbial cellular adaptation mechanisms.
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Irudayaraj, Joseph, Ze'ev Schmilovitch, Amos Mizrach, Giora Kritzman, and Chitrita DebRoy. Rapid detection of food borne pathogens and non-pathogens in fresh produce using FT-IRS and raman spectroscopy. United States Department of Agriculture, October 2004. http://dx.doi.org/10.32747/2004.7587221.bard.

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Rapid detection of pathogens and hazardous elements in fresh fruits and vegetables after harvest requires the use of advanced sensor technology at each step in the farm-to-consumer or farm-to-processing sequence. Fourier-transform infrared (FTIR) spectroscopy and the complementary Raman spectroscopy, an advanced optical technique based on light scattering will be investigated for rapid and on-site assessment of produce safety. Paving the way toward the development of this innovative methodology, specific original objectives were to (1) identify and distinguish different serotypes of Escherichia coli, Listeria monocytogenes, Salmonella typhimurium, and Bacillus cereus by FTIR and Raman spectroscopy, (2) develop spectroscopic fingerprint patterns and detection methodology for fungi such as Aspergillus, Rhizopus, Fusarium, and Penicillium (3) to validate a universal spectroscopic procedure to detect foodborne pathogens and non-pathogens in food systems. The original objectives proposed were very ambitious hence modifications were necessary to fit with the funding. Elaborate experiments were conducted for sensitivity, additionally, testing a wide range of pathogens (more than selected list proposed) was also necessary to demonstrate the robustness of the instruments, most crucially, algorithms for differentiating a specific organism of interest in mixed cultures was conceptualized and validated, and finally neural network and chemometric models were tested on a variety of applications. Food systems tested were apple juice and buffer systems. Pathogens tested include Enterococcus faecium, Salmonella enteritidis, Salmonella typhimurium, Bacillus cereus, Yersinia enterocolitis, Shigella boydii, Staphylococus aureus, Serratiamarcescens, Pseudomonas vulgaris, Vibrio cholerae, Hafniaalvei, Enterobacter cloacae, Enterobacter aerogenes, E. coli (O103, O55, O121, O30 and O26), Aspergillus niger (NRRL 326) and Fusarium verticilliodes (NRRL 13586), Saccharomyces cerevisiae (ATCC 24859), Lactobacillus casei (ATCC 11443), Erwinia carotovora pv. carotovora and Clavibacter michiganense. Sensitivity of the FTIR detection was 103CFU/ml and a clear differentiation was obtained between the different organisms both at the species as well as at the strain level for the tested pathogens. A very crucial step in the direction of analyzing mixed cultures was taken. The vector based algorithm was able to identify a target pathogen of interest in a mixture of up to three organisms. Efforts will be made to extend this to 10-12 key pathogens. The experience gained was very helpful in laying the foundations for extracting the true fingerprint of a specific pathogen irrespective of the background substrate. This is very crucial especially when experimenting with solid samples as well as complex food matrices. Spectroscopic techniques, especially FTIR and Raman methods are being pursued by agencies such as DARPA and Department of Defense to combat homeland security. Through the BARD US-3296-02 feasibility grant, the foundations for detection, sample handling, and the needed algorithms and models were developed. Successive efforts will be made in transferring the methodology to fruit surfaces and to other complex food matrices which can be accomplished with creative sampling methods and experimentation. Even a marginal success in this direction will result in a very significant breakthrough because FTIR and Raman methods, in spite of their limitations are still one of most rapid and nondestructive methods available. Continued interest and efforts in improving the components as well as the refinement of the procedures is bound to result in a significant breakthrough in sensor technology for food safety and biosecurity.
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Chen, Junping, Zach Adam, and Arie Admon. The Role of FtsH11 Protease in Chloroplast Biogenesis and Maintenance at Elevated Temperatures in Model and Crop Plants. United States Department of Agriculture, May 2013. http://dx.doi.org/10.32747/2013.7699845.bard.

