Добірка наукової літератури з теми "MFS proteins"
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Статті в журналах з теми "MFS proteins":
1
Sekhwal, Manoj Kumar, Vinay Sharma, and Renu Sarin. "Identification of MFS proteins in sorghum using semantic similarity." Theory in Biosciences 132, no. 2 (January 9, 2013): 105–13. http://dx.doi.org/10.1007/s12064-012-0174-z.
2
Xiao, Qingjie, Mengxue Xu, Weiwei Wang, Tingting Wu, Weizhe Zhang, Wenming Qin, and Bo Sun. "Utilization of AlphaFold2 to Predict MFS Protein Conformations after Selective Mutation." International Journal of Molecular Sciences 23, no. 13 (June 29, 2022): 7235. http://dx.doi.org/10.3390/ijms23137235.
Анотація:
The major facilitator superfamily (MFS) is the largest secondary transporter family and is responsible for transporting a broad range of substrates across the biomembrane. These proteins are involved in a series of conformational changes during substrate transport. To decipher the transport mechanism, it is necessary to obtain structures of these different conformations. At present, great progress has been made in predicting protein structure based on coevolutionary information. In this study, AlphaFold2 was used to predict different conformational structures for 69 MFS transporters of E. coli after the selective mutation of residues at the interface between the N- and C-terminal domains. The predicted structures for these mutants had small RMSD values when compared to structures obtained using X-ray crystallography, which indicates that AlphaFold2 predicts the structure of MSF transporters with high accuracy. In addition, different conformations of other transporter family proteins have been successfully predicted based on mutation methods. This study provides a structural basis to study the transporting mechanism of the MFS transporters and a method to probe dynamic conformation changes of transporter family proteins when performing their function.
3
Ditty, Jayna L., and Caroline S. Harwood. "Conserved Cytoplasmic Loops Are Important for both the Transport and Chemotaxis Functions of PcaK, a Protein fromPseudomonas putida with 12 Membrane-Spanning Regions." Journal of Bacteriology 181, no. 16 (August 15, 1999): 5068–74. http://dx.doi.org/10.1128/jb.181.16.5068-5074.1999.
Анотація:
ABSTRACT Chemotaxis to the aromatic acid 4-hydroxybenzoate (4-HBA) byPseudomonas putida is mediated by PcaK, a membrane-bound protein that also functions as a 4-HBA transporter. PcaK belongs to the major facilitator superfamily (MFS) of transport proteins, none of which have so far been implicated in chemotaxis. Work with two well-studied MFS transporters, LacY (the lactose permease) and TetA (a tetracycline efflux protein), has revealed two stretches of amino acids located between the second and third (2-3 loop) and the eighth and ninth (8-9 loop) transmembrane regions that are required for substrate transport. These sequences are conserved among most MFS transporters, including PcaK. To determine if PcaK has functional requirements similar to those of other MFS transport proteins and to analyze the relationship between the transport and chemotaxis functions of PcaK, we generated strains with mutations in amino acid residues located in the 2-3 and 8-9 loops of PcaK. The mutant proteins were analyzed in 4-HBA transport and chemotaxis assays. Cells expressing mutant PcaK proteins had a range of phenotypes. Some transported at wild-type levels, while others were partially or completely defective in 4-HBA transport. An aspartate residue in the 8-9 loop that has no counterpart in LacY and TetA, but is conserved among members of the aromatic acid/H+ symporter family of the MFS, was found to be critical for 4-HBA transport. These results indicate that conserved amino acids in the 2-3 and 8-9 loops of PcaK are required for 4-HBA transport. Amino acid changes that decreased 4-HBA transport also caused a decrease in 4-HBA chemotaxis, but the effect on chemotaxis was sometimes slightly more severe. The requirement of PcaK for both 4-HBA transport and chemotaxis demonstrates that P. putida has a chemoreceptor that differs from the classical chemoreceptors described for Escherichia coli and Salmonella typhimurium.
4
Severson, Aaron F., and Bruce Bowerman. "Myosin and the PAR proteins polarize microfilament-dependent forces that shape and position mitotic spindles in Caenorhabditis elegans." Journal of Cell Biology 161, no. 1 (April 14, 2003): 21–26. http://dx.doi.org/10.1083/jcb.200210171.
Анотація:
In Caenorhabditis elegans, the partitioning proteins (PARs), microfilaments (MFs), dynein, dynactin, and a nonmuscle myosin II all localize to the cortex of early embryonic cells. Both the PARs and the actomyosin cytoskeleton are required to polarize the anterior-posterior (a-p) body axis in one-cell zygotes, but it remains unknown how MFs influence embryonic polarity. Here we show that MFs are required for the cortical localization of PAR-2 and PAR-3. Furthermore, we show that PAR polarity regulates MF-dependent cortical forces applied to astral microtubules (MTs). These forces, which appear to be mediated by dynein and dynactin, produce changes in the shape and orientation of mitotic spindles. Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces. Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex. This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.
5
Rizal, S., Masrukhin, H. A. Nugroho, and S. Saputra. "Detection of major facilitator superfamily (MFS) transporter in Enterobacteriaceae isolated from chicken." IOP Conference Series: Earth and Environmental Science 1107, no. 1 (December 1, 2022): 012050. http://dx.doi.org/10.1088/1755-1315/1107/1/012050.
Анотація:
Abstract A major facilitator superfamily (MFS) transporter is an active secondary transport system that uses a chemiosmotic ion gradient to transport small molecules. MFS is part of the efflux pump group of proteins and is responsible for releasing toxic compounds and contributing to the antimicrobial resistance mechanisms. In this study, we aimed to detect MFS genes from a collection of Enterobacteriaceae (n=60) isolated from cloacal swabs of chickens. After DNA extraction and amplification, only four isolates (6.7%) were regarded positive for MFS genes and subjected to Sanger dideoxy sequencing. A BlastX search in GenBank® showed that four positive samples with approximate length 300 bp were identified as MSF transporter from Klebsiella pneumoniae with a similarity of 97-98%. This finding warrants further study to confirm both phenotypic and genotypic resistance profiles that correlate with bacterial multidrug efflux pump mechanism.
6
Varela, Manuel F., Anely Ortiz-Alegria, Manjusha Lekshmi, Jerusha Stephen, and Sanath Kumar. "Functional Roles of the Conserved Amino Acid Sequence Motif C, the Antiporter Motif, in Membrane Transporters of the Major Facilitator Superfamily." Biology 12, no. 10 (October 16, 2023): 1336. http://dx.doi.org/10.3390/biology12101336.
Анотація:
The biological membrane surrounding all living cells forms a hydrophobic barrier to the passage of biologically important molecules. Integral membrane proteins called transporters circumvent the cellular barrier and transport molecules across the cell membrane. These molecular transporters enable the uptake and exit of molecules for cell growth and homeostasis. One important collection of related transporters is the major facilitator superfamily (MFS). This large group of proteins harbors passive and secondary active transporters. The transporters of the MFS consist of uniporters, symporters, and antiporters, which share similarities in structures, predicted mechanism of transport, and highly conserved amino acid sequence motifs. In particular, the antiporter motif, called motif C, is found primarily in antiporters of the MFS. The antiporter motif’s molecular elements mediate conformational changes and other molecular physiological roles during substrate transport across the membrane. This review article traces the history of the antiporter motif. It summarizes the physiological evidence reported that supports these biological roles.
7
Zhang, Jialan, Yingbao Liu, Li Li, and Mengxiang Gao. "iTRAQ-Based Quantitative Proteomic Analysis Reveals Changes in Metabolite Biosynthesis in Monascus purpureus in Response to a Low-Frequency Magnetic Field." Toxins 10, no. 11 (October 29, 2018): 440. http://dx.doi.org/10.3390/toxins10110440.
Анотація:
Background: Low-frequency magnetic fields (LF-MFs) dampen the citrinin output by Monascus purpureus in fermentations. The influence of LF-MFs on biosynthesis by M. purpureus was evaluated at the protein level. Methods: Cultures were treated with a 1.6-mT MF from day 0 to day 2 of incubation, and secondary metabolite production was evaluated on the day 12 of incubation. All proteins were extracted from M. purpureus mycelia and subjected to isobaric tags for relative and absolute quantification (iTRAQ) labeling and subsequent liquid chromatography/mass spectrometry (LC-MS/MS) analysis on day 6 of fermentation. Results: There was no difference in biomass between the treated samples and the control. Citrinin production was 46.7% lower, and the yields of monacolin K and yellow, orange, and red pigment were 29.3%, 31.3%, 41.7%, and 40.3% higher, respectively, in the exposed samples compared to the control. Protein expression in M. purpureus under LF-MF treatment was quantified using iTRAQ technology. Of 2031 detected proteins, 205 were differentially expressed. The differentially-expressed proteins were subjected to Gene Ontology (GO) functional annotation and statistical analysis, which revealed that they mainly refer to biological metabolism, translation, antioxidant, transport and defense pathways. Among all the tagged proteins, emphasis was placed on the analysis of those involved in the synthesis of citrinin, pigment and monacolin K was emphasized. Conclusions: LF-MFs affected Monascus secondary metabolism at the protein level, and aggregate data for all the protein profiles in LF-MF-treated Monascus was obtained.
8
Vardy, Eyal, Sonia Steiner-Mordoch, and Shimon Schuldiner. "Characterization of Bacterial Drug Antiporters Homologous to Mammalian Neurotransmitter Transporters." Journal of Bacteriology 187, no. 21 (November 1, 2005): 7518–25. http://dx.doi.org/10.1128/jb.187.21.7518-7525.2005.
Анотація:
ABSTRACT Multidrug transporters are ubiquitous proteins, and, based on amino acid sequence similarities, they have been classified into several families. Here we characterize a cluster of archaeal and bacterial proteins from the major facilitator superfamily (MFS). One member of this family, the vesicular monoamine transporter (VMAT) was previously shown to remove both neurotransmitters and toxic compounds from the cytoplasm, thereby conferring resistance to their effects. A BLAST search of the available microbial genomes against the VMAT sequence yielded sequences of novel putative multidrug transporters. The new sequences along with VMAT form a distinct cluster within the dendrogram of the MFS, drug-proton antiporters. A comparison with other proteins in the family suggests the existence of a potential ion pair in the membrane domain. Three of these genes, from Mycobacterium smegmatis, Corynebacterium glutamicum, and Halobacterium salinarum, were cloned and functionally expressed in Escherichia coli. The proteins conferred resistance to fluoroquinolones and chloramphenicol (at concentrations two to four times greater than that of the control). Measurement of antibiotic accumulation in cells revealed proton motive force-dependent transport of those compounds.
9
Ginn, Samantha L., Melissa H. Brown, and Ronald A. Skurray. "The TetA(K) Tetracycline/H+ Antiporter from Staphylococcus aureus: Mutagenesis and Functional Analysis of Motif C." Journal of Bacteriology 182, no. 6 (March 15, 2000): 1492–98. http://dx.doi.org/10.1128/jb.182.6.1492-1498.2000.
Анотація:
ABSTRACT Conserved motif C, identified within members of the major facilitator superfamily (MFS) of transport proteins that mediate drug export, was examined in the tetracycline resistance efflux protein TetA(K) from Staphylococcus aureus; motif C is contained within transmembrane segment 5. Using site-directed mutagenesis, the importance of the conserved glycine (G151, G155, G159, and G160) and proline (P156) residues within this motif was investigated. Over 40 individual amino acid replacements were introduced; however, only alanine and serine substitutions for glycine at G151, G155, and G160 were found to retain significant levels of tetracycline resistance and transport activity in cells expressing mutant proteins. Notably, P156 and G159 appear to be crucial, as amino acid replacements at these positions either significantly reduced or abolished tetracycline/H+ activity. The highly conserved nature of motif C and its distribution throughout drug exporters imply that the residues of motif C play a similar role in all MFS proteins that function as antiporters.
