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Artykuły w czasopismach na temat "Prone proteins"
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De Baets, Greet, Joost Schymkowitz i Frederic Rousseau. "Predicting aggregation-prone sequences in proteins". Essays in Biochemistry 56 (18.08.2014): 41–52. http://dx.doi.org/10.1042/bse0560041.
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Owing to its association with a diverse range of human diseases, the determinants of protein aggregation are studied intensively. It is generally accepted that the effective aggregation tendency of a protein depends on many factors such as folding efficiency towards the native state, thermodynamic stability of that conformation, intrinsic aggregation propensity of the polypeptide sequence and its ability to be recognized by the protein quality control system. The intrinsic aggregation propensity of a polypeptide sequence is related to the presence of short APRs (aggregation-prone regions) that self-associate to form intermolecular β-structured assemblies. These are typically short sequence segments (5–15 amino acids) that display high hydrophobicity, low net charge and a high tendency to form β-structures. As the presence of such APRs is a prerequisite for aggregation, a plethora of methods have been developed to identify APRs in amino acid sequences. In the present chapter, the methodological basis of these approaches is discussed, as well as some practical applications.
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Lebendiker, Mario, i Tsafi Danieli. "Production of prone-to-aggregate proteins". FEBS Letters 588, nr 2 (6.11.2013): 236–46. http://dx.doi.org/10.1016/j.febslet.2013.10.044.
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Galves, Margarita, Ritu Rathi, Gali Prag i Avraham Ashkenazi. "Ubiquitin Signaling and Degradation of Aggregate-Prone Proteins". Trends in Biochemical Sciences 44, nr 10 (październik 2019): 872–84. http://dx.doi.org/10.1016/j.tibs.2019.04.007.
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Tartaglia, Gian Gaetano, Amol P. Pawar, Silvia Campioni, Christopher M. Dobson, Fabrizio Chiti i Michele Vendruscolo. "Prediction of Aggregation-Prone Regions in Structured Proteins". Journal of Molecular Biology 380, nr 2 (lipiec 2008): 425–36. http://dx.doi.org/10.1016/j.jmb.2008.05.013.
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Berger, Zdenek, Brinda Ravikumar, Fiona M. Menzies, Lourdes Garcia Oroz, Benjamin R. Underwood, Menelas N. Pangalos, Ina Schmitt i in. "Rapamycin alleviates toxicity of different aggregate-prone proteins". Human Molecular Genetics 15, nr 3 (20.12.2005): 433–42. http://dx.doi.org/10.1093/hmg/ddi458.
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Chennamsetty, Naresh, Vladimir Voynov, Veysel Kayser, Bernhard Helk i Bernhardt L. Trout. "Prediction of Aggregation Prone Regions of Therapeutic Proteins". Journal of Physical Chemistry B 114, nr 19 (20.05.2010): 6614–24. http://dx.doi.org/10.1021/jp911706q.
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Salomons, Florian A., Victoria Menéndez-Benito, Claudia Böttcher, Brett A. McCray, J. Paul Taylor i Nico P. Dantuma. "Selective Accumulation of Aggregation-Prone Proteasome Substrates in Response to Proteotoxic Stress". Molecular and Cellular Biology 29, nr 7 (21.01.2009): 1774–85. http://dx.doi.org/10.1128/mcb.01485-08.
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ABSTRACT Conditions causing an increase in misfolded or aberrant proteins can impair the activity of the ubiquitin/proteasome system (UPS). This observation is of particular interest, given the fact that proteotoxic stress is closely associated with a large variety of disorders. Although impairment of the UPS appears to be a general consequence of proteotoxic insults, the underlying mechanisms remain enigmatic. Here, we show that heat shock-induced proteotoxic stress resulted in conjugation of ubiquitin to detergent-insoluble protein aggregates, which coincided with reduced levels of free ubiquitin and impediment of ubiquitin-dependent proteasomal degradation. Interestingly, whereas soluble proteasome substrates returned to normal levels after a transient accumulation, the levels of an aggregation-prone substrate remained high even when the free ubiquitin levels were restored. Consistently, overexpression of ubiquitin prevented accumulation of soluble but not aggregation-prone substrates in thermally stressed cells. Notably, cells were also unable to resume degradation of aggregation-prone substrates after treatment with the translation inhibitor puromycin, indicating that selective accumulation of aggregation-prone proteins is a consistent feature of proteotoxic stress. Our data suggest that the failure of the UPS to clear aggregated proteins in the aftermath of proteotoxic stress episodes may contribute to the selective deposition of aggregation-prone proteins in conformational diseases.
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Ravikumar, Brinda, Abraham Acevedo-Arozena, Sara Imarisio, Zdenek Berger, Coralie Vacher, Cahir J. O'Kane, Steve D. M. Brown i David C. Rubinsztein. "Dynein mutations impair autophagic clearance of aggregate-prone proteins". Nature Genetics 37, nr 7 (26.06.2005): 771–76. http://dx.doi.org/10.1038/ng1591.
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Knaevelsrud, Helene, i Anne Simonsen. "Fighting disease by selective autophagy of aggregate-prone proteins". FEBS Letters 584, nr 12 (20.04.2010): 2635–45. http://dx.doi.org/10.1016/j.febslet.2010.04.041.
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Källquist, Linda, Markus Hansson, Ann-Maj Persson, Hans Janssen, Jero Calafat, Hans Tapper i Inge Olsson. "The tetraspanin CD63 is involved in granule targeting of neutrophil elastase". Blood 112, nr 8 (15.10.2008): 3444–54. http://dx.doi.org/10.1182/blood-2007-10-116285.
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Abstract Targeting mechanisms of neutrophil elastase (NE) and other luminal proteins stored in myeloperoxidase (MPO)–positive secretory lysosomes/primary granules of neutrophils are unknown. These granules contain an integral membrane protein, CD63, with an adaptor protein-3–dependent granule delivery system. Therefore, we hypothesized that CD63 cooperates in granule delivery of the precursor of NE (proNE). Supporting this hypothesis, an association was demonstrated between CD63 and proNE upon coexpression in COS cells. This also involved augmented cellular retention of proNE requiring intact large extracellular loop of CD63. Furthermore, depletion of CD63 in promyelocytic HL-60 cells with RNA interference or a CD63 mutant caused reduction of cellular NE. However, the proNE steady-state level was similar to wild type in CD63-depleted clones, making it feasible to examine possible effects of CD63 on NE trafficking. Thus, depletion of CD63 led to reduced processing of proNE into mature NE and reduced constitutive secretion. Furthermore, CD63-depleted cells showed a lack of morphologically normal granules, but contained MPO-positive cytoplasmic vacuoles with a lack of proNE and NE. Collectively, our data suggest that granule proteins may cooperate in targeting; CD63 can be involved in ER or Golgi export, cellular retention, and granule targeting of proNE before storage as mature NE.
Rozprawy doktorskie na temat "Prone proteins"
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Malm, Linus. "Size determination of hyaluronan and multivariate analysis of amyloid prone proteins". Doctoral thesis, Umeå universitet, Institutionen för medicinsk kemi och biofysik, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-46601.
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Background.The extracellular matrix surrounds all cells within our bodies. The glycosaminoglycan hyaluronan is a major component in the extracellular matrix. Despite its structural simplicity it has been shown to be involved in several important functions. It is a lubricant and shock absorber, as well as an important player in inflammation and tumor invasion. Many of its functions are closely related to its size and concentration in tissues. Therefore methods for measuring these properties are of great importance to properly understand the role that hyaluronan play in different events. Proteins are found both inside and outside cells, and they have a wide variety of functions. The protein structure and function is determined by the properties of their building blocks, the amino acids. Several diseases have been linked to changes in the amino acid sequence of certain proteins by mutations, causing the proteins to form extracellular deposits of structures called amyloid aggregates. The aim of this thesis is to investigate the function of hyaluronan in cell cultures, develop new methods for size determination hyaluronan and to use multivariate methods to provide prediction and better understanding of factors driving protein amyloid aggregation. Methods.Cardiomyocytes and fibroblast were cultured and stimulated by different growth factors. Hyaluronan was purified and its size and concentration were measured. Crosstalk between cardiomyocytes and fibroblast were investigated and gene expression of hyaluronan synthases was determined. A new method for size measurement of hyaluronan was developed. The amyloid aggregation rate of different mutants of acylphosphatase was predicted by multivariate analysis. Results. Cardiomyocytes stimulated by PDGF-BB produced hyaluronan. Cardiomyocytes could induce fibroblast to increase its hyaluronan production, through an unknown soluble factor. The cardiomyocyte gene expression changed when stimulated by hyaluronan. GEMMA was presented as a new method for size determination of hyaluronan. Amyloid aggregation of different acylphosphatase mutants could be predicted using a multivariate regression model of the physicochemical and structural properties of the amino acid sequence. Conclusion. It was shown that cardiomyocytes are not only able to produce hyaluronan, but also induce an increased hyaluronan production in other cells. GEMMA was proven suitable for size determination of hyaluronan at very low concentrations. Multivariate analysis showed that hydrophobic patterns and charge where the most important factors for amyloid aggregation of acylphosphatase.
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Berger, Z. "The biology of aggregate-prone proteins and possible therapeutic interventions against their toxicity". Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.596587.