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specific objectives of this proposal were to: 1) determine the location, topology, and oligomerization of FtsH11 protease; 2) identify the substrate/s of FtsH11 and the downstream components involved in maintaining thermostability of chloroplasts; 3) identify new elements involved in FtsH11 protease regulatory network related to HT adaptation processes in chloroplast; 4) Study the role of FtsH11 homologs from crop species in HT tolerance. Background to the topic: HT-tolerant varieties that maintain high photosynthetic efficiency at HT, and cope better with daily and seasonal temperature fluctuations are in great need to alleviate the effect of global warming on food production. Photosynthesis is a very complex process requiring accurate coordination of many complex systems and constant adjustments to the changing environments. Proteolytic activities mediated by various proteases in chloroplast are essential part of this process and critical for maintaining normal chloroplast functions under HT. However, little is known about mechanisms that contribute to adaptation of photosynthetic processes to HT. Our study has shown that a chloroplast-targeted Arabidopsis FtsH11 protease plays an essential and specific role in maintaining thermostability of thylakoids and normal photosynthesis at moderate HT. We hypothesized that FtsH11 homologs recently identified in other plant species might have roles similarly to that of AtFtsH1. Thus, dissecting the underlying mechanisms of FtsH11 in the adaptation mechanisms in chloroplasts to HT stress and other elements involved will aid our effort to produce more agricultural products in less favorable environments. Major conclusions, solutions, achievements - Identified the chloroplast inner envelope membrane localization of FtsH11. - Revealed a specific association of FtsH11 with the a and b subunits of CPN60. - Identified the involvement of ARC6, a protein coordinates chloroplast division machineries in plants, in FtsH11 mediated HT adaptation process in chloroplast. -Reveal possible association of a polyribonucleotide nucleotidyltransferase (cpPNPase), coded by At3G03710, with FtsH11 mediated HT adaptation process in chloroplast. - Mapped 4 additional loci in FtsH11 mediated HT adaptation network in chloroplast. - Demonstrated importance of the proteolytic activity of FtsH11 for thermotolerance, in addition to the ATPase activity. - Demonstrated a conserved role of plant FtsH11 proteases in chloroplast biogenesis and in maintaining structural and functional thermostability of chloroplast at elevated temperatures. Implications, both scientific and agricultural:Three different components interacting with FtsH11 were identified during the course of this study. At present, it is not known whether these proteins are directly involved in FtsH11mediated thermotolerance network in chloroplast and/or how these elements are interrelated. Studies aiming to connect the dot among biological functions of these networks are underway in both labs. Nevertheless, in bacteria where it was first studied, FtsH functions in heat shock response by regulating transcription level of σ32, a heat chock factor regulates HSPsexpression. FtsH also involves in control of biosynthesis of membrane components and quality control of membrane proteins etc. In plants, both Arc 6 and CPN60 identified in this study are essential in chloroplast division and developments as mutation of either one impairs chloroplast division in Arabidopsis. The facts that we have found the specific association of both α and β CPN60 with FtsH11 protein biochemically, the suppression/ enhancement of ftsh11 thermosensitive phenotype by arc6 /pnp allele genetically, implicate inter-connection of these networks via FtsH11 mediated network(s) in regulating the dynamic adaptation processes of chloroplast to temperature increases at transcriptional, translational and post-translational levels. The conserved role of FtsH11 proteases in maintaining thermostability of chloroplast at HT demonstrated here provides a foundation for improving crop photosynthetic performance at high temperatures.
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Morrison, Mark, and Joshuah Miron. Molecular-Based Analysis of Cellulose Binding Proteins Involved with Adherence to Cellulose by Ruminococcus albus. United States Department of Agriculture, November 2000. http://dx.doi.org/10.32747/2000.7695844.bard.

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At the beginning of this project, it was clear that R. albus adhered tightly to cellulose and its efficient degradation of this polysaccharide was dependent on micromolar concentrations of phenylacetic acid (PAA) and phenylpropionic acid (PPA). The objectives for our research were: i) to identify how many different kinds of cellulose binding proteins are produced by Ruminococcus albus; ii) to isolate and clone the genes encoding some of these proteins from the same bacterium; iii) to determine where these various proteins were located and; iv) quantify the relative importance of these proteins in affecting the rate and extent to which the bacterium becomes attached to cellulose. BARD support has facilitated a number of breakthroughs relevant to our fundamental understanding of the adhesion process. First, R. albus possesses multiple mechanisms for adhesion to cellulose. The P.I.'s laboratory has discovered a novel cellulose-binding protein (CbpC) that belongs to the Pil-protein family, and in particular, the type 4 fimbrial proteins. We have also obtained genetic and biochemical evidence demonstrating that, in addition to CbpC-mediated adhesion, R. albus also produces a cellulosome-like complex for adhesion. These breakthroughs resulted from the isolation (in Israel and the US) of spontaneously arising mutants of R. albus strains SY3 and 8, which were completely or partially defective in adhesion to cellulose, respectively. While the SY3 mutant strain was incapable of growth with cellulose as the sole carbon source, the strain 8 mutants showed varying abilities to degrade and grow with cellulose. Biochemical and gene cloning experiments have been used in Israel and the US, respectively, to identify what are believed to be key components of a cellulosome. This combination of cellulose adhesion mechanisms has not been identified previously in any bacterium. Second, differential display, reverse transcription polymerase chain reaction (DD RT-PCR) has been developed for use with R. albus. A major limitation to cellulose research has been the intractability of cellulolytic bacteria to genetic manipulation by techniques such as transposon mutagenesis and gene displacement. The P.I.'s successfully developed DD RT- PCR, which expanded the scope of our research beyond the original objectives of the project, and a subset of the transcripts conditionally expressed in response to PAA and PPA have been identified and characterized. Third, proteins immunochemically related to the CbpC protein of R. albus 8 are present in other R. albus strains and F. intestinalis, Western immunoblots have been used to examine additional strains of R. albus, as well as other cellulolytic bacteria of ruminant origin, for production of proteins immunochemically related to the CbpC protein. The results of these experiments showed that R. albus strains SY3, 7 and B199 all possess a protein of ~25 kDa which cross-reacts with polyclonal anti-CbpC antiserum. Several strains of Butyrivibrio fibrisolvens, Ruminococcus flavefaciens strains C- 94 and FD-1, and Fibrobacter succinogenes S85 produced no proteins that cross-react with the same antiserum. Surprisingly though, F. intestinalis strain DR7 does possess a protein(s) of relatively large molecular mass (~200 kDa) that was strongly cross-reactive with the anti- CbpC antiserum. Scientifically, our studies have helped expand the scope of our fundamental understanding of adhesion mechanisms in cellulose-degrading bacteria, and validated the use of RNA-based techniques to examine physiological responses in bacteria that are nor amenable to genetic manipulations. Because efficient fiber hydrolysis by many anaerobic bacteria requires both tight adhesion to substrate and a stable cellulosome, we believe our findings are also the first step in providing the resources needed to achieve our long-term goal of increasing fiber digestibility in animals.