10
Tortosa, Valentina, Maria Carmela Bonaccorsi di Patti, Federico Iacovelli, Andrea Pasquadibisceglie, Mattia Falconi, Giovanni Musci, and Fabio Polticelli. "Dynamical Behavior of the Human Ferroportin Homologue from Bdellovibrio bacteriovorus: Insight into the Ligand Recognition Mechanism." International Journal of Molecular Sciences 21, no. 18 (September 16, 2020): 6785. http://dx.doi.org/10.3390/ijms21186785.
Анотація:
Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer’s disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein’s dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.
Дисертації з теми "MFS proteins":
1
Debbiche, Rim. "Influence des lipides sur la dynamique du transport du fer médié par la ferroportine-1 et sa modulation par des composés amphiphiles." Electronic Thesis or Diss., Brest, 2024. http://www.theses.fr/2024BRES0006.
Анотація:
La ferroportine-1 (FPN1), seul exportateur de fer connu chez les mammifères, est exprimée à la surface de différentes cellules spécialisées du métabolisme de fer. Cette protéine appartient à la famille des protéines MFS « Major Facilitator Superfamily » et libère le fer intracellulaire au travers de changements conformationnels oscillant entre une structure ouverte vers le cytoplasme (« Inward-Facing ») et une structure ouverte vers la circulation sanguine (« Outward-Facing » ; OF). Il a été rapporté que FPN1 est préférentiellement localisée dans les radeaux lipidiques, des microdomaines de la membrane plasmique particulièrement enrichies en cholestérol (CHOL). Au début de la thèse nous avons formulé l’hypothèse que des interactions directes entre FPN1 et les lipides environnants, notamment le CHOL, sont nécessaires pour stabiliser FPN1 dans la conformation OF et/ou favoriser certains changements conformationnels. J’ai confirmé la colocalisation préférentielle de FPN1 dans les radeaux lipidiques des cellules embryonnaires de rein humain. La dépendance de la fonction d’export de fer de FPN1 au CHOL a été examinée par déplétion/réplétion (CHOL/épicholestérol). Des expériences de criblage mutationnel appuyées par des analyses structurelles de la structure expérimentale 3D de FPN1 dans un état OF ont permis d’identifier trois sites de liaison possibles au CHOL (de types CARC/CRAC). Sur la base de simulations de dynamique moléculaire dans un environnement lipidique simplifié de type POPC, nous identifions certaines interactions entre des résidus chargés de la structure 3D FPN1 humaine et les têtes polaires de phospholipides environnants, qui pourraient faciliter les changements conformationnels du transporteur. Je montre également, pour la première fois, que la fonction de FPN1 est modulée par des composés amphiphiles de synthèse, l’ohmline et ses dérivés. Au travers du développement d’une nouvelle approche in vitro (PLA : « Proximity Ligation Assay »), j’indique que l’ohmline délocalise FPN1 des radeaux lipidiques, diminuant ainsi son interaction avec son partenaire fonctionnel, la céruloplasmine (CP), une ferroxydase qui catalyse l'oxydation du fer ferreux, et en conséquence la fonction d’export du ferFerroportin-1(FPN1), the only known mammalian iron exporter, is expressed on the surface of various specialized cells involved in iron metabolism. This protein belongs to the Major Facilitator Superfamily (MFS) and releases intracellular iron through conformational changes oscillating between an open structure towards the cytoplasm (Inward-Facing) and an open structure towards the bloodstream (Outward-Facing; OF). It has been reported that FPN1 is preferentially localized in lipid-rafts, microdomains of the plasma membrane particularly enriched in cholesterol (CHOL). Early in the thesis, we hypothesized that direct interactions between FPN1 and surrounding lipids, notably CHOL, are necessary to stabilize FPN1 in the OF conformation and/or promote certain conformational changes. I confirmed the preferential colocalization of FPN1 in the lipid rafts of human embryonic kidney cells. The dependence of FPN1's iron export function on CHOL was examined by depletion/repletion (CHOL/epicholesterol). Mutational screening experiments supported by structural analyses of the experimental 3D structure of FPN1 in an OF state have identified three possible CHOL-binding sites (of the CARC/CRAC type). Based on molecular dynamics simulations in a simplified POPC-type lipid environment, we identify certain interactions between charged residues of the human FPN1 3D structure and the polar heads of the surrounding phospholipids, which could facilitate conformational changes of the transporter. Besides, I show, for the first time, that FPN1 function is modulated by synthetic amphiphilic compounds, ohmline and its derivatives. Through the development of a novel in vitro approach (PLA: Proximity Ligation Assay), I show that ohmline delocalizes FPN1 from lipid-rafts, thereby decreasing its interaction with its functional partner, ceruloplasmin (CP), a ferroxidase that catalyzes the oxidation of ferrous iron, and consequently its iron export function
2
Yousefian, Narek. "The three-component multidrug MFS-type efflux pump EmrAB-TolC from Escherichia coli : from cloning to structural analysis." Thesis, Bordeaux, 2020. http://www.theses.fr/2020BORD0065.
Анотація:
A l’heure actuelle, suite à une mauvaise utilisation des antibiotiques, nous faisons face à un problème majeur de santé publique. En effet la résistance aux antibiotiques de certaines souches bactériennes rend le traitement des infections très complexe. Dans ce contexte, le présent projet de thèse concerne l'étude d'un complexe d'efflux bactérien capable de transporter des antibiotiques du cytoplasme vers l'extérieur de la cellule. Ce complexe est composé d'un transporteur de la membrane interne appartenant à la Major Facilitator Superfamily (MFS) (EmrB, E. coli multidrug resistance), d'un canal de la membrane externe TolC (Tolerance to Colicin E1) et d'un adaptateur périplasmique (EmrA, E. coli multidrug resistance). Contrairement aux systèmes d'efflux de type RND (tels que AcrAB-TolC), peu de choses sont connues sur le système EmrAB-TolC de type MFS. Il est donc important d'étudier l'ensemble du complexe sur le plan structurale et fonctionnel afin d'identifier les différences entre ces deux types de systèmes d’efflux. L'objectif de mon projet de thèse était d'étudier au moins un complexe EmrAB-TolC d'un point de vue structurale. Ainsi durant mes études, le but était d'isoler le complexe directement des bactéries surexprimant les trois partenaires protéiques. Dans un premier temps, 15 systèmes homologues EmrAB-TolC ont été identifiés et leurs gènes correspondants amplifiés à partir de l'ADN génomique de différentes bactéries à Gram négatif. Parmi les gènes des 15 systèmes, les gènes codant pour les systèmes d’E. coli et de V. cholerae ont été étudiés plus en détail. Les vecteurs d'expression codaient pour des marqueurs fluorescents pour la mesure des niveaux d'expression de différentes protéines et pour l'étude de la formation des complexes. Dans un premier temps, les différents niveaux d'expression des protéines (EmrB-mRFP1 et EmrA-sfGFP) ont été étudiés pour plusieurs souches d'expression d'E. coli en mesurant les niveaux de fluorescence rouge et verte et par Western blot (anti-His, Myc et Strep pour EmrB, EmrA et TolC). La souche d'E. coli C41(DE3) était la mieux adaptée pour la co-expression d’EmrAB-TolC. Dans un deuxième temps, la méthodologie FSEC (Fluorescence detection Size Exclusion Chromatography) a été utilisée pour identifier un complexe adapté à l'étude structurale. Ainsi, cette méthode a permis d'observer que le complexe EmrAB-TolC d'E. coli était produit en plus grande quantité que celui de V. cholerae. Le protocole final de co-purification consiste à effectuer une lyse douce des bactéries à l'aide du lysozyme, puis après solubilisation avec le DDM, la purification est débutée par une étape de chromatographie d'affinité Ni2+-NTA suivie d'une étape de chromatographie d'exclusion stérique. Enfin, les fractions contenant les trois partenaires protéiques sont utilisées pour l'échange de détergent par l'amphipol A8-35 avant l'étude structurale par microscopie électronique. Les images de microscopie électronique en coloration négative montrent des objets allongés d'une longueur de 33 nm en vue de côté. Une image moyenne d'EmrAB-TolC montre des similitudes avec celle du complexe AcrAB-TolC observé dans des conditions similaires. Les similitudes concernent les densités caractéristiques de TolC. Des différences ont été trouvées pour la partie inférieure d'EmrAB qui est plus fine que la partie inférieure d'AcrAB. Les densités visibles au-dessus de l'anneau d'amphipol correspondent à EmrA, qui présente une structure en forme de canal comme observé avec AcrA. Le canal semble cependant s'étendre plus loin vers la ceinture d'amphipol. Comme EmrB n'a pas de domaine périplasmique étendu présent dans le cas des protéines RND, ces densités sont donc uniquement attribuées à EmrA. EmrA, de l'autre côté, contacte TolC de manière similaire à l'interaction d'AcrA/MexA avec leurs canaux de la membrane externe respectifs (TolC/OprM) de façon «tip-to-tip»Currently, due to the misuse of antibiotics, we are facing a major public health problem. The resistance to antibiotics of certain bacterial strains makes the treatment of infections very complex. In this context, the present thesis project concerns the study of a bacterial efflux complex capable of transporting antibiotics from the cytoplasm to the outside of the cell. This complex is composed of an inner-membrane Major Facilitator Superfamily (MFS) transporter (EmrB, E. coli multidrug resistance), a channel of the outer membrane TolC (Tolerance to Colicin E1) and a periplasmic adapter (EmrA, E. coli multidrug resistance). Unlike RND-type efflux systems (such as AcrAB-TolC), little is known about the MFS-type EmrAB-TolC system. It is therefore important to study the entire complex on a structural and functional level, to analyse the marked differences between these two types of transport systems. The goal of my thesis project was to study at least one EmrAB-TolC complex from a structural point of view. For my studies the aim was to isolate the complex directly from bacteria overexpressing the three protein partners. In a first step, 15 homologous EmrAB-TolC systems were identified and their corresponding genes amplified from genomic DNA of different Gram-negative bacteria. Among the genes of the 15 systems, the genes coding for the E. coli and V. cholerae systems were further studied. The expression vectors encoded fluorescent markers for the monitoring of the expression levels of different proteins and for studying the formation of complexes. In a first step, the different protein expression levels (EmrB-mRFP1 and EmrA-sfGFP) were studied for several expression strains of E. coli by measuring the red and green fluorescence levels and by Western blot (anti-His, Myc, and Strep for EmrB, EmrA, and TolC). The E. coli strain C41(DE3) was best suited for co-expression of EmrAB-TolC. In a second step, the FSEC (Fluorescence detection Size Exclusion Chromatography) methodology was used to identify a complex suitable for structural study. Thus this method enabled the observation that the EmrAB-TolC complex of E. coli was produced in higher amount than that of V. cholerae. The final co-purification protocol consists in perfoming a gentle lysis of the bacteria using lysozyme, then after solubilization with DDM, the purification is started by a Ni2+-NTA affinity chromatography step followed by a size exclusion chromatography step. Finally, the fractions containing the three protein partners are used for the detergent-exchange by amphipol A8-35 before the structural study by electron microscopy. Negative stain EM-micrographs displayed elongated objects with a length of 33 nm in side view. An average image of EmrAB-TolC shows similarities to that of the AcrAB-TolC complex observed under similar conditions. Similarities included the characteristic densities of TolC. Whereas differences were found in the lower part of EmrAB which is thinner than the lower part of AcrAB. The densities visible above the amphipol-ring correspond to EmrA, which displays a channel-like structure as in AcrA. The channel however seems to extend further towards the amphipol belt. Since EmrB does not have an extended periplasmic domain as the RND proteins have, these densities are therefore solely assigned to EmrA. EmrA, on the other side, contacts TolC akin to the interaction of AcrA/MexA to their cognate outer membrane channels (TolC/OprM) in a ‘tip-to-tip’ fashion