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The main aim of my project was to investigate pathways involved in protecting against the toxicity caused by different aggregate-prone proteins and to explore possible therapeutic interventions. My strategy was to generate a Drosophila model expressing a stretch of long polyalanines. This model can be used along with models of polyglutamine diseases to study various pathways in the context of different aggregate-prone proteins. In, addition, generation of this model will also help to better understand the toxicity of long polyalanines. My results show that expanded polyalanines are toxic in vivo in Drosophila and this is accompanied by aggregate formation. This suggests that polyalanines can cause diseases by a gain-of-function mechanism in vivo and that this mechanism should be considered for all diseases caused by expanded polyalanines. In order to investigate if manipulation of the Wnt and TOR pathway would be beneficial in the context of different aggregate-prone proteins in vivo, I used fly models of these disorders. Inhibition of GSK3 using lithium and a GSK3-specific inhibitor decreased toxicity of both proteins and similar effects were seen when downstream targets were manipulated in vivo. Inhibition of TOR by rapamycin decreased toxicity of proteins with long polyglutamines and polyalanines in vivo and this in vivo effect can be largely attributed to the induction of autophagy. Rapamycin also reduces toxicity of wild-type and mutant tau in vivo and these effects can be accounted for by reductions in insoluble tau. Thus, lithium, GSK3-specific inhibitors and rapamycin may be a beneficial therapeutic strategy in diseases associated with different aggregate-prone proteins.
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Gallo, Annastassia Dawn. "Homeostasis and trafficking of hydrolysis-prone metals in cells, proteins, and small molecules". Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/568230.
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ChemistryPh.D.
Nature uses inorganic elements for biological processes based on the useful chemistry, abundance, and availability of each metal. Transition metals are critical in the biogeochemical cycling of essential elements and the bioinorganic chemistry of organisms. Hydrolysis-prone metals such as iron and titanium are abundant on Earth but are mostly insoluble in oxic aqueous environments. Nearly every organism requires iron for survival, therefore Nature evolved to stabilize iron from hydrolysis and hydrolytic precipitation through protein and small molecule mechanisms. Like iron, titanium primarily exists as insoluble mineral oxides and is second only to iron as the most abundant transition metal in the Earth’s crust. Despite the reputation as an inert and insoluble metal, titanium can be solubilized and made bioavailable through by chemical and biological weathering. Currently there is no known native role for titanium, however it is quite bioactive. As a stronger Lewis acid, titanium can compete with iron in binding to biomolecules and proteins. It is of interest to investigate the interactions between hydrolysis-prone metals and biological systems, from whole cell organisms to proteins and small molecules. The non-pathogenic bacterium Rhodococcus ruber GIN-1 was isolated for its ability to strongly adhere to titanium dioxide (TiO2) over other metal oxides, providing an opportunity to study the interactions between whole bacterial cells and metal oxides. The GIN-1 strain incorporates Ti(IV) ions into its biomass after adherence to anatase, rutile, and a mixture of the two morphologies. Six metals were quantitated in TiO2-exposed and control (unexposed) cells by inductively coupled plasma optical emission spectroscopy. The exposure to TiO2 caused a significant uptake of titanium with concomitant loss of iron, zinc, and possibly manganese. A collaborative project with the Strongin laboratory at Temple University works to develop stable, biomaterial photocatalysts for environment remediation of toxic inorganic contaminants. Ferritins are a class of proteins that mineralizes and stores iron as a non- toxic ferrihydrite nanoparticle. These proteins can be photoactivated with ultraviolet light to release iron from its core to remediate environmental contaminants. Ferritin can be sensitized with plasmonic gold nanoparticles to extend the photoactivity of the catalyst to the visible spectrum. Work in this thesis highlights the contribution to this collaboration from the Valentine laboratory, included the expression and purification of proteins in E. coli (human H-chain ferritin, human L-chain ferritin, and bacterial DNA protection from starved cells protein), mutation of proteins to improve sensitization of catalyst, and biomineralization with iron and titanium. The trafficking of hydrolysis prone metals is vital for the survival of nearly every organism. Iron transport proteins such as transferrins are studied to understand how nature utilizes a difficult essential metal across the domains of life. Most transferrins have two homologous lobes and are believed to have evolved from a gene duplication of a monolobal transferrin. The ascidian Ciona intestinalis has genes for both a bilobal and monolobal transferrin. Nicatransferrin (nicaTf), the monolobal transferrin from C. intestinalis, is a primitive protein that may provide insight on the evolution of transferrins in higher organisms. It is advantageous to use E. coli expression systems to produce recombinant proteins, however protein misfolding and aggregation can be a concern. To improve expression of nicaTf in E. coli, codon optimization and disulfide bonded protein expression were used. Finally, siderophores are small, high affinity iron-chelating molecules secreted from lower organisms that scavenge iron in iron-limiting conditions. R. ruber GIN-1 and R. ruber DSM 43338 strains both secrete siderophores in artificial seawater media. There are several siderophores identified from Rhodococcus species, however none have been reported from any R. ruber strain. A new siderophore was isolated and preliminary work has been done to purify and characterize the molecule. Understanding the siderophore- metal ion interactions may help elucidate the mechanism of how R. ruber cells obtain titanium from the metal-oxide particles.
Temple University--Theses
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Williams, Andrea. "Characterisation of novel autophagy pathways : implications in the clearance of disease-causing aggregate-prone proteins". Thesis, University of Cambridge, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.612132.
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Vincenz-Donnelly, Lisa Verfasser], i Franz-Ulrich [Akademischer Betreuer] [Hartl. "How the mammalian endoplasmic reticulum handles aggregation-prone β-sheet proteins / Lisa Vincenz-Donnelly ; Betreuer: Franz-Ulrich Hartl". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2016. http://d-nb.info/1122019467/34.
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Born, Ariane. "Etablierung und Optimierung der Error-Prone-PCR und eines Aktivitätsscreenings für Styrol-Monooxygenasen". Master's thesis, Technische Universitaet Bergakademie Freiberg Universitaetsbibliothek "Georgius Agricola", 2011. http://nbn-resolving.de/urn:nbn:de:bsz:105-qucosa-77143.
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Styrol-Monooxygenasen (SMOs) spielen im bakteriellen Abbau von Styrol eine wichtige Rolle. Sie epoxidieren den Kohlenwasserstoff zu (S)-Styroloxid und waren bis vor kurzem vor allem aus Gram-negativen Vertretern wie Pseudomonaden bekannt. Das Grampositive nocardioforme Bodenbakterium Rhodococcus opacus 1CP kann Styrol als Energie- und Kohlenstoffquelle nutzen und verfügt über zwei Typen von SMOs. Neben StyA2B, einer fusionierten FAD:NADH-Oxidoreduktase (StyB) und Monooxygenase (StyA2) findet sich eine weitere Monooxygenase StyA1, deren Gen direkt stromaufwärts zu styA2B lokalisiert ist. Zusätzlich zum natürlichen Fusionsprotein StyA2B gelang kürzlich die Konstruktion künstlicher Fusionen StyAL1B und StyAL2B aus Pseudomonas fluorescens ST. Um sowohl StyA1/StyA2B als auch die künstlichen Fusionen StyAL1B und StyAL2B für eine biotechnologische Anwendung nutzen zu können, wurde im Rahmen dieser Arbeit angestrebt, ihre spezifische Oxygenierungsaktivität (StyA1/StyA2B: 0,24 U/mg) mit Hilfe der error prone PCR zu erhöhen. Um Veränderungen der katalytischen Aktivität in einer großen Zahl von Mutanten schnell zu erkennen, ist ein einfacher Screeningtest erforderlich. Die Fähigkeit von SMOs zur Oxidation von Indol zu blauem Indigo bietet diese Möglichkeit. Allerdings ist hierfür die Expression löslicher Proteine eine wesentliche Voraussetzung. Versuche zur Veränderung der Gene styA2B und styA1A2B mit Hilfe eines kommerziellen error prone PCR Kits lieferten ca. 300 bis 1.200 mutmaßlich veränderte Klone, welche jedoch keinerlei Aktivität für den Indolumsatz zeigten. Als Ursache wurde eine Expression der Proteine in Form inaktiver Inclusion Bodies vermutet. Die Fusionsproteine StyAL1B und StyAL2B bilden lösliches Protein, welche Indol zum blauen Farbstoff Indigo umsetzen. Verschiedene Kultivierungsbedingungen wurden auf den Umsatz von Indol untersucht. Dabei wurde erkannt, dass die Klone sich nicht identisch bezüglich ihrer Proteinlöslichkeit verhalten. Mit Hilfe dieser Ergebnisse wurde ein Test für das Aktivitätsscreening von Styrol-Monooxygenasen auf Platte entwickelt. Die Erhöhung der NaCl-Konzentration im Medium steigerte die Indoloxidation, welche sich jedoch durch zusätzliche physiologisch Faktoren schwer beeinflussen lassen. Auch für die Fusionsproteine erfolgte die Durchführung einer error prone PCR. Der Schritt der error prone PCR stellte kein Problem dar, jedoch die Einbindung des veränderten Genfragmentes in den Vektor, beziehungsweise dessen Transformation in E. coli. Alternative Strategien, wie die Nutzung alternativer DNA Polymerasen und eines konventionellen Konzepts, bei dem veränderte Gene in geschnittene Expressionsvektoren ligiert werden, führte zu keinen detektierbaren Klonen. Die Kultivierung von identischen Klonen auf Festmedium wirkte sich aufgrund nicht näher identifizierter Einflüsse auf das Verhalten bezüglich der Indoloxidation sehr unterschiedlich aus. Um diese Einflüsse zu minimieren, erfolgte die Untersuchung des Systems in einer Flüssigkultur. Im Blickpunkt stand hierbei die Indigoproduktion von E. coli BL21 (pET_StyAL2B) die in Abhängigkeit der optischen Dichte der Kultur untersucht wurdeStyrene monooxygenases (SMOs) play an important role in the bacterial degradation of styrene. They epoxidize the hydrocarbon highly enantioselective to (S)-styrene oxide. Most of the styrene monooxygenases known so far were identified in Gram-negative microorganisms like pseudomonads. Rhodococcus opacus 1CP, a Gram-positive nocardioform actinobacterium, which uses styrene as energy and carbon source was recently found to possess a novel type of SMO, StyA2B. This protein represents a natural fusion between an FAD:NADH oxidoreductase (StyB) and a single monooxygenase subunit (StyA2) and might act in combination with another single oxygenase StyA1 in strain 1CP. Two artificial analogs to StyA2B, designated StyAL1B and StyAL2B, were recently prepared by a fusion of styA and styB of Pseudomonas fluorescens ST and both showed oxygenating activity. For StyA1/StyA2B as well as the artificial fusion proteins StyAL1B and StyAL2B, it was tried to enhance the specific oxygenation activity in order to support their biotechnological applicability. The method of error prone PCR was used for that purpose. In order to identify favorable modifications with increased catalytic activity from a high number of mutants, an easy and simple screening test is necessary. Therefore, it is reasonable to use the ability of SMOs to oxidize indole to the blue dye indigo. However, the expression of SMOs as soluble proteins is an important requirement for any activity screening. Attempts to modify the genes styA2B and styA1/styA2B by means of a commercial error prone PCR kit yielded 300 to 1,200 potential mutants. Unfortunately, none of the obtained colonies showed any indole-oxidizing activity and the formation of insoluble inclusion bodies was assumed to be a likely explanation. In contrast to StyA2B and StyA1, recombinant expression of the artificial fused SMOs StyAL1B und StyAL2B should yield detectable amounts of active proteins. In fact, cultivation of clones expressing both types of proteins showed a blue coloration. Since the coloration of clones from one single solid medium evolved in a non-uniform manner, cultivation conditions were varied in order to identify factors which promote a more uniform tendency for indole oxidation. Although a high NaCl concentration in the medium was shown to favor indole oxidation, the latter one seems to be influenced by additional physiological factors, hardly to control. For the artificially fused proteins an error prone PCR was carried out, too. Although the initial step of mutagenic PCR was found to be successful, completing the vector system by a second ll-up PCR reaction failed. Alternative strategies like the usage of alternative DNA polymerases as well as a conventional cloning approach of various genes into a digested expression vector did not lead to detectable clones. The cultivation of identical clones on petri dishes provided no uniform tendency for indole oxidation and thus did not allow the reliable comparison of mutants in respect of their specific SMO activities. Cultivation of mutants in liquid medium should lead to more reproducible conditions and for that purpose a method was successfully established to quantify indigo formation and cell density
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Liu, Xueyun, i Xueyun Liu. "Probe Ca2+/Camodulin reguation of membrane proteins engineering". Digital Archive @ GSU, 2013. http://digitalarchive.gsu.edu/chemistry_theses/59.