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Guy, Charles, Gozal Ben-Hayyim, Gloria Moore, Doron Holland, and Yuval Eshdat. Common Mechanisms of Response to the Stresses of High Salinity and Low Temperature and Genetic Mapping of Stress Tolerance Loci in Citrus. United States Department of Agriculture, May 1995. http://dx.doi.org/10.32747/1995.7613013.bard.

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The objectives that were outlined in our original proposal have largely been achieved or will be so by the end of the project in February 1995 with one exception; that of mapping cold tolerance loci based on the segregation of tolerance in the BC1 progeny population. Briefly, our goals were to 1) construct a densely populated linkage map of the citrus genome: 2) map loci important in cold and/or salt stress tolerance; and 3) characterize the expression of genes responsive to cold land salt stress. As can be seen by the preceding listing of accomplishments, our original objectives A and B have been realized, objective C has been partially tested, objective D has been completed, and work on objectives E and F will be completed by the end of 1995. Although we have yet to map any loci that contribute to an ability of citrus to maintain growth when irrigated with saline water, our very encouraging results from the 1993 experiment provides us with considerable hope that 1994's much more comprehensive and better controlled experiment will yield the desired results once the data has been fully analyzed. Part of our optimism derives from the findings that loci for growth are closely linked with loci associated with foliar Cl- and Na+ accumulation patterns under non-salinization conditions. In the 1994 experiment, if ion exclusion or sequestration traits are segregating in the population, the experimental design will permit their resolution. Our fortunes with respect to cold tolerance is another situation. In three attempts to quantitatively characterize cold tolerance as an LT50, the results have been too variable and the incremental differences between sensitive and tolerant too small to use for mapping. To adequately determine the LT50 requires many plants, many more than we have been able to generate in the time and space available by making cuttings from small greenhouse-grown stock plants. As it has turned out, with citrus, to prepare enough plants needed to be successful in this objective would have required extensive facilities for both growing and testing hardiness which simply were not available at University of Florida. The large populations necessary to overcome the variability we encountered was unanticipated and unforeseeable at the project's outset. In spite of the setbacks, this project, when it is finally complete will be exceedingly successful. Listing of Accomplishments During the funded interval we have accomplished the following objectives: Developed a reasonably high density linkage map for citrus - mapped the loci for two cold responsive genes that were cloned from Poncirus - mapped the loci for csa, the salt responsive gene for glutathione peroxidase, and ccr a circadian rhythm gene from citrus - identified loci that confer parental derived specific DNA methylation patterns in the Citrus X Poncirus cross - mapped 5 loci that determine shoot vigor - mapped 2 loci that influence leaf Na+ accumulation patterns under non-saline conditions in the BC1 population - mapped 3 loci that influence leaf Na+ accumulation paterns during salt sress - mapped 2 loci that control leaf Cl- accumulation patterns under non-saline conditions - mapped a locus that controls leaf Cl- accumulation patterns during salt stress Screened the BC1 population for growth reduction during salinization (controls and salinized), and cold tolerance - determined population variation for shoot/root ratio of Na+ and Cl- - determined levels for 12 inorganic nutrient elements in an effort to examine the influence of salinization on ion content with emphasis on foliar responses - collected data on ion distribution to reveal patterns of exclusion/sequestration/ accumulation - analyzed relationships between ion content and growth Characterization of gene expression in response to salt or cold stress - cloned the gene for the salt responsive protein csa, identified it as glutathione peroxidase, determined the potential target substrate from enzymatic studies - cloned two other genes responsive to salt stress, one for the citrus homologue of a Lea5, and the other for an "oleosin" like gene - cold regulated (cor) genes belonging to five hybridization classes were isolated from Poncirus, two belonged to the group 2 Lea superfamily of stress proteins, the others show no significant homology to other known sequences - the expression of csa during cold acclimation was examined, and the expression of some of the cor genes were examined in response to salt stress - the influence of salinization on cold tolerance has been examined with seedling populations - conducted protein blot studies for expression of cold stress proteins during salt stress and vice versa
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