Aufgrund des Missbrauchs von Antibiotika stehen wir derzeit vor einem großen Problem deröffentlichen Gesundheit. Die Antibiotikaresistenz bestimmter Bakterienstämme macht die Behandlungvon Infektionen sehr komplex.In diesem Zusammenhang befasst sich diese Arbeit mit der Untersuchung eines bakteriellenEffluxkomplexes, der Antibiotika vom Zytoplasma zur Außenseite der Zelle transportieren kann. DieserKomplex besteht aus einem Major Facilitator Superfamily (MFS) Transporter der inneren Membran(EmrB, E. coli multidrug resistance), einem Kanal der äußeren Membran TolC (Tolerance to Colicin E1)und einem periplasmatischen Adapter (EmrA, E. coli multidrug resistance).Im Gegensatz zu Effluxsystemen vom RND-Typ (wie AcrAB-TolC) ist über das EmrAB-TolCSystemvom MFS-Typ wenig bekannt. Es ist daher wichtig, den gesamten Komplex auf struktureller undfunktioneller Sicht zu untersuchen, um die deutlichen Unterschiede zwischen diesen beiden Arten vonEffluxsystemen zu analysieren.Ziel meiner Doktorarbeit war es, mindestens einen EmrAB-TolC-Komplex aus struktureller Sichtzu untersuchen. Ziel meiner Studien war es, den Komplex direkt aus Bakterien, die die dreiProteinpartner überexprimieren, zu isolieren. In einem ersten Schritt wurden 15 homologe EmrAB-TolCSystemeidentifiziert und ihre entsprechenden Gene aus der genomischen DNA verschiedenergramnegativer Bakterien amplifiziert. Unter den Genen der 15 Systeme wurden die Gene, die für die E.coli und V. cholerae Systeme kodieren, weiter untersucht. Die Expressionsvektoren codiertenfluoreszierende Marker zur Untersuchung der Expression verschiedener Proteine und zur Untersuchungder Komplexbildung. In einem ersten Schritt wurden die verschiedenen Niveaus der Proteinexpression(EmrB-mRFP1 und EmrA-sfGFP) für mehrere E. coli Expressionsstämme untersucht durch Messen derroten und grünen Fluoreszenzniveaus und durch Western Blot (Anti-His, Myc und Strep für EmrB, EmrAund TolC). Der Stamm von E. coli C41(DE3) war am besten für die Koexpression von EmrAB-TolC14 geeignet. In einem zweiten Schritt wurde die FSEC-Methode (Fluorescence Detection Size ExclusionChromatography) verwendet, um einen für Strukturuntersuchungen geeigneten Komplex zuidentifizieren. Somit konnte mit dieser Methode festgestellt werden, dass der EmrAB-TolC-Komplex vonE. coli in größerer Menge als der von V. cholerae produziert wurde.Das endgültige Ko-Reinigungsprotokoll besteht darin, eine sanfte Lyse der Bakterien unterVerwendung von Lysozym durchzuführen. Nach der Solubilisierung mit DDM wird die Reinigung durcheinen Ni2+-NTA Affinitätschromatographieschritt gefolgt von einemGrößenausschlusschromatographieschritt gestartet. Schließlich werden die Fraktionen, die die dreiProteinpartner enthalten, für den Detergensaustausch durch Amphipol A8-35 vor derStrukturuntersuchung durch Elektronenmikroskopie verwendet.EM-Aufnahmen mit negativer Kontrastierung zeigten längliche Objekte mit einer Länge von 33nm in Seitenansicht. Ein durch Mittlung der Partikel erhaltenes Bild von EmrAB-TolC zeigt Ähnlichkeitenmit dem des AcrAB-TolC-Komplexes, der unter ähnlichen Bedingungen beobachtet wurde.Ähnlichkeiten schlossen die charakteristischen Dichten von TolC ein. Während im unteren Teil vonEmrAB Unterschiede festgestellt wurden, der dünner ist als der untere Teil von AcrAB. Die über demAmphipolring sichtbaren Dichten entsprechen EmrA, das wie bei AcrA eine kanalartige Strukturaufweist. Der Kanal scheint sich jedoch weiter in Richtung des Amphipolgürtels zu erstrecken. Da EmrBkeine erweiterte periplasmatische Domäne aufweist wie die RND-Proteine, werden diese Dichten daherausschließlich EmrA zugeordnet. Auf der anderen Seite kontaktiert EmrA TolC, ähnlich der Interaktionvon AcrA/MexA mit ihren jeweiligen Außenmembrankanälen (TolC/OprM), von “tip-to-tip”
3
Symington, Vicki F. "Structure and function of nitrate and nitrite transporters, NrtA and NitA, from Aspergillus nidulans." Thesis, University of St Andrews, 2009. http://hdl.handle.net/10023/748.
Анотація:
Membrane proteins play an integral role in the control of ion transport across the cell membrane in biological systems. However, due to experimental constraints, structural and functional data available for these proteins is limited, especially considering their importance. In this study, two membrane proteins which transport nitrate and nitrate into the model filamentous ascomycete Aspergillus nidulans were investigated. Work on the twelve trans-membrane domain nitrate transport protein NrtA is well established. As a member of the major facilitator super family (MFS) the role of signature sequences characteristic of this family have previously been studied. Here, a series of point mutations were made to facilitate an understanding of key residues in the nitrate binding domain, the first nitrate signature motif and residues of the unique fungal central-loop domain. Using an expanded alignment package, the proposed secondary structure of NrtA was enhanced and used as a starting point for mutagenesis. Alanine scanning mutagenesis showed that glycine residues in the conserved nitrate nitrite porter (NNP) motif were critical for NrtA function. Two asparagines in the NNP were investigated; N160 and N168. N168 was found to be critical for NrtA function as all mutants were devoid of growth on nitrate solid agar medium though they expressed in the membrane to varying degrees. The nitrate binding site has been studied previously, revealing the interaction of conserved arginine residues with the anion as it traverses the bilayer. Though it was thought that mutations of residue T83 to a small, charge neutral, amino acid would substitute for no alteration to enzyme kinetics in mutant T83S was found when using ¹³NO₃⁻. Another major part of this thesis examined NitA which is part of a distinct nitrite transport family to NrtA (the Formate Nitrite Transporters, FNT). A mutagenesis approach targeted NitA residues conserved amongst homologous proteins. Residues in position D88 in an alignment of homologues were conserved in terms of charge. Mutagenesis of D88 revealed that maintaining charge at this position was essential for NitA function, likely due to a role in salt-bridge formation during conformational changes. Mutations to asparagine, glutamine, serine and valine showed reduced growth on agar though the protein was expressed to approximately wild-type levels. Nitrite uptake assays using a ¹³NO₂⁻ tracer were performed on D88N, D88E and D88Q and all showed wild-type Km and Vmax. Finally, the role of conserved asparagine residues found throughout NitA was investigated by mutagenesis. Expression studies revealed that mutants created in N122 and N246, changed to aspartic acid, lysine, glutamine and serine were generally not present in the membrane and thus did not grow on nitrite agar. However, mutations in N173 (in Tm 4) and N214 (in Tm 5), which are conserved in > 95 % of NitA homologues, showed varying degrees of growth and expression. Both of these residues are located in FNT signature motifs, so it is likely that they are involved with conformational changes or protein dynamics.
4
Bayro, Marvin J. "Protein MAS NMR methodology and structural analysis of protein assemblies." Thesis, Massachusetts Institute of Technology, 2010. http://hdl.handle.net/1721.1/57800.
Анотація:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2010.Vita. Cataloged from PDF version of thesis.
Includes bibliographical references.
Methodological developments and applications of solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy, with particular emphasis on the analysis of protein structure, are described in this thesis. MAS NMR studies of biomolecules ranging from model peptides and proteins in crystalline form to amyloid fibrils and whole bacterial organelles are reported. The methods presented include novel pulse sequences and optimized pulse sequence elements, experimental approaches designed for multiple-spin systems, a protocol for efficient sequential resonance assignment of proteins in the solid state, and techniques to determine the inter-molecular organization of amyloid fibrils formed by moderately sized proteins. Notably, an efficient dipolar recoupling technique, bandselective radio frequency-driven recoupling (BASE RFDR), is introduced and combined with alternating 13C-12C labeling to yield highly sensitive 13C-13C correlation spectra between distant nuclei in proteins. Various applications of the BASE RFDR scheme are presented, including protein resonance assignment, determination of tertiary structure of amyloid fibrils, and variable-temperature studies of protein dynamics. The main biological systems analyzed are amyloid fibrils formed by the SH3 domain of P13 kinase (P13-SH3) and intact gas vesicles from anabaena flos-aquae, for which atomic-level structural information was previously unavailable. P13-SH3 (86 residues) is a system thoroughly studied as a model of protein misfolding and amyloid formation by a natively globular protein. Gas vesicles are bacterial buoyancy organelles, with walls composed almost entirely by a single protein (GvpA, 70 residues), whose formation and structure constitute a highly intriguing biophysical problem. Nearly complete 13C and 'IN resonance assignments and the molecular conformations of the polypeptide backbones of both P13-SH3 and GvpA have been obtained via MAS NMR spectroscopy, enabling the proposal of models for the structure of these two protein assembly systems. In addition, the tertiary structure of P13-SH3 amyloid fibrils has been elucidated by the application of novel methodology introduced in this thesis. Finally, investigations regarding the effects of temperature and protein dynamics on MAS NMR experiments and biomolecular dynamic nuclear polarization studies are presented.
by Marvin J. Bayro.
Ph.D.
5
Hunnewell, Mary E. "Probing for Conformational Changes in the Repair Enzyme Mfd Using Mutant Protein Constructs." Connect to this title, 2008. https://scholarworks.umass.edu/theses/154.
Анотація:
DNA repair is essential for survival, as damage to the genome can interrupt the precarious balance of cell functions, causing further mutations and possibly leading to cancer. The bacterial transcription repair coupling factor, Mfd, is capable of recognizing a stalled RNA polymerase at a site of DNA damage. The Mfd works both to remove the RNA polymerase through its motor function (utilizing the energy of ATP to translocate along DNA), and to recruit the DNA repair complex UvrA/B/C. To study conformational changes in the protein, we are creating multiple mutants of the full length Mfd protein. My approach is to use a cleavable mutant of full-length Mfd as a template for further mutations. This will allow us to probe for conformational changes by changing interactions at the interface of the two halves of Mfd, and then using the ability to cut with TEV protease as a sensor to identify and characterize the open state of the protein. By introducing this TEV protease cut site at residue 450 in the protein linker region between the N (amino-) and C (carboxy-) terminal domains, we can then assess the conformational changes Mfd must undergo to obtain activity. We can study the effect of further mutations on the full length and cut versions of the protein. Another approach attempted in this study involves using cysteine modification of the full length Mfd protein as a sensor for these conformational changes. Mfd acts as a model system for studying the DNA repair mechanisms found in humans, and the elucidation of functional and conformational changes in Mfd contributes to studying disease phenotypes resulting from aberrant transcription coupled repair.
6
Agarwal, Vipin. "Development and application of MAS solid state NMR methodologies to biomolecules." Berlin mbv, 2009. http://d-nb.info/998718602/04.
7
Vahl, Martin [Verfasser]. "Identifizierung von Liganden des G-Protein gekoppelten Rezeptors Mas und Mas-Sequenz-ähnlicher Rezeptoren / Martin Vahl." Berlin : Medizinische Fakultät Charité - Universitätsmedizin Berlin, 2010. http://d-nb.info/102410494X/34.
8
Raynor, James E. Jr. "Characterization of the mas protein as an angiotensin ii receptor." DigitalCommons@Robert W. Woodruff Library, Atlanta University Center, 1994. http://digitalcommons.auctr.edu/dissertations/2831.
Анотація:
The mas proto-oncogene encodes a seven transmembrane protein (MAS) which is suggested to function as a receptor for angiotensin. It (MAS) was initially identified in NIH-3T3 cells that were transformed with DNA isolated from a human epidermoid carcinoma. These cells formed foci in culture and tumors when injected into nude mice. On the other hand, untransformed cells did not. Further analysis of these cells showed that transformed cells bind increased levels of angiotensin when compared to untransformed cells. These studies also demonstrated that the Mas protein was structurally similar to the angiotensin receptor transmembrane proteins, AT1 and AT2 . This investigation was undertaken to examine the ability of the Mas protein to function as an receptor for angiotensin and promote cell proliferation. To this end, quantitation of mas genes by Polymerase Chain Reaction (PCR) and serial dilutions, and Southern blot analysis support an increased in mas genes in transformed cells. Northern blot analysis demonstrated an increased expression of the mas gene in transformed cells. No changes in the level of the AT2 angiotensin receptor gene expression was observed in the transformed and untransformed cell lines. Expression of the AT1 angiotensin receptor gene was not observed in these cell lines. Anti-peptide antibodies were generated against the 1st and 2nd extracellular regions of the Mas protein. Flow cytometric analysis using these antibodies indicated an increased presence of the Mas protein on the surf ace of transformed cells recognized by anti-peptide antibodies.