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Calmodulin (CaM) is a eukaryotic Ca2+ signaling protein which can interact with more than 300 enzymes in the cell including membrane proteins Ryanodine receptor1 (RyR1) and gap junction protein connexin 43 (Cx43). By binding to Ca2+, CaM undergoes a conformational change which exposed the hydrophobic patch that access to the target protein. wt-CaM and three mutant CaM (isolate C and N domain of CaM, deletion of five residues from the central linker of CaM) are designed for studying the specific contributions to calcium binding affinity and calcium induced conformational change. wt-CaM exhibits metal binding affinity to calcium analog Tb3+ with a Kd of 3.97 nM using FRET assay and metal-buffer system and activates target protein phosphodiesterase assay. The Kd values of domain specific calcium binding affinity of CaM probed by intrinsic Phe or Tyr are 12.2 and 2.77 uM, respectively. In addition, Ca2+ also induces helicity for both w.t. CaM and C-terminal domain variant. Further, conditions such as medium and Glucose amount for isotopic labeling of CaM by 15N, 13C and D2O have been optimized with relatively high yield of hetero-isotopic labeled CaM. This prepared us to probe the detailed interaction of CaM and its target protein and calcium induced conformational change by high resolution NMR. Furthermore, RyR1 mini domain, which contains two CaM binding regions of RyR1 was designed to study the binding mode of the two regions with CaM. Obtaining bacterial expressed and purified RyR1 mini domain was achieved by engineered with a His-Tag which overcomes the insoluble issue that occurred in the initial study of expression and purification with a GST tag. Moreover, to probe the interaction of CaM to the cytosolic loop of Cx43 that contains two putative CaM binding sites as well as the role of transmembrane region of Cx43, we have successfully expressed and purified fragments Cx4388-154 and Cx4399-154 as a His-tag protein encompassing regions 88-154 and 99-154 of Cx43 with predicted CaM binding sites with and without additional transmembrane region from Cx43. Both fragments were obtained with high yield after expressed as inclusion body and His-tag purification. Fragment Cx4388-154 was shown to bind dansylated CaM with a Kd of 0.107 μM using florescence spectroscopy.
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Mossuto, Maria Francesca. "Protein amyloidogenesis: characterization of aggregation prone conformations and fibrils structure". Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3425566.
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Current interest in studying amyloid fibrils arises from their involvement in different fields (Chiti and Dobson, 2006). First, they play a crucial role in disorders such as Alzheimer's and Parkinson's diseases. Second, since it has been demonstrated that all polypeptide chains form fibrils under appropriate conditions, the understanding of why and how this process happens has become central problem in protein knowledge. Last, the ordered ultrastructure characterizing amyloid fibrils may be thought as a basis for nanomaterials with possible technological applications. However, despite the ability of most proteins to form amyloid fibrils, very little is known about their structures and the factors that govern their formation.
The process of amyloid formation requires the partial unfolding of protein molecules into such conformations able to interact to each others and reorganize into well-ordered structured aggregates, named amyloid fibrils.
In this Thesis the amyloid formation by globular proteins has been analyzed from different points of view, focusing in the first part of the research work on the elucidation of some conformational features promoting protein aggregation. The second part was concentrated on the characterization of the final supramolecular structure of amyloid fibrils.
In order to study the partially folded state, two globular proteins have been analyzed, alpha-lactalbumin (LA) and HypF-N. Indeed, under specific conditions, these proteins populate a not fully folded state previously shown to play an important role in the amyloid formation (Uversky, 2002; Chiti et al., 2001).
The study conducted on LA has been based on the effects of the proteolytic dissection of the molecule on its conformational features and aggregation properties. It was previously shown that LA is able to form fibrils morphologically indistinct from the pathological ones (Uversky et al., 2002). Here, we have studied the aggregation propensities of LA derivatives characterized by a single peptide bond fission (1-40/41-123, named Th1-LA) or a deletion of a chain segment of 12 amino acid residues located at the level of the ?-subdomain of the native protein (1-40/53-123, named des?-LA). We have also compared the early stages of the aggregation process of these LA derivatives with those of intact LA. The main conclusion of this work was that the inherent flexibility of the LA derivatives allows the large conformational changes required to form the cross-?-structure of the amyloid fibrils. It has been emphasized that proteolysis can be considered a causative mechanism of protein aggregation and fibrillogenesis (Polverino de Laureto et al., 2005).
In the other case, the conformational characterization of an amyloidogenic state of HypF-N has been performed at acid pH, in order to allow the protein to populate a partially unfolded ensemble. Combining different biophysical and biochemical techniques, it has been shown that this partially unfolded structure has all the hallmarks of a pre-molten globule state, i.e. it is more compact than a random coil-like state but less organized than a native-like intermediate or a MG state (Uversky, 2002). Furthermore, it is shown that a modulation of the total ionic strength of the solution allows enhancing the apparent rate of aggregation of HypF-N under these conditions. This increased rate of aggregation has been shown to be mediated by the interaction of monomers to form initial oligomers, through a particular region in the sequence, corresponding to the sequence part having highly hydrophobicity, the highest beta-sheet propensity and with no net charge at acid pH, representing the ideal segment suitable to mediate protein oligomerization.
From all these studies, it is clear that, except the unique native state of globular proteins wherein the side chains pack together in a unique manner, every state of a polypeptide molecule is a broad ensemble of often diverse conformations. It is not surprising, therefore, that even the fibrillar products of aggregation processes are characterized by morphological and structural diversity, representing variations on a common theme.
The second part of my PhD Thesis deals with the structural characterization of fibrils. Many studies have been conducted on amyloid aggregates formed under different conditions by peptides, such as A?, TTR and prion fragments (Kodali and Wetzel, 2007). Indeed, the problem of amyloid formation by a full-length protein is more complex, since the dense packing reachable in amyloid fibrils made of peptides (10-40 residues) could not be accomplished in all the amino acid residues of a full-length protein, except in the core regions (Chatani and Goto, 2005). The object of my study was human lysozyme due, most of all, to the fact that some natural variants of human lysozyme (HuL) are responsible for the formation of amyloid plaques in vivo, in a so called familial non-neuropathic systemic amyloidosis (Pepys et al., 1993; Booth et al., 1997). Moreover, it has been possible to exploit the available wealth of structural and folding information about wild type HuL, since it has been shown to be able to form fibrils quite similar to the pathological ones, under acidic conditions and high temperature (Morozova-Roche et al, 2000). With respect to fibrils made of peptides, besides, studying amyloid fibrils conformations from HuL is more challenging because it is a 130 amino acid chain with the structural constrains given by the four disulfide bridges present in the lysozyme molecule. This study can also give some insights into the complex problem of strains diversity, such as for prion diseases, helping the clarification of the structural principles of amyloid fibrils which can produce multiple and distinct amyloid conformations from one protein sequence.
In the presented study, fibrils of wild-type HuL formed at low pH have been analyzed by limited proteolysis experiments and Fourier-transform infrared (FTIR) spectroscopy, in order to map conformational features of the 130 residue chain of lysozyme when embedded in the amyloid aggregates (Frare et al., 2006). After digestion with pepsin at low pH, the lysozyme fibrils were found to be composed primarily of N and C-terminally truncated protein species encompassing residues 26-123 and 32-108, although a minority of molecules was found to be completely resistant to proteolysis under these conditions. FTIR spectra provide evidence that lysozyme fibrils contain extensive ?-sheet structure and substantial elements of non ?-sheet or random structure that are reduced significantly in the fibrils after digestion. The sequence 32-108 includes the ?-sheet and helix C of the native protein, previously found to be prone to unfold locally in human lysozyme and its pathogenic variants. Moreover, this core structure of the lysozyme fibrils encompasses the highly aggregation-prone region of the sequence recently identified in hen lysozyme (Frare et al., 2004). The present data indicate that the region of the lysozyme molecule that unfolds and aggregates most readily corresponds to the most highly protease-resistant and thus highly structured region of the majority of mature amyloid fibrils. Overall, the data show that amyloid formation does not require the participation of the entire lysozyme chain.