Western blot analysis showed two cross-reacting proteins of approximately ll0kd and 66kd in transformed cells; whereas, only a 66kd protein was found in untransformed cells.
Transformed cells exposed to mas antisense oligos greatly reduced the synthesis of Mas, decreased cell proliferation and the binding of angiotensin. Binding studies using [3H]-DUP-
753 (a non-peptidyl ligand which recognizes Ang subtype AT1 receptors) showed little binding to transformed cells.
Similar studies using PD-123319 (a non-peptidyl ligand that recognizes AT2 subtype receptors) indicated that approximately 60% of [125I]-Ang II was displaced using PD-123319. Further binding analysis of transformed cells suggests that [Sarl]-Ang II (an Ang II antagonist) could not completely displace [ 125I]-Ang II. Taken together, these data suggest that Mas protein is an Ang receptor which functions in the regulation of cell proliferation. Mas appears to be a member of a subtype different from AT1 or AT2.
9
Jordan, Katherine Jo. "Guanidine-stable chymoelastase : a comparative study of its hydrolytic specificity in the presence and absence of denaturant." Virtual Press, 1987. http://liblink.bsu.edu/uhtbin/catkey/481688.
Анотація:
Guanidine-stable chymoelastase (GUSCE), a component of the protease mixture known as Pronase, has been shown to be stable and active under conditions which denature and inactivate most proteins. In the absence of denaturant, this enzyme has shown proteolytic specificity for phenylalanyl, tyrosyl, and leucyl peptide bonds. In the presence of denaturant, however, the cleavage specificity has not been defined.In order to determine the effects of denaturant on the cleavage specificity of GUSCE, six small peptides of known amino acid sequence were hydrolyzed by GUSCE in the presence and absence of 6.OM guanidinium chloride. The site of GUSCE cleavage was acertained by dansylation of the new N-terminal amino acids, which were produced by proteolysis, followed by thin layer chromatographic identification of the resulting dansylated amino acids.The results indicate that GUSCE catalyzed the hydrolysis of phenylalanyl and tyrosyl peptide bonds in the absence as well as the presence of 6.OM guanidinium chloride. Of the tyrosyl and phenylalanyl peptide bonds hydrolyzed, all were between non-terminal amino acids, which illistrates the endo-peptidase characteristics of GUSCE. With one exception, only those peptide bonds cleaved by GUSCE in the absence of denaturant were cleaved in the presence of denaturant. In the case of oxytocin, the presence of denaturant was actually required for the cleavage of the Tyr(2)-Ile(3) peptide bond. The demonstrated predictablilty of GUSCE cleavage in the presence of denaturant should greatly enhance its utility in the sitespecific proteolysis of insoluble or otherwise proteolysisresistant protein substrates.Ball State UniversityMuncie, IN 47306
10
Shevelkov, Veniamin. "Development of MAS solid state NMR methods for structural and dynamical characterization of biomolecules." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16260.
Анотація:
Das Verständnis der Mechanismen, nach denen biologische Systeme ablaufen, ist ein wichtiger Fokus der aktuellen Strukturbiologie. Kernmagnetische Resonanzspektroskopie (NMR) ist eine geeignete Technik, um solche Ziele anzustreben sowie Struktur und Dynamik von Biomolekülen zu erforschen, um komplementäre Informationen zum Verständnis von Proteinfunktionalität zu erhalten. Rasante Fortschritte sind vor nicht langer Zeit auf dem Gebiet biologischer Festkörper-NMR (ssNMR) erzielt worden, was zu vollständiger Strukturaufklärung zahlreicher Peptide und kleiner Proteine, der Beschreibung von Protein-Komplexbildung sowie der der dynamischen Eigenschaften kleiner Proteine geführt hat. Festkörper-NMR ist die Methode der Wahl bei struktureller und dynamischer Charakterisierung von Membranproteinen und aggregierten amyloidogenen Systemen, die schwer löslich und kaum mit Lösungs-NMR oder Röntgenkristallographie zugänglich sind. Moderne Festkörper-NMR ist noch immer limitiert, was Auflösung und Empfindlichkeit betrifft, und macht weitere Entwicklungen auf den Gebieten der Probenpräparation und des Pulssequenz-Designs erforderlich. In meiner Arbeit untersuche ich die potenzielle Verwendung von Deuterierung in der Protein Festkörper-NMR zur Erhöhung von Empfindlichkeit und Auflösung in 15N-1H Korrelationsexperimenten. Der erzielte Fortschritt auf diesen Gebieten erlaubt die Verfolgung von Proteinrückgratbewegungen mit hoher Genauigkeit, die vorher nicht verfügbar war. Wir zeigen zum ersten Mal, dass TROSY Experimente für Festkörper-NMR gewinnbringend sind. Außerdem wurde eine Pulssequenz für 13C-13C J Kopplung zur Erhöhung der Auflösung in der Kohlenstoff-Dimension entwickelt.Understanding the mechanisms how biological systems work is an important objective of current structural biology. Nuclear magnetic resonance (NMR) spectroscopy is a well suited technique to approach these goals and to study structure and dynamics of biomolecules in order to obtain complimentary information for understanding functionality of proteins. Recently, rapid progress has been made in the field of biological solid state NMR (ssNMR), which resulted in complete structure elucidation of several peptides and small proteins, the characterization of protein complex formation and the characterization of dynamic properties of small proteins. Solid state NMR is the method of choice for structural and dynamic characterization of membrane proteins and aggregated amyloidogenic systems, which are poorly soluble and can not be easily studied by solution state NMR and X-ray spectroscopy. Modern solid state NMR is still limited in resolution and sensitivity, and requires developments in sample preparation and pulse sequence design. In my thesis, I study the potential use of deuteration in protein solid state NMR for sensitivity, as well as for resolution enhancement in 15N-1H correlation experiments. Achieved progress in these fields allows to monitor backbone motion with high accuracy, which has not been available before. We show for the first time that TROSY type experiments can be beneficial for solid state NMR. In addition, a pulse sequence for 13C-13C J decoupling was developed to increase resolution in the carbon dimension.
Книги з теми "MFS proteins":
1
L, Burlingame A., ed. Biological mass spectrometry. Amsterdam: Elsevier Academic Press, 2005.
2
(Editor), Regina Murphy, and Amos Tsai (Editor), eds. Misbehaving Proteins: Protein (Mis)Folding, Aggregation, and Stability. Springer, 2006.
3
Hakim, Alan J., and Rodney Grahame. Hypermobility syndromes. Oxford University Press, 2013. http://dx.doi.org/10.1093/med/9780199642489.003.0159.
Анотація:
Hypermobility-related syndromes constitute a family of heritable disorders of connective tissue (HDCT) that derive from abnormalities affecting genes that encode for the connective tissue matrix proteins such as collagen, fibrillin, and tenascin. They range from such commonplace though poorly recognized conditions such as the joint hypermobility syndrome (JHS) to the better-known, if more rare, eponymous syndromes such as Marfan's syndrome (MFS) and the different types of the Ehlers-Danlos syndrome (EDS). The more common presentations are with skin pathology (bruising, scaring), joint or spinal and/or muscle pain and instability with vulnerability to injury and chronic widespread pain, cardiac valve pathologies, and in MFS and vascular EDS, arterial dilatation with the risk of dissection and rupture. JHS (widely considered synonymous with the EDS hypermobility type) is further complicated by cardiovascular autonomic dysfunction such as orthostatic intolerance, palpitations, and syncope, and the recently described and commonly encountered pangastrointestinal dysmotility. The latter can manifest as gastro-oesophageal reflux, gastroparesis, slow-transit constipation, or rectal evacuatory dysfunction with rectal intussusception. In addition, HDCT are associated with bladder and uterine problems as a consequence of pelvic floor weakness. Such multisystemic conditions need to be managed by a multidisciplinary team able to draw on medical, surgical, physical, and psychological interventions by appropriately experienced specialists and therapists. This chapter introduces the reader to the epidemiology, genetics, classification, and clinical presentation of JHS, EDS, and MFS. It also describes the key investigations required to support a diagnosis and assess complications of an HDCT, as well as the multidisciplinary approach to management.
4
Perez, Carlos. Mis Recetas Paleovida: 100 Recetas Paleo para Recuperar una Vida Saludable / Paleo Recipes. Ediciones B, 2017.
5
Albo, Carmen. Adelgaza sin hambre y con humor con mis recetas proteicas / Lose weight without hunger and humor with my protein recipes: Guisándome la vida. Grijalbo Mondadori Sa, 2013.
6
Pérez Jiménez, María José. Caracterización del perfil de disfunción mitocondrial en fibroblastos de pacientes con enfermedad de Alzheimer. Universidad Autónoma de Chile, 2018. http://dx.doi.org/10.32457/20.500.12728/87492018dcbm9.
Анотація:
La enfermedad de Alzheimer (EA) es la patología neurodegenerativa más común en todo el mundo y se le considera la principal causa de demencia en la población adulta. El principal rasgo neuropatológico de esta enfermedad es la acumulación de proteínas mal plegadas en el cerebro de los pacientes. Estos agregados proteicos conducen a la oxidación progresiva y daño inflamatorio, lo que contribuye al proceso neurodegenerativo y al daño cerebral irreversible. Por otro lado, la evidencia sugiere que la disfunción mitocondrial es un elemento importante en la patogénesis de la EA y que su aparición precede al establecimiento de los agregados proteicos en el cerebro.Interesantemente, diversos estudios en el tejido periférico de modelos animales y pacientes con EA, sugieren que la función mitocondrial también podría estar afectada, lo que indicaría la existencia de un novedoso blanco para la búsqueda de un biomarcador temprano en la EA. En este contexto, evidencia reciente indica que los fibroblastos extraídos de biopsia de piel pueden ser los candidatos adecuados para el escrutinio de nuevos biomarcadores en la EA. Esto ya que existe evidencia de que fibroblastos de pacientes con EA familiar presentan desregulaciones metabólicas que reflejarán alteraciones neuronales que ocurren en el cerebro. Más allá de lo prometedor de estas evidencias actualmente no existen estudios profundos que demuestren y/o caractericen el daño mitocondrial en fibroblastos de pacientes con EA y su potencial uso en la búsqueda de un biomarcador.Dado los antecedentes anteriores, en esta tesis se propone que los fibroblastos de pacientes con la EA presentan un perfil de disfunción mitocondrial característico que los diferencia de los pacientes normales. Para esto se analizaron cultivos primarios de fibroblastos de pacientes controles y EA con diferentes niveles de deterioro cognitivo. Se realizaron análisis de microscopía de fluorescencia en vivo, PCR en tiempo real, Western blot y determinaciones de ATP. Nuestros resultados sugieren que los fibroblastos de pacientes con EA presentan defectos específicos en la morfología y bioenergética mitocondrial que se asemejan a las desregulaciones cerebrales observadas en la EA.Esta disfunción mitocondrial podría estar desencadenada por la formación y apertura del poro de transición de membrana mitocondrial, un complejo proteico que cumple funciones en la regulación de la producción de energía y procesos de muerte celular. El presente estudio presenta la primera caracterización del estado mitocondrial de los fibroblastos de pacientes con EA y sugiere que la evaluación de la función mitocondrial podría diferenciar el envejecimiento normal del envejecimiento patológico relacionado con esta enfermedad. Nuestro trabajo abre la posibilidad de un nuevo blanco para el desarrollo de biomarcadores de EA y presenta una nueva estrategia para estudios epidemiológicos en esta enfermedad.
7
Jara Orellana,, Claudia. Efectos de la proteína Tau sobre la disfunción mitocondrial y el deterioro cognitivo en el envejecimiento. Universidad Autónoma de Chile, 2018. http://dx.doi.org/10.32457/20.500.12728/87452018dcbm6.
Анотація:
El envejecimiento es un proceso complejo e irreversible que afecta el funcionamiento del cerebro y se considera el principal factor de riesgo para las patologías neurodegenerativas. Las mitocondrias son organelos fundamentales para la generación de energía, el equilibrio oxidativo y la regulación del calcio. Actualmente, se considera a la disfunción mitocondrial como un factor importante que contribuye al envejecimiento cerebral y a la patogénesis de diversas enfermedades neurodegenerativas. Este daño mitocondrial se ve reflejado en una disminución del metabolismo cerebral, aumentando el daño oxidativo y disminuyendo la formación de ATP. Estos daños alteran el normal funcionamiento neuronal y juegan un papel importante en la pérdida de la función cognitiva. Durante el envejecimiento, una serie de agregados proteicos se acumulan regularmente en el cerebro, como por ejemplo la proteína tau. Interesantemente, investigaciones de nuestro grupo y otros han determinado que bajo condiciones patológicas tau puede afectar la función sináptica debido a su capacidad para facilitar la falla mitocondrial. Dentro de este contexto, nuestro grupo recientemente ha encontrado que la ausencia de la proteína tau mejora la salud mitocondrial y las capacidades cognitivas en ratones jóvenes. Ya que las modificaciones patológicas de tau pueden afectar la función mitocondrial y la falla mitocondrial contribuye al envejecimiento cerebral, es interesante poder estudiar los efectos que la ausencia de tau podría tener sobre la función mitocondrial y las habilidades cognitivas durante el envejecimiento. En el desarrollo de esta tesis, observamos que la ausencia de tau previene la disfunción mitocondrial y el deterioro cognitivo asociado al envejecimiento. Se realizaron pruebas cognitivas, bioquímicas e histológicas utilizando ratones Wild Type (WT) y Knockout para la proteína tau (tau-/-) de 18 meses de edad. Nuestros resultados indican que la ausencia de tau previene la pérdida de la función cognitiva durante el envejecimiento, incluido la memoria de reconocimiento, la memoria espacial y las capacidades sociales. Además, los ratones tau-/- mostraron una mejor bioenergética mitocondrial, que fue evidenciada por una mayor producción de ATP y una menor sensibilidad a la sobrecarga de calcio en mitocondrias aisladas. También observamos elevados niveles de proteína ciclofilina-D(CypD) en el cerebro de ratones WT; de manera interesante, los ratones tau-/- envejecidos mostraron una disminución significativa de esta proteína en comparación con los ratones WT. CypD es una proteína fundamental en la formación del poro de transición de permeabilidad mitocondrial (mPTP). Para determinar si la disminución de CypD jugó un papel en la bioenergética mitocondrial mejorada de ratones tau-/- envejecidos, se realizó la sobreexpresión de CypD en el hipocampo de estos ratones utilizando la técnica de inyección estereotáxica de vectores lentivirales que codifican para CypD. Tres semanas después de la infección hipocampal, se realizaron pruebas conductuales y, posteriormente, se utilizó el hipocampo para evaluar la función mitocondrial. En estos estudios observamos que la expresión de CypD en ratones tau -/- redujo la producción de ATP y afecto la regulación del calcio produciendo un aumento de la sensibilidad al calcio mitocondrial, lo que sugiere apertura del mPTP. Más interesante es que los ratones tau-/- envejecidos infectados con lentivirus CypD mostraron una disminución significativa en sus capacidades cognitivas en comparación con los ratones tau-/- no infectados. En conclusión, nuestros resultados sugieren que la ausencia de tau previene la pérdida de habilidades cognitivas a través de la mejora de la función mitocondrial durante el envejecimiento. Estos efectos beneficiosos implican la regulación de la expresión de CypD y posiblemente la formación de mPTP. Esto también propone un nuevo y potencial objetivo terapéutico para prevenir alteraciones relacionadas con la edad.
8
Joseph Correa (Nutricionista Deportivo Certificado). Barras de Proteina Caseras para Acelerar el Desarrollo de Musculo para Hockey: Aumente naturalmente el crecimiento de musculo y disminuya la grasa para ganar mas y durar mas tiempo. CreateSpace Independent Publishing Platform, 2015.
9
Armendano, Andrea, Alda González, and Sergio Roberto Martorelli. Conceptos claves en Biología. Editorial de la Universidad Nacional de La Plata (EDULP), 2016. http://dx.doi.org/10.35537/10915/54497.
Анотація:
El conocimiento de la organización de la materia es indispensable para comprender la estructura y función de los seres vivos. Justamente las interrelaciones entre los átomos y las moléculas, son las que permiten el desarrollo de todas las funciones vitales de los organismos animales. Estos están constituidos por miles de moléculas orgánicas diferentes, que se agrupan en cuatro categorías principales: carbohidratos, lípidos, proteínas o ácidos nucleicos. La información genética que controla la vida de cada célula está contenida en sus cromosomas, más específicamente en su ADN, codificándose y transfiriéndose de generación en generación. A lo largo de miles de millones de años surgieron las especies a partir de otras preexistentes por un proceso llamado de "descendencia con modificación" o "evolución". Una de las características de la naturaleza es la gran diversidad de organismos que la componen. La taxonomía, es la ciencia que se encarga de la búsqueda del orden natural, mediante la que se obtienen las distintas clasificaciones animales.
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Placencia López, Bárbara Miladys, Fernanda Vanessa Alcívar Macías, José Aníbal Sánchez Saltos, Mayra Alejandra Cedeño Mera, Silvia Beatriz Alarcón Barreiro, María Gabriela Pertuz Alarcón, Jacqueline Beatriz Delgado Molina, José Roberto Rodríguez Mera, Agustina Elizabeth Cedeño Casanova, and Christian Paúl Vera Zambrano. Covid 19. Mawil Publicaciones de Ecuador, 2022, 2022. http://dx.doi.org/10.26820/978-9942-602-37-4.
Анотація:
El siglo XXI se ha caracterizado desde sus inicios por una problemática de salud que ha afectado al mundo, que va desde un incremento de la resistencia microbiana, aumento de las enfermedades oncológicas hasta la aparición de nuevas enfermedades infecciosas emergentes y reemergentes, como ha sido la aparición de la COVID-19 a finales del pasado año. Los coronavirus son una extensa familia de virus que pueden causar enfermedades tanto en animales como en humanos. En los humanos, se sabe que varios coronavirus causan infecciones respiratorias que pueden ir desde el resfriado común hasta enfermedades más graves como el síndrome respiratorio de Oriente Medio (MERS) y el síndrome respiratorio agudo severo (SRAS). La COVID-19 (coronavirus disease 2019) también conocida como enfermedad por nuevo coronavirus es causada por el coronavirus del síndrome respiratorio agudo severo (SARS-CoV-2), su forma es redonda u ovalada y a menudo polimórfica, tiene un diámetro de 60 a 140 nm, la proteína espiga que se encuentra en la superficie del virus y forma una estructura en forma de barra, es la estructura principal utilizada para la tipificación, la proteína de la nucleocápside encapsula el genoma viral y puede usarse como antígeno de diagnóstico. Produce síntomas similares a los de la gripe, entre los que se incluyen fiebre, tos, disnea, mialgia y fatiga. También se ha observado la pérdida súbita del olfato y el gusto (sin que la mucosidad fuese la causa). En casos graves se caracteriza por producir neumonía, síndrome de dificultad respiratoria aguda, sepsis y choque séptico que conduce a alrededor del 3 % de los infectados a la muerte, aunque la tasa de mortalidad se encuentra en 4,48 % y sigue ascendiendo.
Частини книг з теми "MFS proteins":
1
Saidijam, Massoud, Kim E. Bettaney, Dong Leng, Pikyee Ma, Zhiqiang Xu, Jeffrey N. Keen, Nicholas G. Rutherford, et al. "The MFS Efflux Proteins of Gram-Positive and Gram-Negative Bacteria." In Advances in Enzymology - and Related Areas of Molecular Biology, 147–66. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9780470920541.ch4.
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Yang, Ziping, Wenkui Li, Harold T. Smith, and Francis L. S. Tse. "LC-MS Bioanalysis of Proteins." In Handbook of LC-MS Bioanalysis, 601–5. Hoboken, NJ, USA: John Wiley & Sons Inc., 2013. http://dx.doi.org/10.1002/9781118671276.ch47.
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Hjernø, Karin, and Ole N. Jensen. "MALDI-MS in Protein Chemistry and Proteomics." In MALDI MS, 105–31. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2013. http://dx.doi.org/10.1002/9783527335961.ch3.
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Pang, Yongle, Chuan Shi, and Wenying Jian. "Targeted Protein Biomarker Quantitation by LC-MS." In Targeted Biomarker Quantitation by LC-MS, 227–44. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2017. http://dx.doi.org/10.1002/9781119413073.ch15.
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Popletaeva, Sofya, Denis Erokhin, and Vitaly Dzhavakhiya. "Comparison of the Protective Activity of Elicitor Proteins MF2 and MF3 Applied Individually or in Combination Against Tobacco Mosaic Virus on Tobacco Leaves." In Agriculture Digitalization and Organic Production, 225–34. Singapore: Springer Nature Singapore, 2023. http://dx.doi.org/10.1007/978-981-99-4165-0_21.
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Haselberg, Rob, and Govert W. Somsen. "CE-MS for the analysis of intact proteins." In Capillary Electrophoresis-Mass Spectrometry (CE-MS): Principles and Applications, 159–92. Weinheim, Germany: Wiley-VCH Verlag GmbH & Co. KGaA, 2016. http://dx.doi.org/10.1002/9783527693801.ch7.
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Carter, J. Mark. "Conjugation of Peptide to Carrier Proteins via m-Maleimidobenzoyl-N-Hydroxysuccinimide Ester (MBS)." In Springer Protocols Handbooks, 689–92. Totowa, NJ: Humana Press, 1996. http://dx.doi.org/10.1007/978-1-60327-259-9_118.
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Reif, Bernd. "Deuterated Peptides and Proteins: Structure and Dynamics Studies by MAS Solid-State NMR." In Methods in Molecular Biology, 279–301. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-480-3_16.
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Tiwari, Vineeta, Vinod Tiwari, Shaoqiu He, Tong Zhang, Srinivasa N. Raja, Xinzhong Dong, and Yun Guan. "Mas-Related G Protein-Coupled Receptors Offer Potential New Targets for Pain Therapy." In Advances in Experimental Medicine and Biology, 87–103. Dordrecht: Springer Netherlands, 2016. http://dx.doi.org/10.1007/978-94-017-7537-3_7.
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Van Remoortel, Samuel, and Jean-Pierre Timmermans. "Mas-Related G Protein-Coupled Receptors (Mrgprs) as Mediators of Gut Neuro-Immune Signaling." In Advances in Experimental Medicine and Biology, 259–69. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-031-05843-1_25.
Тези доповідей конференцій з теми "MFS proteins":
1
Júnior, Nino Sérgio Lemos De Oliveira, Keilane Silva Farias, and Carlos Priminho Pirovani. "AVALIAÇÃO DO EFEITO DE UMA CANDIDATA A EFETORA EM ALPISTE (PHALARIS CANARIENSIS) E EM TOMATE (SOLANUM LYCOPERSICUM)." In II Congresso Brasileiro de Biotecnologia On-line. Revista Multidisciplinar de Educação e Meio Ambiente, 2022. http://dx.doi.org/10.51189/conbiotec/30.