HuL variants, however, aggregate in a physiological environment, roughly at pH 7-7.5 at 37 °C, because of their instability (Dumoulin et al., 2005). In my work, it has been demonstrated that also HuL is able to aggregate under conditions similar to the pathological ones, presumably neutral pH and 37 °C. Considering that HuL forms amyloid fibrils in such different conditions (pH 2.0 50°C and pH 7.5, 60°C), a comparison of the structure and the stability of fibrils obtained under these different conditions has been conducted. In this study HuL fibrils were produced at acidic and at neutral pH, leading both to the formation of fibrils having the three hallmarks of amyloid, that are cross-beta structure, binding of ThT and an overall amyloid fiber morphology. These fibrils have been studied by means of ANS binding, FTIR and X-ray fiber diffraction in order to characterize the differences in the structure. Guanidinium-induced fibrils dissociation, instead, has been applied in order to test the chemical stability of the two kinds of fibrils. The results clearly indicate that the solution conditions used for lysozyme aggregation promote the formation of fibrils with different structural features and stability properties, due to the diverse rearrangements of the lysozyme polypeptide chain into the fibril structure.
In conclusion, the research work conducted in this Thesis allowed the comprehension of important aspects of the unfolding of some globular proteins leading to amyloid fibrils. In addition, original data have been obtained on the structural polymorphism of amyloid fibrils.
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SOUZA, Renata Silva Cabral de. "Avalia??o do potencial antioxidante e antimicrobiano de prote?nas do soro de leite concentradas por membranas e hidrolisadas por diferentes enzimas comerciais". Universidade Federal Rural do Rio de Janeiro, 2013. https://tede.ufrrj.br/jspui/handle/jspui/2534.
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The aim of this study was to evaluate the process of protein concentration in bovine whey proteins by ultrafiltration process and subsequently the protein hydrolysate obtained by enzymatic hydrolysis to produce bioactive peptides with potential antimicrobial and antioxidant activities. For concentration process was used a ceramic ultrafiltration membrane with a molecular range cut-off of 10-20 kDa, transmembrane pressure of 5 bar and, temperature of 30 ?C, 40 ?C and 50 ?C . The optimum temperature condition was at 40 ?C. The Volume Concentrate Factor (VCF) parameter was used as a end-point of the ultrafiltration process and fixed at 2, corresponding on concentrating the initial volume twice, in volume. At the temperature of 40 ?C, VCF had a correspondence on final protein concentration on the concentrated fraction by ultrafiltration and confirmed by Bradford method. Two commercial enzymes were tested Alcalase, Flavourzyme and an equivalent mixture of both 50:50 (w/w) in the hydrolysis reaction. The hydrolysis conditions were determined according to manufacturer instructions and confirmed by other studies: 60 ?C and pH 8 for Alcalase; 50 ?C and pH 7 for Flavourzyme; 50 ?C and pH 8 for enzyme mixture with enzyme / substrate ratio (w / w) 5/100 for all enzymes. The reaction was monitored by pH Stat method. The final Degree of Hydrolysis (DH) achieved was 15%, 52% and 63% for Flavourzyme, Alcalase and enzyme mixture, respectively. Five aliquots were collected along the hydrolysis for each enzyme reaction corresponding to differents DH in order to evaluatethe antioxidant activity by ORAC and ABTS assays with final values between 597- 1092 m? TE (ABTS) and from 1615 to 2920 ?M TE (ORAC) for Flavourzyme; 998-6290 ?M TE (ABTS) and 3092-7567 ?M TE ( ORAC) for Alcalase and finally 913-2678 ?M TE (ABTS) and 2547-5903 ?M TE (ORAC) for the enzyme mixture. The samples from all hydrolysates showed no antimicrobial activity against strains of Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) and Listeria monocytogenes (ATCC 9117).
A proposta do presente trabalho foi avaliar a concentra??o das prote?nas do soro de leite bovino por ultrafiltra??o e posterior obten??o de hidrolisados proteicos deste concentrado via hidr?lise enzim?tica visando obter pept?deos bioativos com potencial atividade antimicrobiana e antioxidante. Para concentra??o das prote?nas do soro foi utilizada membrana cer?mica de ultrafiltra??o com massa molar de corte de 10-20 kDa, press?o aplicada ? membrana de 5 bar, temperaturas testadas (30 ?C, 40 ?C e 50 ?C) . A temperatura ?tima selecionada foi de 40 ?C. O Fator de Concentra??o Volum?trica foi o par?metro utilizado para indicar o final do processo de ultrafiltra??o sendo fixado em duas vezes o volume inicial da alimenta??o. Na temperatura de 40 ?C foi obtida correspond?ncia entre a concentra??o volum?trica e a concentra??o proteica final na fra??o retida pela UF, que tamb?m foi o dobro da encontrada na fra??o alimenta??o, avaliada pelo m?todo de Bradford. Foram testadas duas enzimas comerciais: Alcalase, Flavourzyme e uma mistura equivalente de ambas, na propor??o 50:50 (m/m) na rea??o de hidr?lise. As condi??es de rea??o enzim?tica foram determinadas de acordo com instru??es do fabricante e corroboradas por outros estudos em: 60 ?C, pH 8 para Alcalase; 50 ?C, pH 7 para Flavourzyme; 50 ?C, pH 8 para mistura enzim?tica e rela??o enzima/substrato (g/g) foi de 5/100 para todas as enzimas. A rea??o de hidr?lise foi monitorada pelo m?todo pH Stat. Os Graus de Hidr?lise (GH) finais alcan?ados foram de 15 %, 52 % e 63 % para Flavourzyme, mistura enzim?tica e Alcalase, respectivamente. Foram coletadas cinco al?quotas correspondentes a diferentes GH ao longo da rea??o para cada condi??o enzim?tica utilizada e avaliadas quanto a atividade antioxidante pelos m?todos ABTS e ORAC tendo valores entre 597 a 1092 ?M TE (ABTS) e 1615 a 2920 ?M TE (ORAC) para Flavourzyme, 998 a 6290 ?M TE (ABTS) e 3092 a 7567 ?M TE (ORAC) para Alcalase e por fim, 913 a 2678 ?M TE (ABTS) e 2547 a 5903 ?M TE (ORAC) para a mistura enzim?tica. Nenhuma das amostras de hidrolisado com diferentes GH apresentou atividade antimicrobiana contra cepas de Salmonella choleraesuis subsp. Enteritidis (ATCC 13076) e Listeria monocytogenes (ATCC 9117).
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Wikström, Malin. "Synthesis and protein curing abilities of membrane glycolipids". Doctoral thesis, Stockholm University, Department of Biochemistry and Biophysics, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-1361.
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There are many types of membrane lipids throughout Nature. Still little is known about synthesizing pathways and how different lipids affect the embedded membrane proteins. The most common lipids are glycolipids since they dominate plant green tissue. Glycolipids also exist in mammal cells as well as in most Gram-positive bacteria. Glycosyltransferases (GTs) catalyze the final enzymatic steps for these glycolipids. In the bacteria Acholeplasma laidlawii and Streptococcus pneumonie and in the plant Arabidopsis thaliana, GTs for mono-/di-glycosyl-diacylglycerol (-DAG) are suggested to be regulated to keep a certain membrane curvature close to a bilayer/nonbilayer phase transition. The monoglycosylDAGs are nonbilayer-prone with small headgroups, hence by themselves they will not form bilayer structures.
Here we have determined the genes encoding the main glycolipids of A. laidlawii and S. pneumonie. We have also shown that these GTs belong to a large enzyme group widely spread in Nature, and that all four enzymes are differently regulated by membrane lipids. The importance of different lipid properties were traced in a lipid mutant of Escherichia coli lacking the major (75 %), nonbilayer-prone/zwitterionic, lipid phosphatidylethanolamine. Introducing the genes for the GTs of A. laidlawii and two analogous genes from A. thaliana yielded new strains containing 50 percent of glyco-DAG lipids. The monoglyco-DAG strains contain significant amounts of nonbilayer-prone lipids while the diglyco-DAG strains contain no such lipids. Comparing these new strains for viability and the state of membrane-associated functions made it possible to connect different functions to certain lipid properties. In summary, a low surface charge density of anionic lipids is important in E.coli membranes, but this fails to be supportive if the diluting species have a too large headgroup. This indicates that a certain magnitude of the curvature stress is crucial for the membrane bilayer in vivo.
Książki na temat "Prone proteins"
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R, Ansari Rafat, i United States. National Aeronautics and Space Administration., red. A fiber optic probe for monitoring protein aggregation, nucleation, and crystallization. Washington, DC: National Aeronautics and Space Administration, 1997.
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Keromnes, Anne. Synthesis of a novel chemically modified tetrasaccharide to probe carbohydrate-protein interactions. Birmingham: University of Birmingham, 1996.
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Prof, Miller Lawrence W., red. Probes and tags to study biomolecular function: For proteins, RNA, and membranes. Weinheim: Wiley-VCH Verlag, 2008.
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Pieribone, Vincent. Aglow in the dark: The revolutionary science of biofluorescence. Cambridge, Mass: Belknap Press of Harvard University Press, 2005.
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Min, Zhang, Yin Bin-Cheng i SpringerLink (Online service), red. Nano-Bio Probe Design and Its Application for Biochemical Analysis. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012.