Анотація:
Introdução - O fungo Moniliophthora perniciosa (Mp), causador da vassoura-de-bruxa no cacaueiro é hemibiotrófico. Sua infecção inicia com a germinação dos esporos na superfície do tecido vegetal, seguido da penetração nos espaços intercelulares, estabelecendo a fase biotrófica da doença. Essa fase pode durar de 60-90 dias, evoluindo para uma fase saprofítica. Em ambas as fases, o fungo utiliza proteínas efetoras que manipulam a fisiologia da célula hospedeira, por meio da modulação ou supressão da resposta imune. O estudo do modo de ação dessas proteínas pode levar a compreensão dos mecanismos infecciosos utilizados pelo fungo. Objetivo - O objetivo do trabalho foi compreender o efeito de uma potencial proteína efetora de Mp em plantas mono e dicotiledôneas. Metodologia - A proteína candidata a efetora Mp4145-3305 foi expressa em bactéria e foi testada em bioensaios com plantas dicotiledôneas. Nesse contexto, foi analisado o efeito da proteína recombinante na espécie Phalaris canariensis (alpiste) que é uma monocotiledônea. Para isso, foram feitos teste de atividade de necrose e também foi avaliado o envolvimento da proteína na geração de espécies reativas de oxigénio em folhas do alpiste. Resultados - Foi analisado em revisão que os fungos de mono e dicotiledônea podem apresentar diferenças nos tamanhos dos genomas e nas especificidades dos genes e efetores. Porém, existem evidências de efetores homólogos em espécies de patógenos em monocotiledônea e dicotiledôneas distantemente relacionados. A proteína efetora MpNEP (Necrosis and ethylene induced protein) promove morte celular em dicotiledôneas, mas não afeta monocotiledôneas. Tal fato aventa hipóteses que indicam a falta de receptores nas superfícies da célula de monocotiledôneas que possam reconhecer tal proteína efetora. Os resultados mostraram alterações fisiológicas nas plantas de alpiste induzida pela proteína. Isto promoveu um melhor entendimento dos sistemas evolutivos envolvidos com a relação planta patógeno. Conclusões - Os resultados sugerem que a proteína possui potencial uso como indutora de crescimento e resistência em plantas cultivadas.
2
Mukai, Masahiro, and Ayae Honda. "Analysis of promoter binding proteins of Ebp1 that is inhibitor protein of influenza virus RNA polymerase." In 2009 International Symposium on Micro-NanoMechatronics and Human Science (MHS). IEEE, 2009. http://dx.doi.org/10.1109/mhs.2009.5351934.
3
Dodge, Taylor N., Jeana L. Drake, Paul Falkowski, and Liesl Hotaling. "Evolution of biomineralization proteins in Cnidaria." In OCEANS 2015 - MTS/IEEE Washington. IEEE, 2015. http://dx.doi.org/10.23919/oceans.2015.7404525.
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Athayde, Natália Merten, and Alzira Alves de Siqueira Carvalho. "The heart of myofibrillary myopathy." In XIII Congresso Paulista de Neurologia. Zeppelini Editorial e Comunicação, 2021. http://dx.doi.org/10.5327/1516-3180.457.
Анотація:
Context: Myofibrillar myopathies (MFM) represent a heterogeneous group of disorders of skeletal and cardiac muscle caused by mutations in genes that encode proteins of sarcomere. Diagnosis is a challenge due to clinical and genetic variability. Case report: Woman, 36 years old, presenting stumbles and falls for 3 years evolving with proximal limb weakness. At age 30, she fainted and a cardiac pacemaker was implanted. Non-consanguineous parents. Neurological exam: proximal and distal weakness in lower limbs and distal atrophy; osteotendinous reflexes normal. Bilateral scapula alata. Exams: CPK = 457 U / l; EMG: myopathic pattern. Muscle MRI: diffuse and heterogeneous fatty degeneration, marked in sartorius, gracilis and semitedinous. Panel NGS myopathies: pathogenic variant, c.1175T> C, missense in heterozygosis in desmin gene. CONCLUSION: The diagnosis of MFM is based on the morphological findings of muscle biopsy with the presence of protein aggregates as a determining factor. Currently, genetic testing by NGS has facilitated early diagnosis allowing for a more appropriate clinical approach. The desmin gene was the first one described to be associated with this group of myopathies. It encodes the desmin protein, a member of the intermediate filament family present in cardiac and skeletal muscle. Several phenotypes are related to desmin gene: isolated dilated cardiomyopathy; scapuloperoneal weakness and distoproximal weakness with cardiac alterations. Desminopathy is a rare cause of cardiomyopathy and / or myopathy. The diagnosis should be thought in patient with muscle weakness and cardiac changes.
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Sidhu, A. S., T. S. Dillon, B. S. Sidhu, and E. Chang. "Protein Ontology Project: 2006 updates." In DATA MINING AND MIS 2006. Southampton, UK: WIT Press, 2006. http://dx.doi.org/10.2495/data060301.
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Sousa-Santos, Patrick, Tarcísio Alvarenga, Pedro Pozzobon, Ana Beatriz Baston, Ana Flavia Lemos, Maria C. Foloni, Itamar Andrade, Luis Lopes, and Igor Teixeira. "Miller-Fisher syndrome in puerperium." In XIV Congresso Paulista de Neurologia. Zeppelini Editorial e Comunicação, 2023. http://dx.doi.org/10.5327/1516-3180.141s1.703.
Анотація:
Introduction: Miller-Fisher syndrome (MFS) is recognized as a variant of Guillain-Barré syndrome, composed of the clinical triad of ataxia, areflexia and ophtalmoplegia. The association between MFS and pregnancy/postpartum has been described in few cases previously. Case presentation: Woman, 28-year-old, nine days after normal partum present paresthesia and weakness in arms, a day later presenting with abnormal gate and diplopia. No recent infections or vaccination was described. The examination showed gait ataxia, right sixty nerve palsy, muscle strength in legs MRC grade 4 and hypotonia. The Patellar and Achilles tendon reflex was absent. No alterations in sensitive examination. Cerebrospinal fluid (CSF) showed 1 cell, protein 41 mg/dL, with infectious analysis negative. Laboratorial tests and magnetic resonance imaging were unmarkable. Electroneuromyography (ENMG) showed reduced amplitude of the sensory action potential in bilateral sural and ulnar nerves. The set of clinical and ENMG findings diagnosed MFS. She was admitted to the intensive care and realized five plasmapheresis sessions, showing significant improvement of symptoms. Four months later, she was asymptomatic of the neurological condition. Discussion: In PubMed, we found a total of four cases in pregnancy were reported, and no case in puerperium. The effect of hormonal changes of pregnancy/puerperium presents on the immune system make these periods special situations in the understanding of the MFS. A particularity of our case is the normal CSF count of cells and protein levels, which may be normal in 30–50% of patients in first week of symptoms. Anti-GQ1b antibodies are found in up to 90% of patients with MFS, however it is not necessary to establish the diagnosis. When MFS is suspected, the recommendation is not wait for antibody tests to starting treatment. Conclusion: The relationship of MSF and pregnancy/puerperium is few described, but the evolution of these cases seems to be like the other patients.
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Nitta, Takahiro. "In silico design of guiding tracks for molecular shuttles powered by motor proteins." In 2011 International Symposium on Micro-NanoMechatronics and Human Science (MHS). IEEE, 2011. http://dx.doi.org/10.1109/mhs.2011.6102250.
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Kuriki, Hiroki, Sou Takasawa, Shinya Sakuma, Genji Kurisu, Fumihito Arai, and Yoko Yamanishi. "Electrically-induced bubble knife for protein crystallization and processing." In 2013 International Symposium on Micro-NanoMechatronics and Human Science (MHS). IEEE, 2013. http://dx.doi.org/10.1109/mhs.2013.6710474.
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XAVIER, WILLIAM, FRANCISCO ERNANI ALVES MAGALHãES, ANTONIO EUFRáSIO VIEIRA NETO, ANTONIO ROMáRIO COELHO ALCâNTARA, KALINA KELMA OLIVEIRA DE SOUSA, MICHELINE SOARES COSTA OLIVEIRA, and ANA CRISTINA DE OLIVEIRA MONTEIRO MOREIRA. "ESTUDO IN SILICO DAS BASES MOLECULARES DE INTERAÇÃO ENTRE O ÁCIDO OLEANÓLICO E O RECEPTOR TGR5." In II Brazilian Congress of Development. DEV2021, 2021. http://dx.doi.org/10.51162/brc.dev2021-0056.
Анотація:
Os estudos sobre metabolismo vêm sendo potencializados com as investigações sobre produtos naturais e suas atividades biotecnológicas. Com base nisto, o trabalho investiga a interação molecular do ácido oleanólico com o receptor TGR5 (receptor 1 de ácido biliar acoplado à proteína G), também conhecido como receptor 19 acoplado à proteína G, relatado em diversos modelos experimentais como integrante das moléculas responsáveis por regulações metabólicas capazes de exercer efeitos benéficos contra o diabetes e síndrome metabólica. O ácido oleanólico é um componente natural de muitos alimentos vegetais e ervas medicinais, já o TGR5 é um receptor proteico endógeno ligado à membrana plasmática, expresso em todo o corpo, mas apresenta níveis elevados de expressão no fígado, intestino, estômago, baço, e tecido adiposo marrom. Para promover esta interação in silico, foram obtidas as estruturas tridimensionais das duas moléculas em bancos de dados estruturais e foi realizado o docking molecular, que consiste numa complexação guiada por algoritmo e software especializado, para obtenção de complexos estruturais estáveis. Os resultados descrevem que o ácido oleanólico possui afinidade por um sítio específico do receptor TGR5, se complexando a partir de 6 ligações químicas e as atividades biotecnológicas promovidas por ele possuem alta reprodutibilidade e afinidade, o que é capaz de promover estabilização energética do receptor envolvido, no contexto metabólico, atuando como um aliado no combate à obesidade.,
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Katayama, Yoshiki, Hirotaro Kitazaki, Jeong-Hun Kang, Xiaoming Han, Takeshi Mori, and Takuro Niidome. "High-throughput Detection of Protein Kinase Activities in Cell Lysate Based on the Aggregation of Gold Nanoparticles with Peptides." In 2009 MRS Spring Meet. Materials Research Society, 2009. http://dx.doi.org/10.1557/proc-1241-xx08-08.
Звіти організацій з теми "MFS proteins":
1
Yalovsky, Shaul, and Julian Schroeder. The function of protein farnesylation in early events of ABA signal transduction in stomatal guard cells of Arabidopsis. United States Department of Agriculture, January 2002. http://dx.doi.org/10.32747/2002.7695873.bard.
Анотація:
Loss of function mutations in the farnesyltransferase β subunit gene ERA1 (enhanced response to abscisic acid), cause abscisic acid hypersensitivity in seedlings and in guard cells. This results in slowed water loss of plants in response to drought. Farnesyltransferase (PFT) catalyses the attachment of the 15-carbon isoprenoid farnesyl to conserved cysteine residues located in a conserved C-terminal domain designated CaaX box. PFT is a heterodimeric protein comprised of an a and b sununits. The a subunit is shared between PFT and geranylgeranyltransferase-I (PGGTI) which catalyses the attachemt of the 20-carbon isoprenoid geranylgeranyl to CaaX box proteins in which the last amino acid is almost always leucine and in addition have a polybasic domain proximal to the CaaL box. Preliminary data presented in the proposal showed that increased cytoplasmic Ca2+ concentration in stomal guard cells in response to non-inductive ABA treatements. The goals set in the proposal were to characterize better how PFT (ERA1) affects ABA induced Ca2+ concentrations in guard cells and to identify putative CaaX box proteins which function as negative regulators of ABA signaling and which function is compromised in era1 mutant plants. To achieve these goals we proposed to use camelion Ca2+ sensor protein, high throughput genomic to identify the guard cell transcriptome and test prenylation of candidate proteins. We also proposed to focus our efforts of RAC small GTPases which are prenylated proteins which function in signaling. Our results show that farnesyltransferaseprenylates protein/s that act between the points of ABA perception and the activation of plasma membrane calcium influx channels. A RAC protein designated AtRAC8/AtRop10 also acts in negative regulation of ABA signaling. However, we discovered that this protein is palmitoylated and not prenylated although it contains a C-terminal CXXX motif. We further discovered a unique C-terminal sequence motif required for membrane targeting of palmitoylatedRACs and showed that their function is prenylation independent. A GC/MS based method for expression in plants, purification and analysis of prenyl group was developed. This method would allow highly reliable identification of prenylated protein. Mutants in the shared α subunit of PFT and PGGT-I was identified and characterized and was shown to be ABA hypersensitive but less than era1. This suggested that PFT and PGGT-I have opposing functions in ABA signaling. Our results enhanced the understanding of the role of protein prenylation in ABA signaling and drought resistance in plants with the implications of developing drought resistant plants. The results of our studies were published 4 papers which acknowledge support from BARD.
2
Epel, Bernard, and Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, November 2005. http://dx.doi.org/10.32747/2005.7695874.bard.
Анотація:
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.
3
Czosnek, Henryk Hanokh, Dani Zamir, Robert L. Gilbertson, and Lucas J. William. Resistance to Tomato Yellow Leaf Curl Virus by Combining Expression of a Natural Tolerance Gene and a Dysfunctional Movement Protein in a Single Cultivar. United States Department of Agriculture, June 2000. http://dx.doi.org/10.32747/2000.7573079.bard.
Анотація:
Background The tomato yellow leaf curl disease (TYLCV) has been a major deterrent to tomato production in Israel for the last 20 years. This whitefly-transmitted viral disease has been found in the Caribbean Island in the early 1990s, probably as an import from the Middle East. In the late 1990s, the virus has spread to the US and is now conspicuous in Florida and Georgia. Objectives Because of the urgency facing the TYLCV epidemics, there was a compelling need to mobilize scientists to develop tomato variety resistant to TYLCV. The major goal was to identify the virus movement protein (MP) and to express a defective from of MP in a cultivar that contained the natural Ty-1 resistance gene. The research included 1. cloning of the TYLCV isolate from the Dominican Republic (DR) which is (or a close variant) also present in the continental USA; 2. ddefining the role of the MP; 3. mutating the putative MP gene; 4. introducing the modified gene into an advance Ty-1 line; 5. testing the transgenic plants in the field. The pressing threat to tomato production in the US resulted in an extension of the objectives: more emphasis was placed on characterization of TYLCV i the DR, on determination of the epidemiology of the virus in the DR, and on using new TYLCV resistance sources for tomato breeding. Achievements and signification 1. The characterization of TYLCV-DR allowed for more effective TYLCV management strategies that are now implemented in the DR. 2. The identification of the TYLCV MPs and, more importantly, insight into their function has provided a model for how these proteins function in TYLCV movement and support the targeting of one or more of these proteins in a dominant lethal strategy to engineer plants for TYLCV resistance. 3. The transgenic plants that are being generated with wild-type and mutated TYLCV MPs will serve to test the hypothesis that interference with one or more of the TYLCV movement proteins will be a strategy for generating TYLCV-resistant plants. 4. The fine mapping of the resistance Ty-1 gene allowed eliminating deleterious chromosome segments from the wild tomato genitor L. chilense. It may in a near future allow the cloning of the first geminivirus resistance gene. 5. Another resistance source from the wild tomato species L. hirsitum was introgressed into the domesticated tomato, resulting in the production of resistant breeding lines. Implications 1. The monitoring of TYLCV in whiteflies has been applied in the DR. These tools are presently being used to assist in the evaluation of the host-free period and to help select the appropriate locations for growing tomatoes in the DR. 2. An overall strategy to obtain resistance against TYLCV has been used. The expression of wild-type or mutated TYLCV MPs in transgenic tomato is another addition to the arsenal used to fight TYLCV, together with marker assisted breeding and mobilization of additional resistant genes from the wild.
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Fluhr, Robert, and Maor Bar-Peled. Novel Lectin Controls Wound-responses in Arabidopsis. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7697123.bard.
Анотація:
Innate immune responses in animals and plants involve receptors that recognize microbe-associated molecules. In plants, one set of this defense system is characterized by large families of TIR–nucleotide binding site–leucine-rich repeat (TIR-NBS-LRR) resistance genes. The direct interaction between plant proteins harboring the TIR domain with proteins that transmit and facilitate a signaling pathway has yet to be shown. The Arabidopsis genome encodes TIR-domain containing genes that lack NBS and LRR whose functions are unknown. Here we investigated the functional role of such protein, TLW1 (TIR LECTIN WOUNDRESPONSIVE1). The TLW1 gene encodes a protein with two domains: a TIR domain linked to a lectin-containing domain. Our specific aim in this proposal was to examine the ramifications of the TL1-glycan interaction by; A) The functional characterization of TL1 activity in the context of plant wound response and B) Examine the hypothesis that wounding induced specific polysaccharides and examine them as candidates for TL-1 interactive glycan compounds. The Weizmann group showed TLW1 transcripts are rapidly induced by wounding in a JA-independent pathway and T-DNA-tagged tlw1 mutants that lack TLW1 transcripts, fail to initiate the full systemic wound response. Transcriptome methodology analysis was set up and transcriptome analyses indicates a two-fold reduced level of JA-responsive but not JA-independent transcripts. The TIR domain of TLW1 was found to interact directly with the KAT2/PED1 gene product responsible for the final b-oxidation steps in peroxisomal-basedJA biosynthesis. To identify potential binding target(s) of TL1 in plant wound response, the CCRC group first expressed recombinant TL1 in bacterial cells and optimized conditions for the protein expression. TL1 was most highly expressed in ArcticExpress cell line. Different types of extraction buffers and extraction methods were used to prepare plant extracts for TL1 binding assay. Optimized condition for glycan labeling was determined, and 2-aminobenzamide was used to label plant extracts. Sensitivity of MALDI and LC-MS using standard glycans. THAP (2,4,6- Trihydroxyacetophenone) showed minimal background peaks at positive mode of MALDI, however, it was insensitive with a minimum detection level of 100 ng. Using LC-MS, sensitivity was highly increased enough to detect 30 pmol concentration. However, patterns of total glycans displayed no significant difference between different extraction conditions when samples were separated with Dionex ICS-2000 ion chromatography system. Transgenic plants over-expressing lectin domains were generated to obtain active lectin domain in plant cells. Insertion of the overexpression construct into the plant genome was confirmed by antibiotic selection and genomic DNA PCR. However, RT-PCR analysis was not able to detect increased level of the transcripts. Binding ability of azelaic acid to recombinant TL1. Azelaic acid was detected in GST-TL1 elution fraction, however, DHB matrix has the same mass in background signals, which needs to be further tested on other matrices. The major findings showed the importance of TLW1 in regulating wound response. The findings demonstrate completely novel and unexpected TIR domain interactions and reveal a control nexus and mechanism that contributes to the propagation of wound responses in Arabidopsis. The implications are to our understanding of the function of TIR domains and to the notion that early molecular events occur systemically within minutes of a plant sustaining a wound. A WEB site (http://genome.weizmann.ac.il/hormonometer/) was set up that enables scientists to interact with a collated plant hormone database.
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Tucker, Mark L., Shimon Meir, Amnon Lers, Sonia Philosoph-Hadas, and Cai-Zhong Jiang. Elucidation of signaling pathways that regulate ethylene-induced leaf and flower abscission of agriculturally important plants. United States Department of Agriculture, January 2012. http://dx.doi.org/10.32747/2012.7597929.bard.
Анотація:
The Problem: Abscission is a highly regulated process, occurring as a natural terminal stage of development, in which various organs are separated from the parent plant. In most plant species, the process is initiated by a decrease in active auxin in the abscission zone (AZ) and an increase in ethylene, and may be accelerated by postharvest or environmental stresses. Another potential key regulator in abscission is IDA (Inflorescence Deficient in Abscission), which was identified as an essential peptide signal for floral organ abscission in Arabidopsis. However, information is still lacking regarding the molecular mechanisms integrating all these regulators. In our previous BARD funded research we made substantial progress towards understanding these molecular events in tomato, and the study is still in progress. We established a powerful platform for analysis of genes for regulatory proteins expressed in AZ. We identified changes in gene expression for several transcription factors (TFs) directly linked to ethylene and auxin signaling and several additional regulatory proteins not so obviously linked to these hormones. Moreover, we demonstrated using a virus-induced gene silencing (VIGS) assay that several play a functional role in the onset of abscission. Based on these results we have selected 14 genes for further analysis in stably transformed tomato plants. All 14 genes were suppressed by RNA interference (RNAi) using a constitutive promoter, and 5 of them were also suppressed using an abscission-specific promoter. Transformations are currently at different stages of progress including some lines that already display an abscission phenotype. Objectives: We propose here to (1) complete the functional analysis of the stably transformed tomato plants with T2 lines and perform transcriptome analysis using custom abscission-specific microarrays; (2) conduct an indepth analysis of the role of IDA signaling in tomato leaf and flower abscission; (3) perform transcriptome and proteome analyses to extend the earlier gene expression studies to identify transcripts and proteins that are highly specific to the separation layer (i.e., target cells for cell separation) prior to the onset of abscission; (4) extend and compliment the work in tomato using a winnowed set of genes in soybean. Methodology: Next Generation Sequencing (NGS) of mRNA will be used to further increase the list of abscission-associated genes, and for preparation of a custom tomato abscission microarray to test altered gene expression in transgenic plants. Tandem mass spectrometry (LC-MS/MS) of protein extracts from leaf petiole, flower pedicel and their AZ tissues will be used to identify the proteome of the AZ before and during abscission. AZ-specific gene promoters will be used in stably transformed tomato plants to reduce non-target phenotypes. The bean pod mottle virus (BPMV) plasmid vectors will be used for VIGS analysis in soybean. Expected Contribution: Our study will provide new insights into the regulation of ethylene-induced abscission by further revealing the role of key regulators in the process. This will permit development of novel techniques for manipulating leaf and flower abscission, thereby improving the postharvest performance of agriculturally important crops.
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Blumwald, Eduardo, and Avi Sadka. Citric acid metabolism and mobilization in citrus fruit. United States Department of Agriculture, October 2007. http://dx.doi.org/10.32747/2007.7587732.bard.
Анотація:
Accumulation of citric acid is a major determinant of maturity and fruit quality in citrus. Many citrus varieties accumulate citric acid in concentrations that exceed market desires, reducing grower income and consumer satisfaction. Citrate is accumulated in the vacuole of the juice sac cell, a process that requires both metabolic changes and transport across cellular membranes, in particular, the mitochondrial and the vacuolar (tonoplast) membranes. Although the accumulation of citrate in the vacuoles of juice cells has been clearly demonstrated, the mechanisms for vacuolar citrate homeostasis and the components controlling citrate metabolism and transport are still unknown. Previous results in the PIs’ laboratories have indicated that the expression of a large number of a large number of proteins is enhanced during fruit development, and that the regulation of sugar and acid content in fruits is correlated with the differential expression of a large number of proteins that could play significant roles in fruit acid accumulation and/or regulation of acid content. The objectives of this proposal are: i) the characterization of transporters that mediate the transport of citrate and determine their role in uptake/retrieval in juice sac cells; ii) the study of citric acid metabolism, in particular the effect of arsenical compounds affecting citric acid levels and mobilization; and iii) the development of a citrus fruit proteomics platform to identify and characterize key processes associated with fruit development in general and sugar and acid accumulation in particular. The understanding of the cellular processes that determine the citrate content in citrus fruits will contribute to the development of tools aimed at the enhancement of citrus fruit quality. Our efforts resulted in the identification, cloning and characterization of CsCit1 (Citrus sinensis citrate transporter 1) from Navel oranges (Citrus sinesins cv Washington). Higher levels of CsCit1 transcripts were detected at later stages of fruit development that coincided with the decrease in the juice cell citrate concentrations (Shimada et al., 2006). Our functional analysis revealed that CsCit1 mediates the vacuolar efflux of citrate and that the CsCit1 operates as an electroneutral 1CitrateH2-/2H+ symporter. Our results supported the notion that it is the low permeable citrateH2 - the anion that establishes the buffer capacity of the fruit and determines its overall acidity. On the other hand, it is the more permeable form, CitrateH2-, which is being exported into the cytosol during maturation and controls the citrate catabolism in the juice cells. Our Mass-Spectrometry-based proteomics efforts (using MALDI-TOF-TOF and LC2- MS-MS) identified a large number of fruit juice sac cell proteins and established comparisons of protein synthesis patterns during fruit development. So far, we have identified over 1,500 fruit specific proteins that play roles in sugar metabolism, citric acid cycle, signaling, transport, processing, etc., and organized these proteins into 84 known biosynthetic pathways (Katz et al. 2007). This data is now being integrated in a public database and will serve as a valuable tool for the scientific community in general and fruit scientists in particular. Using molecular, biochemical and physiological approaches we have identified factors affecting the activity of aconitase, which catalyze the first step of citrate catabolism (Shlizerman et al., 2007). Iron limitation specifically reduced the activity of the cytosolic, but not the mitochondrial, aconitase, increasing the acid level in the fruit. Citramalate (a natural compound in the juice) also inhibits the activity of aconitase, and it plays a major role in acid accumulation during the first half of fruit development. On the other hand, arsenite induced increased levels of aconitase, decreasing fruit acidity. We have initiated studies aimed at the identification of the citramalate biosynthetic pathway and the role(s) of isopropylmalate synthase in this pathway. These studies, especially those involved aconitase inhibition by citramalate, are aimed at the development of tools to control fruit acidity, particularly in those cases where acid level declines below the desired threshold. Our work has significant implications both scientifically and practically and is directly aimed at the improvement of fruit quality through the improvement of existing pre- and post-harvest fruit treatments.