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Les recettes Dukan: Mon re gime en 350 recettes. Paris: J'ai lu, 2008.
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Les recettes Dukan: Mon régime en 350 recettes. [Paris]: Flammarion, 2011.
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Nanomedicine. Austin, Tex: Landes Bioscience, 1999.
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Anna, Brajter-Toth, i Chambers James Q, red. Electroanalytical methods for biological materials. New York: Marcel Dekker, 2002.
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Tinker-Mill, Claire Louisa. Nanoscale Imaging and Characterisation of Amyloid-β. Springer International Publishing AG, 2016.
Znajdź pełny tekst źródłaCzęści książek na temat "Prone proteins"
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Kumar, Sandeep, Xiaoling Wang i Satish K. Singh. "Identification and Impact of Aggregation-Prone Regions in Proteins and Therapeutic Monoclonal Antibodies". W Aggregation of Therapeutic Proteins, 103–18. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2010. http://dx.doi.org/10.1002/9780470769829.ch3.
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Ravikumar, Brinda, Sovan Sarkar i David C. Rubinsztein. "Clearance of Mutant Aggregate-Prone Proteins by Autophagy". W Autophagosome and Phagosome, 195–211. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-157-4_13.
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Needle, Danielle, i David S. Waugh. "Rescuing Aggregation-Prone Proteins in Escherichia coli with a Dual His6-MBP Tag". W Protein Affinity Tags, 81–94. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1034-2_7.
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Sinnige, Tessa, Anan Yu i Richard I. Morimoto. "Challenging Proteostasis: Role of the Chaperone Network to Control Aggregation-Prone Proteins in Human Disease". W Advances in Experimental Medicine and Biology, 53–68. Cham: Springer International Publishing, 2020. http://dx.doi.org/10.1007/978-3-030-40204-4_4.
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Kronqvist, Nina, Anna Rising i Jan Johansson. "A Novel Approach for the Production of Aggregation-Prone Proteins Using the Spidroin-Derived NT* Tag". W Methods in Molecular Biology, 113–30. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-1859-2_6.
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Ranganathan, Umarani, i Steven P. C. Groot. "Seed Longevity and Deterioration". W Seed Science and Technology, 91–108. Singapore: Springer Nature Singapore, 2023. http://dx.doi.org/10.1007/978-981-19-5888-5_5.
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AbstractThe fundamental deteriorative processes that lead to loss of seed viability contrastingly vary between desiccation insensitive (orthodox) and desiccation sensitive seeds (recalcitrant). Orthodox seeds which undergo maturation drying are bestowed with protective mechanisms which guard the seeds against deterioration. They include the accumulation of antioxidants, non-reducing sugars, protective proteins such as late embryogenesis abundant proteins, heat-shock proteins, lipocalins, hormones and chemical protectants (raffinose family oligosaccharides, flavonoids, lignins, vitamin E). The nuclear DNA is packed denser and chlorophyll is degraded. Besides, the cytoplasm is capable of transitioning between liquid and glassy state depending on the moisture content of the seeds aiding in the maintenance of seed viability potential. In the dry seeds, the glassy state of the cytoplasm ensures the stabilization of cellular components by arresting cell metabolism. However, even with low moisture content and a glassy state of cytoplasm, reactive oxygen species generated due to the presence of oxygen in the storage atmosphere may cause the ageing of seed. As the seed moisture content increases, mitochondrial respiration gets activated, also leading to increased production of reactive oxygen species, owing to inefficient mitochondrial activity. The reactive oxygen species lead to the oxidation of essential molecules such as DNA, RNA, proteins and lipids. Further, mitochondrial membranes also get oxidized, leading to reduced aerobic respiration potential. When the damage is not substantial, orthodox seeds are capable of repairing the molecular damages that accumulate during storage, enabling the seeds to partially overcome the damages and extend their longevity. This includes activation of repair of cell membranes, DNA, RNA, proteins and mitochondria as the seeds imbibe water.Unlike the orthodox seeds, the recalcitrant seeds are largely devoid of protective mechanisms which guard the seeds against rapid deterioration. The recalcitrant seeds are shed from the mother tree at high moisture content while they are metabolically active. After dispersal, the seeds undergo deteriorative changes during drying due to the damage to the cytoskeleton (physical damage), besides reactive oxygen species-induced damage due to lack of antioxidant activity (metabolism-induced damage). Even when maintained under high moisture content, seeds exhibit dysfunction of the cell organelles and extensive vacuolization predisposing the seeds to deterioration. Thus, recalcitrant seeds are prone to deterioration either under low or high moisture content.
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Sljoka, Adnan. "Structural and Functional Analysis of Proteins Using Rigidity Theory". W Sublinear Computation Paradigm, 337–67. Singapore: Springer Singapore, 2021. http://dx.doi.org/10.1007/978-981-16-4095-7_14.
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AbstractOver the past two decades, we have witnessed an unprecedented explosion in available biological data. In the age of big data, large biological datasets have created an urgent need for the development of bioinformatics methods and innovative fast algorithms. Bioinformatics tools can enable data-driven hypothesis and interpretation of complex biological data that can advance biological and medicinal knowledge discovery. Advances in structural biology and computational modelling have led to the characterization of atomistic structures of many biomolecular components of cells. Proteins in particular are the most fundamental biomolecules and the key constituent elements of all living organisms, as they are necessary for cellular functions. Proteins play crucial roles in immunity, catalysis, metabolism and the majority of biological processes, and hence there is significant interest to understand how these macromolecules carry out their complex functions. The mechanical heterogeneity of protein structures and a delicate mix of rigidity and flexibility, which dictates their dynamic nature, is linked to their highly diverse biological functions. Mathematical rigidity theory and related algorithms have opened up many exciting opportunities to accurately analyse protein dynamics and probe various biological enigmas at a molecular level. Importantly, rigidity theoretical algorithms and methods run in almost linear time complexity, which makes it suitable for high-throughput and big-data style analysis. In this chapter, we discuss the importance of protein flexibility and dynamics and review concepts in mathematical rigidity theory for analysing stability and the dynamics of protein structures. We then review some recent breakthrough studies, where we designed rigidity theory methods to understand complex biological events, such as allosteric communication, large-scale analysis of immune system antibody proteins, the highly complex dynamics of intrinsically disordered proteins and the validation of Nuclear Magnetic Resonance (NMR) solved protein structures.
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Grubmüller, Helmut. "Force Probe Molecular Dynamics Simulations". W Protein-Ligand Interactions, 493–515. Totowa, NJ: Humana Press, 2005. http://dx.doi.org/10.1385/1-59259-912-5_493.
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Peterson, Cynthia B., i Michael N. Blackburn. "Localization and Interaction of Functional Sites on Antithrombin III. Use of an Anti-Hapten Antibody as a Structural Probe". W Proteins, 665–72. Boston, MA: Springer US, 1987. http://dx.doi.org/10.1007/978-1-4613-1787-6_67.
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Frauenfelder, Hans. "The Nucleus as a Probe (C. E. Schulz1)". W The Physics of Proteins, 393–414. New York, NY: Springer New York, 2010. http://dx.doi.org/10.1007/978-1-4419-1044-8_28.
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Abakumets, V. Y., i K. Ya Bulanava. "THE INFLUENCE OF INSULIN FIBRILLATION". W SAKHAROV READINGS 2021: ENVIRONMENTAL PROBLEMS OF THE XXI CENTURY. International Sakharov Environmental Institute of Belarusian State University, 2021. http://dx.doi.org/10.46646/sakh-2021-2-7-10.
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Violation of protein folding leads to the development of a number of systemic and neurodegenerative diseases-proteinopathy. In these pathologies, proteins acquire an incorrect conformation that differs from the native one, become functionally inactive, toxic, and prone to aggregation and deposition in various organs and tissues. There is a widespread hypothesis that the primary cytotoxic agents in the development of proteinopathies are protein oligomers that are prone to aggregation. These diseases include Parkinson’s disease, Creutzfeldt-Jakob disease, type 2 diabetes, and many others.
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Golmohamadi, Farzin Ghane, Amna Mehmood, Franz-Josef Schmitt i Jan Laufer. "Pump-probe photoacoustic spectroscopy of red fluorescent proteins". W European Conference on Biomedical Optics. Washington, D.C.: Optica Publishing Group, 2021. http://dx.doi.org/10.1364/ecbo.2021.em2d.2.
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Photoacoustic pump-probe excitation was used to generate a fluorophore-specific contrast in fluorescent proteins. The measured signals using pump-probe spectroscopy were found to correlate with the absorption and emission spectra of the protein.
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Reed, Scott M., Min S. Wang i Erica L. Curello. "Electrophoretic Mobility of Lipid Coated Nanoparticles: Understanding the Influence of Size and Charge on a Lipoprotein Particle Mimic". W ASME 2011 International Mechanical Engineering Congress and Exposition. ASMEDC, 2011. http://dx.doi.org/10.1115/imece2011-64158.
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Elevated levels of low-density lipoprotein (LDL) are associated with increased risk of coronary heart disease (CHD). Although smaller LDL particles are more atherogenic, it is not clear how LDL particle size influences atherogenesis. Smaller particles may be more prone to macrophage uptake and plaque formation. Alternatively, increased rates of lipid oxidation may explain the atherogenic effects of smaller LDL. We have developed a mimic of LDL that allows independent examination of the effect of LDL size and oxidation. We have engineered LDL mimics using liposome-encapsulated gold nanoparticles, in which the size and surface charge are independently controlled during synthesis. Here we examine the effects of lipid composition on zeta potential and electrophoretic mobility of LDL mimics. Using these mimics, we explored the effect of the lipid coating on the nanoparticles including anionic lipids and oxidized lipids. Dynamic light scattering was used to determine the size of the mimics and gel electrophoresis was used to measure the mobility and calculate zeta potential. The charge of the lipid coating influenced the mobility and we anticipate this will influence how the mimics interacts with proteins.
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Church, W., T. Messier, P. Howard, J. Amiral, D. Meyer i K. Mam. "A SHARED EPITOPE ON HUMAN PROTEIN C, FACTOR X, FACTOR VII, AND PROTTOBIN DEFINED BY A MONOCLONAL ANTIBODY". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643937.
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A monoclonal antibody prepared against hunan protein C (HPC) was found to react with several other vitamin K-dependent blood proteins. Using a competitive inhibition solid-phase radioinminoassay with HPC, binding of 125I-HPC to the antibody was inhibited by purified prothrombin, Factor X, and Factor VII in addition to protein C. Other vitamin K-dependent proteins including Factor IX, protein S, and bone-GLA protein did not compete for binding of 125I-HPC to the antibody. The effect of calciun ion on the binding of antibody to 125I-HPC was examined in a solid-phase imnunoassay system with the antibody bound to rabbit anti-mouse inminoglobulin adsorbed to microtiter plates. In the presence of 5 mM calciun ion, radiolabeled protein C did not bind to the antibody; radiolabeled protein C did bind, however, in the presence of 5 nM EDTA suggesting that the epitope is expressed only after removal of calciun ion. The antibody bound to prothrombin and to decarboxylated prothrombin after adsorption of the antigens onto nitrocellulose indicating that the presence of GLA was not required for antibody binding. Iimunoblotting of proteins which were reduced, the peptides separated by SDS-PAGE, and transferred to nitrocellulose showed that the antibody reacts with a determinant found on the light chains of protein C and Factor X and with prothrombin Fragment 1. Comparison of the protein sequences of protein C light chain, Factor X light chain, Factor VII, and prothrombin Fragment 1 identified a segment of amino acid sequence that is highly conserved in all four proteins and might contain the antigenic site. The monoclonal antibody thus defines an antigenic determinant which is masked by calcium ion and is found on the surface of several related, yet different coagulation proteins. This antibody should prove useful in understanding the evolutionary relationships amongst the vitamin K-dependent proteins and also in understanding the effect of calcium ion on the structure of protein C, Factor X, prothrombin, Factor VII and possibly other related proteins. (Supported by NIH grant MHLBI HL35058)
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Aleixo, Frederico, Matthias Knorr i João Leite. "Revising Boolean Logical Models of Biological Regulatory Networks". W 20th International Conference on Principles of Knowledge Representation and Reasoning {KR-2023}. California: International Joint Conferences on Artificial Intelligence Organization, 2023. http://dx.doi.org/10.24963/kr.2023/2.
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Boolean regulatory networks are used to represent complex biological processes, modelling the interactions of biological compounds, such as proteins or genes, with each other and with other substances in a cell. Creating and maintaining computational models of these networks is crucial for comprehending corresponding cellular processes, as they allow reproducing known behaviours and testing new hypotheses and predictions in silico. In this context, model revision focuses on validating and (if necessary) repairing existing models based on new experimental data. However, model revision is commonly performed manually, which is inefficient and prone to error, and the few existing automated solutions either only apply to simpler networks or are limited in their revision process, since they may not be able to produce a solution within a reasonable time frame or miss the optimal solution. In this paper, we develop a solution for revising logical models of Boolean regulatory networks, able to find repairs that are consistent with provided, possibly incomplete experimental data, and minimal w.r.t. the differences to the original network. We show that our solution can be used to revise different real-world Boolean logical models very efficiently, surpassing a previous solution in terms of solved instances and with a considerable margin w.r.t. processing time.
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Villeneuve, Pierre, Jeanne Duplessis-Kergomard, Melina Robert, Gilles Paboeuf, Nathalie Barouh, Olivier Schafer, Tim Wooster, Claire Bourlieu-Lacanal i Veronique Vie. "Effect of Processing and Fat Content on the Oxidative Stability and Interfacial Behavior of Tree Nut Oil-bodies". W 2022 AOCS Annual Meeting & Expo. American Oil Chemists' Society (AOCS), 2022. http://dx.doi.org/10.21748/djlm1220.
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Oil bodies (OB) are the natural form of energy storage in oilseeds. They consist of a triacylglyceride core stabilized by proteins (mainly oleosins) embedded in a phospholipid membrane. In some tree nut species, OB contain an important amount of polyunsaturated fatty acids that make them interesting food ingredients but prone to oxidation. Due to the growing interest in minimally-processed nut-based beverages, the understanding and the preservation of the physico-chemical properties of these lipoprotein assemblies are of great interest. In this context, previous interfacial studies have led to the proposition of a new model of adsorption for minimally-processed OB that is useful to design functional emulsion or foam in which OB act as emulsifiers. The objective of the present study was to determine the impact of processing and fat content on the physico-chemical behavior of tree nut-based matrixes. The interfacial and oxidative behaviors of minimally-processed isolated OB were compared with processed complex nut-based matrixes (i.e. beverages) of different fat content. Processing operations (heat-treatment, homogenization) affected OB interfacial properties and oxidative stability but fat content had even stronger impact on this last parameter, with more concentrated systems more stable to oxidation. The results underline the good physico-chemical stability of tree nut OB, and raise the interest of their use in the minimally-processed form to fully exploit their beneficial properties.
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Kanchanawong, Pakorn (Tony). "Interference-Based Techniques in Superresolution Microscopy". W JSAP-OSA Joint Symposia. Washington, D.C.: Optica Publishing Group, 2017. http://dx.doi.org/10.1364/jsap.2017.6p_a409_1.
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Many complex biological functions are performed by supramolecular assemblies selforganized from a diverse ensemble of proteins. The spatial organizations of protein building blocks within these structures are key to their functions, but have been challenging to determine experimentally. Due to the nanometer size scale of proteins, the nanoscale is the salient length scale for structural organization in cells. Thanks to the recent development of superresolution microscopy, it has become possible to decipher the intricate nanoscale architecture of several cellular structures. For these structural cell biology applications, high spatial resolution, ideally approaching the sub-10 nm scale of protein-size is desirable. Interference-based strategies have been particularly useful for achieving such molecular-scale resolution[1-4]. I will discuss examples of interference-based techniques in superresolution microscopy and their applications to decipher nanoscale organization of cell adhesions machinery. Of particular biological relevance, cell adhesions such as the cadherin-mediated cell-cell junctions are multi-protein assemblies known to transmit, sustain, sense, and respond to mechanical force. The knowledge of their physical organization is therefore essential for molecular mechanistic insights into their mechanobiological functions. I will discuss our recent study whereby interference-based superresolution strategy was employed to elucidate the nanoscale architecture of the cadherin-based cell adhesions and to probe conformational transitions of the mechanotransducer protein vinculin, revealing how they are regulated by mechanical tension together with specific tyrosine kinase and phosphatase [4].
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Hansen, John E., Sandra J. Rosenthal i Graham R. Fleming. "Subpicosecond Fluorescence Anisotropy Measurements of Tryptophanyl Residues in Proteins". W International Conference on Ultrafast Phenomena. Washington, D.C.: Optica Publishing Group, 1990. http://dx.doi.org/10.1364/up.1990.pdp2.
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The fluorescence anisotropy of tryptophanyl residues in proteins is used as a probe of protein motion. We have recently shown(1) that the short life time (200 fs - 10 ps) region of the fluorescence anisotropy of tryptophan in solution is dominated by an electronic relaxation process, which has the potential to obscure molecular motion. The question arises as to the relevance of this result for the behavior of tryptophanyl residues in proteins. To address this question we have examined the subpicosecond fluorescence anisotropy of tryptophanyl residues in two very different enviroments - exposed to the solvent in melittin, a 26 amino acid peptide, and buried in a hydrophobic region in apoazurin purified from the bacteria Pseudomonas aeruginosa. Measurements were made by ultraviolet fluorescence upconversion described elsewhere(1). Samples were degassed and flowed in absence of air. Concentrations were adjusted so the optical density at the excitation wavelength was between 0.6 and 1.0 for a 1mm pathlength.
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de Vries, C. J. M., N. K. Veerman i H. Pannekoek. "ARTIFICIAL EXON SHUFFLING: CONSTRUCTION OF HYBRID cDNAS CONTAINING DOMAINS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (T-PA) AND UROKINASE (u-PA)". W XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643940.
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The intriguing finding that functions of t-PA coincide with structural domains and that these domains occur in related proteins, has been the basis to construct hybrid proteins by artificial exon shuffling to prove the conservation of functions in the shuffled domains. The heavy chain (Hch) of t-PA mediates both binding to fibrin and stimulation of plasminogen activator activity via its Finger- and Kringle-2 domain, whereas the light chain (Lch) contains the serine protease moiety of the protein. The Hch of u-PA is very homologous to the Lch of t-PA, but exhibits a higher plasminogen activator activity. This activity of u-PA is not stimulated by fibrin. We employed the ‘M13 in vitro outlooping’ technique to fuse the Hch of t-PA cDNA and the Hch of u-PA cDNA, to create two different hybrid cDNAs. On one hybrid cDNA, the t-PA and the u-PA sequences are coupled precisely at the exon-intron boundaries of the corresponding genes, while the other hybrid cDNA lacks a u-PA segment at the junction, encoding 13 amino acids of u-PA. The hybrid cDNAs were transiently expressed in mouse Ltk- cells and the recombinant proteins were characterized. The plasminogen activator activity of these proteins was determined in an indirect amidolytic assay, using plasminogen and the chromogenic substrate S2251. As anticipated, the activity of both t-PA/u-PA hybrid proteins is stimulated by fibrin, however, not to the same extent as t-PA. Remarkably, we found a decreased inhibition of the hybrid proteins by the endothelial plasminogen activator inhibitor (PAI-1) as compared to t-PA and u-PA, although stable complexes between the hybrid proteins and the inhibitor are formed. We conclude that functions of structural domains are maintained during exon shuffling
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Nomura, Wataru, Nami Ohashi, Tetsuo Narumi i Hirokazu Tamamura. "Tag-Probe System for Imaging of Intracellular Proteins". W The Twenty-Third American and the Sixth International Peptide Symposium. Prompt Scientific Publishing, 2013. http://dx.doi.org/10.17952/23aps.2013.174.