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MARTÍNEZ LEAL, LAURA DE LA CRUZ, and Carlos Romá Mateo. Preliminary proteomics analysis of the potential use of HMGB1 as sepsis biomarker. Fundación Avanza, May 2023. http://dx.doi.org/10.60096/fundacionavanza/2312022.
Анотація:
With this study, we evaluate the first steps into the development of a new possible biomarker for sepsis disease consisting in detection of HGMB1 protein in plasma, using mass spectrometry (MRM-MS), that could improve sepsis clinical management.
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Sengupta-Gopalan, Champa, Shmuel Galili, and Rachel Amir. Improving Methionine Content in Transgenic Forage Legumes. United States Department of Agriculture, February 2001. http://dx.doi.org/10.32747/2001.7580671.bard.
Анотація:
Leguminous forage crops are high in proteins but deficient in S- amino acids. It has been shown that both wool quality and milk production can be limited by the post-ruminal supply of sulfur-containing amino acids. Efforts to use conventional plant breeding and cell selection techniques to increase the S-amino acid content of alfalfa have met with little success. With the objective to increase the S-amino acid content of forage legumes, the goal of this project was to co- express the methionine rich zein genes from corn along with a gene for a key enzyme in methionine biosynthesis, aspartate kinase(AK). The zeins are seed storage proteins from corn and are groupec into four distinct classes based on their amino acid sequence homologies. The b-zein (15kd) and the 6zein (10kD and 18kD) have proportionately high levels of methionine (10%, 22% and 28%, respectively). Initial studies from our lab had shown that while the 15kD zein accumulated to high levels in vegetative tissues of transgenic tobacco the l0kD zein did not. However, co-expression of the 10kD zein with the 15kD zein genes in tobacco showed stabilization of the 10kD zein and the co-localization of the 10kD and 15kD zein proteins in unique ER derived protein bodies. AK is the key enzyme for producing carbon skeletons for all amino acids of the aspartate family including methionine. It is, however, regulated by end-product feedback inhibition. The specific objectives of this proposal were: i. to co-express the 15kD zein with the 10/18kD zein genes in alfalfa in order to enhance the level of accumulation of the 10/18kD zein; ii. to increase methionine pools by expressing a feedback insensitive AK gene in transformants co-expressing the 15kD and 10/18kD zein genes. The Israeli partners were successful in expressing the AK gene in alfalfa which resulted in an increase in free and bound threonine but not in methionine (Galili et al., 2000). Since our target was to increase methionine pools, we changed our second objective to replace the AK gene with the gene for cystathionine gamma synthase (CGS) in the co-expression studies. The first methionine specific reaction is catalyzed by CGS. An additional objective was to develop a transformation system for Berseem clover, and to introduce the appropriate gene constructs into it with the goal of improving their methionine content. Genes for the 15kD zein along with the genes for either the 10kD or 18kD zein have been introduced into the same alfalfa plant both by sexual crosses and by re-transformation. Analysis of these zein co-expressors have shown that both the IOkD and 18kD zein levels go up 5 to 10 fold when co-expressed with the 15kD zein (Bagga et al., MS in preparation). Incubation of the leaves of transgenic alfalfa co-expressing the 15kD and 10kD zein genes, in the rumen of cows have shown that the zein proteins are stable in the rumen. To increase the level of zein accumulation in transgenic alfalfa different promoters have been used to drive the zein genes in alfalfa and we have concluded that the CaMV 35S promoter is superior to the other strong leaf -specific promoters. By feeding callus tissue of alfalfa plants co-expressing the 15kD and 10kD zein genes with methionine and its precursors, we have shown that the zein levels could be significantly enhanced by increasing the methionine pools. We have now introduced the CGS gene (from Arabidopsis; kindly provided to us by Dr. Leustek), into the 15kD zein transformants and experiments are in progress to check if the expression of the CGS gene indeed increases the level of zein accumulation in alfalfa. We were not successful in developing a transformation protocol for Berseem clover.
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Whitham, Steven A., Amit Gal-On, and Victor Gaba. Post-transcriptional Regulation of Host Genes Involved with Symptom Expression in Potyviral Infections. United States Department of Agriculture, June 2012. http://dx.doi.org/10.32747/2012.7593391.bard.
Анотація:
Understanding how RNA viruses cause disease symptoms in their hosts is expected to provide information that can be exploited to enhance modern agriculture. The helper component-proteinase (HC-Pro) protein of potyviruses has been implicated in symptom development. Previously, we demonstrated that symptom expression is associated with binding of duplex small-interfering-RNA (duplex-siRNA) to a highly conserved FRNK amino acid motif in the HC-Pro of Zucchini yellow mosaic virus (ZYMV). This binding activity also alters host microRNA (miRNA) profiles. In Turnip mosaic virus (TuMV), which infects the model plant Arabidopsis, mutation of the FRNK motif to FINK was lethal providing further indication of the importance of this motif to HC-Pro function. In this continuation project, our goal was to further investigate how ZYMV and TuMV cause the mis-expression of genes in cucurbits and Arabidopsis, respectively, and to correlate altered gene expression with disease symptoms. Objective 1 was to examine the roles of aromatic and positively charged residues F164RNH and K215RLF adjacent to FR180NK in small RNA binding. Objective 2 was to determine the target genes of the miRNAs which change during HC-Pro expression in infected tissues and transgenic cucumber. Objective 3 was to characterize RNA silencing mechanisms underlying differential expression of host genes. Objective 4 was to analyze the function of miRNA target genes and differentially expressed genes in potyvirus-infected tissues. We found that the charged K/R amino acid residues in the FKNH and KRLF motifs are essential for virus viability. Replacement of K to I in FKNH disrupted duplex-siRNA binding and virus infectivity, while in KRLF mutants duplex-siRNA binding was maintained and virus infectivity was limited: symptomless following a recovery phenomenon. These findings expanded the duplex-siRNA binding activity of HC-Pro to include the adjacent FRNK and FRNH sites. ZYMV causes many squash miRNAs to hyper-accumulate such as miR166, miR390, mir168, and many others. Screening of mir target genes showed that only INCURVATA-4 and PHAVOLUTA were significantly upregulated following ZYMVFRNK infection. Supporting this finding, we found similar developmental symptoms in transgenic Arabidopsis overexpressing P1-HC-Pro of a range of potyviruses to those observed in miR166 mutants. We characterized increased transcription of AGO1 in response to infection with both ZYMV strains. Differences in viral siRNA profiles and accumulation between mild and severe virus infections were characterized by Illumina sequencing, probably due to the differences in HC-Pro binding activity. We determined that the TuMV FINK mutant could accumulate and cause symptoms in dcl2 dcl4 or dcl2 dcl3 dcl4 mutants similar to TuMV FRNK in wild type Arabidopsis plants. These dcl mutant plants are defective in antiviral defenses, and the results show that factors other than HC-ProFRNK motif can induce symptoms in virus-infected plants. As a result of this work, we have a better understanding of the FRNK and FKNH amino acid motifs of HC-Pro and their contributions to the duplex-siRNA binding functions. We have identified plant genes that potentially contribute to infectivity and symptoms of virus infected plants when they are mis-expressed during potyviral infections. The results establish that there are multiple underlying molecular mechanisms that lead viral pathogenicity, some dependent on HC-Pro. The potential benefits include the development of novel strategies for controlling diseases caused by viruses, methods to ensure stable expression of transgenes in genetically improved crops, and improved potyvirus vectors for expression of proteins or peptides in plants.
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Medrano, Juan, Adam Friedmann, Moshe (Morris) Soller, Ehud Lipkin, and Abraham Korol. High resolution linkage disequilibrium mapping of QTL affecting milk production traits in Israel Holstein dairy cattle. United States Department of Agriculture, March 2008. http://dx.doi.org/10.32747/2008.7696509.bard.
Анотація:
Original objectives: To create BAC contigs covering two QTL containing chromosomal regions (QTLR) and obtain BAC end sequence information as a platform for SNP identification. Use the SNPs to search for marker-QTL linkage disequilibrium (LD) in the test populations (US and Israel Holstein cattle). Identify candidate genes, test for association with dairy cattle production and functional traits, and confirm any associations in a secondary test population. Revisions in the course of the project: The selective recombinant genotyping (SRG) methodology which we implemented to provide moderate resolution QTL mapping turned out to be less effective than expected, due to problems introduced by incomplete marker informativity. This required a no-cost one-year extension of the project. Aside from this, the project was implemented essentially as envisaged, but only with respect to a single QTLR and single population association-test. Background to the topic. Dairy cattle breeders are looking to marker-assisted selection (MAS) as a means of identifying genetically superior sires and dams. MAS based on population-wide LD can be many times more effective than MAS based on within-family linkage mapping. In this proposal we developed a protocol leading from family based QTL mapping to population-wide LD between markers and the QTL Major conclusions, solutions, achievements. The critical importance of marker informativity for application of the SRG design in outcrossing random mating populations was identified, and an alternative Fractioned Pool Design (FPD) based on selective DNA pooling was developed. We demonstrated the feasibility of constructing a BAC contig across a targeted chromosomal region flanking the marker RM188 on bovine chromosome BTA4, which was shown in previous work to contain a QTL affecting milk production traits. BAC end sequences were obtained and successfully screened for SNPs. LD studies of these SNPs in the Israel population, and of an independent set of SNPs taken across the entire proximal region of BTA4 in the USA population, showed a much lower degree of LD than previously reported in the literature. Only at distances in the sub-cM level did an appreciable fraction of SNP marker-pairs show levels of LD useful for MAS. In contrast, studies in the Israel population using microsatellite markers, presented an equivalent degree of LD at a 1-5 separation distance. SNP LD appeared to reflect historical population size of Bostaurus (Ne=5000- 10,000), while microsatellite LD appeared to be in proportion to more recent effective population size of the Holstein breed (Ne=50-100). An appreciable fraction of the observed LD was due to Family admixture structure of the Holstein population. The SNPs MEOX2/IF2G (found within the gene SETMAR at 23,000 bp from RM188) and SNP23 were significantly associated with PTA protein, Cheese dollars and Net Merit Protein in the Davis bull resource population, and were also associated with protein and casein percentages in the Davis cow resource population. Implications. These studies document a major difference in degree of LD presented by SNPs as compared to microsatellites, and raise questions as to the source of this difference and its implications for QTL mapping and MAS. The study lends significant support to the targeted approach to fine map a previously identified QTL. Using high density genotyping with SNP discovered in flanking genes to the QTL, we have identified important markers associated with milk protein percentage that can be tested in markers assisted selection programs.