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Asenath-Smith, Emily, Emily Jeng, Emma Ambrogi, Garrett Hoch i Jason Olivier. Investigations into the ice crystallization and freezing properties of the antifreeze protein ApAFP752. Engineer Research and Development Center (U.S.), wrzesień 2022. http://dx.doi.org/10.21079/11681/45620.
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Antifreeze proteins (AFPs) allow biological organisms, including insects, fish, and plants, to survive in freezing temperatures. While in solution, AFPs impart cryoprotection by creating a thermal hysteresis (TH), imparting ice recrystallization inhibition (IRI), and providing dynamic ice shaping (DIS). To leverage these ice-modulating effects of AFPs in other scenarios, a range of icing assays were performed with AFPs to investigate how AFPs interact with ice formation when tethered to a surface. In this work, we studied ApAFP752, an AFP from the beetle Anatolica polita, and first investigated whether removing the fusion protein attached during protein expression would result in a difference in freezing behavior. We performed optical microscopy to examine ice-crystal shape, micro-structure, and the recrystallization behavior of frozen droplets of AFP solutions. We developed a surface chemistry approach to tether these proteins to glass surfaces and conducted droplet-freezing experiments to probe the interactions of these proteins with ice formed on those surfaces. In solution, ApAFP752 did not show any DIS or TH, but it did show IRI capabilities. In surface studies, the freezing of AFP droplets on clean glass surfaces showed no dependence on concentration, and the results from freezing water droplets on AFP-decorated surfaces were inconclusive.
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Epel, Bernard L., Roger N. Beachy, A. Katz, G. Kotlinzky, M. Erlanger, A. Yahalom, M. Erlanger i J. Szecsi. Isolation and Characterization of Plasmodesmata Components by Association with Tobacco Mosaic Virus Movement Proteins Fused with the Green Fluorescent Protein from Aequorea victoria. United States Department of Agriculture, wrzesień 1999. http://dx.doi.org/10.32747/1999.7573996.bard.
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The coordination and regulation of growth and development in multicellular organisms is dependent, in part, on the controlled short and long-distance transport of signaling molecule: In plants, symplastic communication is provided by trans-wall co-axial membranous tunnels termed plasmodesmata (Pd). Plant viruses spread cell-to-cell by altering Pd. This movement scenario necessitates a targeting mechanism that delivers the virus to a Pd and a transport mechanism to move the virion or viral nucleic acid through the Pd channel. The identity of host proteins with which MP interacts, the mechanism of the targeting of the MP to the Pd and biochemical information on how Pd are alter are questions which have been dealt with during this BARD project. The research objectives of the two labs were to continue their biochemical, cellular and molecular studies of Pd composition and function by employing infectious modified clones of TMV in which MP is fused with GFP. We examined Pd composition, and studied the intra- and intercellular targeting mechanism of MP during the infection cycle. Most of the goals we set for ourselves were met. The Israeli PI and collaborators (Oparka et al., 1999) demonstrated that Pd permeability is under developmental control, that Pd in sink tissues indiscriminately traffic proteins of sizes of up to 50 kDa and that during the sink to source transition there is a substantial decrease in Pd permeability. It was shown that companion cells in source phloem tissue export proteins which traffic in phloem and which unload in sink tissue and move cell to cell. The TAU group employing MP:GFP as a fluorescence probe for optimized the procedure for Pd isolation. At least two proteins kinases found to be associated with Pd isolated from source leaves of N. benthamiana, one being a calcium dependent protein kinase. A number of proteins were microsequenced and identified. Polyclonal antibodies were generated against proteins in a purified Pd fraction. A T-7 phage display library was created and used to "biopan" for Pd genes using these antibodies. Selected isolates are being sequenced. The TAU group also examined whether the subcellular targeting of MP:GFP was dependent on processes that occurred only in the presence of the virus or whether targeting was a property indigenous to MP. Mutant non-functional movement proteins were also employed to study partial reactions. Subcellular targeting and movement were shown to be properties indigenous to MP and that these processes do not require other viral elements. The data also suggest post-translational modification of MP is required before the MP can move cell to cell. The USA group monitored the development of the infection and local movement of TMV in N. benthamiana, using viral constructs expressing GFP either fused to the MP of TMV or expressing GFP as a free protein. The fusion protein and/or the free GFP were expressed from either the movement protein subgenomic promoter or from the subgenomic promoter of the coat protein. Observations supported the hypothesis that expression from the cp sgp is regulated differently than expression from the mp sgp (Szecsi et al., 1999). Using immunocytochemistry and electron microscopy, it was determined that paired wall-appressed bodies behind the leading edge of the fluorescent ring induced by TMV-(mp)-MP:GFP contain MP:GFP and the viral replicase. These data suggest that viral spread may be a consequence of the replication process. Observation point out that expression of proteins from the mp sgp is temporary regulated, and degradation of the proteins occurs rapidly or more slowly, depending on protein stability. It is suggested that the MP contains an external degradation signal that contributes to rapid degradation of the protein even if expressed from the constitutive cp sgp. Experiments conducted to determine whether the degradation of GFP and MP:GFP was regulated at the protein or RNA level, indicated that regulation was at the protein level. RNA accumulation in infected protoplast was not always in correlation with protein accumulation, indicating that other mechanisms together with RNA production determine the final intensity and stability of the fluorescent proteins.
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Ghanim, Murad, Joe Cicero, Judith K. Brown i Henryk Czosnek. Dissection of Whitefly-geminivirus Interactions at the Transcriptomic, Proteomic and Cellular Levels. United States Department of Agriculture, luty 2010. http://dx.doi.org/10.32747/2010.7592654.bard.
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Our project focuses on gene expression and proteomics of the whitefly Bemisia tabaci (Gennadius) species complex in relation to the internal anatomy and localization of expressed genes and virions in the whitefly vector, which poses a major constraint to vegetable and fiber production in Israel and the USA. While many biological parameters are known for begomovirus transmission, nothing is known about vector proteins involved in the specific interactions between begomoviruses and their whitefly vectors. Identifying such proteins is expected to lead to the design of novel control methods that interfere with whitefly-mediated begomovirus transmission. The project objectives were to: 1) Perform gene expression analyses using microarrays to study the response of whiteflies (B, Q and A biotypes) to the acquisition of begomoviruses (Tomato yellow leaf curl (TYLCV) and Squash leaf curl (SLCV). 2) Construct a whitefly proteome from whole whiteflies and dissected organs after begomovirus acquisition. 3) Validate gene expression by q-RTPCR and sub-cellular localization of candidate ESTs identified in microarray and proteomic analyses. 4) Verify functionality of candidate ESTs using an RNAi approach, and to link these datasets to overall functional whitefly anatomical studies. During the first and second years biological experiments with TYLCV and SLCV acquisition and transmission were completed to verify the suitable parameters for sample collection for microarray experiments. The parameters were generally found to be similar to previously published results by our groups and others. Samples from whole whiteflies and midguts of the B, A and Q biotypes that acquired TYLCV and SLCV were collected in both the US and Israel and hybridized to B. tabaci microarray. The data we analyzed, candidate genes that respond to both viruses in the three tested biotypes were identified and their expression that included quantitative real-time PCR and co-localization was verified for HSP70 by the Israeli group. In addition, experiments were undertaken to employ in situ hybridization to localize several candidate genes (in progress) using an oligonucleotide probe to the primary endosymbiont as a positive control. A proteome and corresponding transcriptome to enable more effective protein identification of adult whiteflies was constructed by the US group. Further validation of the transmission route of begomoviruses, mainly SLCV and the involvement of the digestive and salivary systems was investigated (Cicero and Brown). Due to time and budget constraints the RNAi-mediated silencing objective to verify gene function was not accomplished as anticipated. HSP70, a strong candidate protein that showed over-expression after TYLCV and SLCV acquisition and retention by B. tabaci, and co-localization with TYLCV in the midgut, was further studies. Besides this protein, our joint research resulted in the identification of many intriguing candidate genes and proteins that will be followed up by additional experiments during our future research. To identify these proteins it was necessary to increase the number and breadth of whitefly ESTs substantially and so whitefly cDNAs from various libraries made during the project were sequenced (Sanger, 454). As a result, the proteome annotation (ID) was far more successful than in the initial attempt to identify proteins using Uniprot or translated insect ESTs from public databases. The extent of homology shared by insects in different orders was surprisingly low, underscoring the imperative need for genome and transcriptome sequencing of homopteran insects. Having increased the number of EST from the original usable 5500 generated several years ago to >600,000 (this project+NCBI data mining), we have identified about one fifth of the whitefly proteome using these new resources. Also we have created a database that links all identified whitefly proteins to the PAVEdb-ESTs in the database, resulting in a useful dataset to which additional ESTS will be added. We are optimistic about the prospect of linking the proteome ID results to the transcriptome database to enable our own and other labs the opportunity to functionally annotate not only genes and proteins involved in our area of interest (whitefly mediated transmission) but for the plethora of other functionalities that will emerge from mining and functionally annotating other key genes and gene families in whitefly metabolism, development, among others. This joint grant has resulted in the identification of numerous candidate proteins involved in begomovirus transmission by B. tabaci. A next major step will be to capitalize on validated genes/proteins to develop approaches to interfere with the virus transmission.
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Buck, D. R. Theoretical Simulations and Ultrafast Pump-probe Spectroscopy Experiments in Pigment-protein Photosynthetic Complexes. Office of Scientific and Technical Information (OSTI), wrzesień 2000. http://dx.doi.org/10.2172/764683.
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Porat, Ron, Gregory T. McCollum, Amnon Lers i Charles L. Guy. Identification and characterization of genes involved in the acquisition of chilling tolerance in citrus fruit. United States Department of Agriculture, grudzień 2007. http://dx.doi.org/10.32747/2007.7587727.bard.
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Citrus, like many other tropical and subtropical fruit are sensitive to chilling temperatures. However, application of a pre-storage temperature conditioning (CD) treatment at 16°C for 7 d or of a hot water brushing (HWB) treatment at 60°C for 20 sec remarkably enhances chilling tolerance and reduces the development of chilling injuries (CI) upon storage at 5°C. In the current research, we proposed to identify and characterize grapefruit genes that are induced by CD, and may contribute to the acquisition of fruit chilling tolerance, by two different molecular approaches: cDNA array analysis and PCR cDNA subtraction. In addition, following the recent development and commercialization of the new Affymetrix Citrus Genome Array, we further performed genome-wide transcript profiling analysis following exposure to CD and chilling treatments. To conduct the cDNA array analysis, we constructed cDNA libraries from the peel tissue of CD- and HWB-treated grapefruit, and performed an EST sequencing project including sequencing of 3,456 cDNAs from each library. Based on the obtained sequence information, we chose 70 stress-responsive and chilling-related genes and spotted them on nylon membranes. Following hybridization the constructed cDNA arrays with RNA probes from control and CD-treated fruit and detailed confirmations by RT-PCR analysis, we found that six genes: lipid-transfer protein, metallothionein-like protein, catalase, GTP-binding protein, Lea5, and stress-responsive zinc finger protein, showed higher transcript levels in flavedo of conditioned than in non-conditioned fruit stored at 5 ᵒC. The transcript levels of another four genes: galactinol synthase, ACC oxidase, temperature-induced lipocalin, and chilling-inducible oxygenase, increased only in control untreated fruit but not in chilling-tolerant CD-treated fruit. By PCR cDNA subtraction analysis we identified 17 new chilling-responsive and HWB- and CD-induced genes. Overall, characterization of the expression patterns of these genes as well as of 11 more stress-related genes by RNA gel blot hybridizations revealed that the HWB treatment activated mainly the expression of stress-related genes(HSP19-I, HSP19-II, dehydrin, universal stress protein, EIN2, 1,3;4-β-D-glucanase, and SOD), whereas the CD treatment activated mainly the expression of lipid modification enzymes, including fatty acid disaturase2 (FAD2) and lipid transfer protein (LTP). Genome wide transcriptional profiling analysis using the newly developed Affymetrix Citrus GeneChip® microarray (including 30,171 citrus probe sets) revealed the identification of three different chilling-related regulons: 1,345 probe sets were significantly affected by chilling in both control and CD-treated fruits (chilling-response regulon), 509 probe sets were unique to the CD-treated fruits (chilling tolerance regulon), and 417 probe sets were unique to the chilling-sensitive control fruits (chilling stress regulon). Overall, exposure to chilling led to expression governed arrest of general cellular metabolic activity, including concretive down-regulation of cell wall, pathogen defense, photosynthesis, respiration, and protein, nucleic acid and secondary metabolism. On the other hand, chilling enhanced various adaptation processes, such as changes in the expression levels of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.
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Epel, Bernard, i Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, listopad 2005. http://dx.doi.org/10.32747/2005.7695874.bard.
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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.
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Terzyan, Aram. Belarus in the Wake of a Revolution: Domestic and International Factors. Eurasia Institutes, grudzień 2020. http://dx.doi.org/10.47669/eea-3-2020.
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This paper explores the political landscape of Belarus in the aftermath of the 2020 presidential elections, with a focus on both domestic and international factors behind the ongoing crisis. Lukashenko’s regime has a long record of sustaining its power by preserving elite unity, controlling elections, and/or using force against opponents. Therefore, massive fraud characterizing the 2020 presidential elections and brutal suppression of peaceful protests in its aftermath came as no surprise. Against this backdrop, the anti-government protests following the presidential elections raised a series of unanswered questions regarding both their domestic and foreign policy implications. The biggest question is whether the Belarusian civil society and opposition will prove powerful enough to overcome state repression and change the status quo in Europe’s “last dictatorship”. Worries remain about the Belarusian opposition’s emphasis on foreign policy continuity, meaning that Belarus is bound to remain in the orbit of the Russian authoritarian influence. The total fiasco of post-Velvet Revolution Armenian government both in terms of domestic and foreign policies, among others, further reveals the excruciating difficulties of a democratic state-building within the Russia-led socio-political order.
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Braun, Alexander. The Interaction between a Thiol Specific Probe (OPA) and the Single Channel Characteristics of the Reconstituted Ca++ Release Protein from Skeletal Muscle Sarcoplasmic Reticulum. Portland State University Library, styczeń 2000. http://dx.doi.org/10.15760/etd.6745.
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Rafaeli, Ada, Russell Jurenka i Daniel Segal. Isolation, Purification and Sequence Determination of Pheromonotropic-Receptors. United States Department of Agriculture, lipiec 2003. http://dx.doi.org/10.32747/2003.7695850.bard.
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Moths constitute a major group of pest insects in agriculture. Pheromone blends are utilised by a variety of moth species to attract conspecific mates, which is under circadian control by the neurohormone, PBAN (pheromone-biosynthesis-activating neuropeptide). Our working hypothesis was that, since the emission of sex-pheromone is necessary to attract a mate, then failure to produce and emit pheromone is a potential strategy for manipulating adult moth behavior. The project aimed at identifying, characterising and determining the sequence of specific receptors responsible for the interaction with pheromonotropic neuropeptide/s using two related moth species: Helicoverpa armigera and H. lea as model insects. We established specific binding to a membrane protein estimated at 50 kDa in mature adult females using a photoaffinity-biotin probe for PBAN. We showed that JH is required for the up-regulation of this putative receptor protein. In vitro studies established that the binding initiates a cascade of second messengers including channel opening for calcium ions and intracellular cAMP production. Pharmacological studies (using sodium fluoride) established that the receptor is coupled to a G-protein, that is, the pheromone-biosynthesis-activating neuropeptide receptor (PBAN-R) belongs to the family of G protein-coupled receptor (GPCR)'s. We showed that PBAN-like peptides are present in Drosophila melanogaster based on bioassay and immunocytochemical data. Using the annotated genome of D. melanogaster to search for a GPCR, we found that some were similar to neuromedin U- receptors of vertebrates, which contain a similar C-terminal ending as PBAN. We established that neuromedin U does indeed induce pheromone biosynthesis and cAMP production. Using a PCR based cloning strategy and mRNA isolated from pheromone glands of H. zea, we successfully identified a gene encoding a GPCR from pheromone glands. The full-length PBAN-R was subsequently cloned and expressed in Sf9 insect cells and was shown to mobilize calcium in response to PBAN in a dose-dependent manner. The successful progress in the identification of a gene, encoding a GPCR for the neurohormone, PBAN, provides a basis for the design of a novel battery of compounds that will specifically antagonize pheromone production. Furthermore, since PBAN belongs to a family of insect neuropeptides with more than one function in different life stages, this rationale may be extended to other physiological key-regulatory processes in different insects.
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Shapira, Roni, Judith Grizzle, Nachman Paster, Mark Pines i Chamindrani Mendis-Handagama. Novel Approach to Mycotoxin Detoxification in Farm Animals Using Probiotics Added to Feed Stuffs. United States Department of Agriculture, maj 2010. http://dx.doi.org/10.32747/2010.7592115.bard.
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T-2 toxin, a toxic product belongs to the trichothecene mycotoxins, attracts major interest because of its severe detrimental effects on the health of human and farm animals. The occurrence of trichothecenes contamination is global and they are very resistant to physical or chemical detoxification techniques. Trichothecenes are absorbed in the small intestine into the blood stream. The hypothesis of this project was to develop a protecting system using probiotic bacteria that will express trichothecene 3-O-acetyltransferase (Tri101) that convert T-2 to a less toxic intermediate to reduce ingested levels in-situ. The major obstacle that we had faced during the project is the absence of stable and efficient expression vectors in probiotics. Most of the project period was invested to screen and isolate strong promoter to express high amounts of the detoxify enzyme on one hand and to stabilize the expression vector on the other hand. In order to estimate the detoxification capacity of the isolated promoters we had developed two very sensitive bioassays.The first system was based on Saccharomyces cerevisiae cells expressing the green fluorescent protein (GFP). Human liver cells proliferation was used as the second bioassay system.Using both systems we were able to prove actual detoxification on living cells by probiotic bacteria expressing Tri101. The first step was the isolation of already discovered strong promoters from lactic acid bacteria, cloning them downstream the Tri101 gene and transformed vectors to E. coli, a lactic acid bacteria strain Lactococcuslactis MG1363, and a probiotic strain of Lactobacillus casei. All plasmid constructs transformed to L. casei were unstable. The promoter designated lacA found to be the most efficient in reducing T-2 from the growth media of E. coli and L. lactis. A prompter library was generated from L. casei in order to isolate authentic probiotic promoters. Seven promoters were isolated, cloned downstream Tri101, transformed to bacteria and their detoxification capability was compared. One of those prompters, designated P201 showed a relatively high efficiency in detoxification. Sequence analysis of the promoter region of P201 and another promoter, P41, revealed the consensus region recognized by the sigma factor. We further attempted to isolate an inducible, strong promoter by comparing the protein profiles of L. casei grown in the presence of 0.3% bile salt (mimicking intestine conditions). Six spots that were consistently overexpressed in the presence of bile salts were isolated and identified. Their promoter reigns are now under investigation and characterization.