Academic literature on the topic 'Mutants de type-1'
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Journal articles on the topic "Mutants de type-1"
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Nikolaitchik, Olga, Terence D. Rhodes, David Ott, and Wei-Shau Hu. "Effects of Mutations in the Human Immunodeficiency Virus Type 1 gag Gene on RNA Packaging and Recombination." Journal of Virology 80, no. 10 (2006): 4691–97. http://dx.doi.org/10.1128/jvi.80.10.4691-4697.2006.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) recombination occurs during reverse transcription when parts of the two copackaged RNAs are used as templates for DNA synthesis. It was previously hypothesized that HIV-1 Gag polyproteins preferentially encapsidate the RNA from which they were translated (cis-packaging hypothesis). This hypothesis implies that mutants encoding Gag that cannot efficiently package viral RNA are selected against at two levels: these mutants do not generate infectious virus, and these mutants are not efficiently rescued by the wild-type virus because the mutant RNAs are packaged at much lower levels than are those of the wild-type genome. Therefore, genetic information encoded by gag mutants can be rapidly lost in the viral population. To test this prediction of the cis-packaging hypothesis, we examined several gag mutants by measuring the efficiencies of the mutant RNAs in being packaged in trans in the presence of wild-type virus and determining the rates of recombination between gag mutants and wild-type viruses. We observed that the viral RNAs from the nucleocapsid zinc finger or the capsid truncation mutant were packaged efficiently in trans, and these mutant viruses also frequently recombined with the wild-type viruses. In contrast, viral RNAs from mutants containing a 6-nucleotide substitution encompassing the gag AUG were not efficiently encapsidated, resulting in a low rate of recombination between the mutants and wild-type viruses. Further analyses revealed that other, more subtle mutations changing the gag AUG and abolishing Gag translation did not interfere with efficient encapsidation of the mutant RNA. Our results indicated that neither the gag AUG sequence nor Gag translation is essential for viral RNA encapsidation, and Gag can package both wild-type and gag mutant RNAs with similar efficiencies. Therefore, we propose that HIV-1 RNA encapsidation occurs mainly in trans, and most gag mutants can be rescued by wild-type virus; therefore, they are unlikely to face the aforementioned double-negative selection.
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Operario, Darwin J., Mini Balakrishnan, Robert A. Bambara, and Baek Kim. "Reduced dNTP Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase Promotes Strand Transfer." Journal of Biological Chemistry 281, no. 43 (2006): 32113–21. http://dx.doi.org/10.1074/jbc.m604665200.
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We have recently demonstrated that HIV-1 RT mutants characterized by low dNTP binding affinity display significantly reduced dNTP incorporation kinetics in comparison to wild-type RT. This defect is particularly emphasized at low dNTP concentrations where WT RT remains capable of efficient synthesis. Kinetic interference in DNA synthesis can induce RT pausing and slow down the synthesis rate. RT stalling and slow synthesis rate can enhance RNA template cleavage by RT-RNase H, facilitating transfer of the primer to a homologous template. We therefore hypothesized that reduced dNTP binding RT mutants can promote template switching during minus strand synthesis more efficiently than WT HIV-1 RT at low dNTP concentrations. To test this hypothesis, we employed two dNTP binding HIV-1 RT mutants, Q151N and V148I. Indeed, as the dNTP concentration was decreased, the template switching frequency progressively increased for both WT and mutant RTs. However, as predicted, the RT mutants promoted more transfers compared with WT RT. The WT and mutant RTs were similar in their intrinsic RNase H activity, supporting that the elevated template switching efficiency of the mutants was not the result of the mutations enhancing RNase H activity. Rather, kinetic interference leading to stalled DNA synthesis likely enhanced transfers. These results suggest that the RT-dNTP substrate interaction mechanistically influences strand transfer and recombination of HIV-1 RT.
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LA RAGIONE, R. M., A. R. SAYERS, and M. J. WOODWARD. "The role of fimbriae and flagella in the colonization, invasion and persistence of Escherichia coli O78[ratio ]K80 in the day-old-chick model." Epidemiology and Infection 124, no. 3 (2000): 351–63. http://dx.doi.org/10.1017/s0950268899004045.
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To understand the role of flagella and fimbriae of Escherichia coli O78[ratio ]K80 in avian colibacillosis, day-old chicks were dosed orally with defined afimbriate and or aflagellate mutants and colonization, invasion and persistence compared with that of the wild-type. In an invasion model, chicks were dosed with 1 × 105 c.f.u. of a single strain and mutants defective for type 1 fimbriae, curli fimbriae or flagella colonized livers by 24 h although the numbers of bacteria present were significantly less than the wild-type. Mutants colonized between 50 and 75% of spleens whereas the wild-type colonized 100% of spleens. Additionally, the numbers of mutant bacteria in colonized spleens were significantly less than the wild-type. Surprisingly, mutants defective for the elaboration of more than one appendage were no more attenuated than single mutants. In a persistence model, chicks were dosed with 1 × 102 c.f.u. of a single strain and mutants defective for type 1 or curli or flagella or any combination thereof persisted as assessed by cloacal swabbing for 5 weeks of the experiment less well than the wild-type. In an additional persistence model, chicks were dosed with 5 × 102 c.f.u. of each of wild-type and one mutant together. All mutants were significantly less persistent than the wild-type (P < 0·001) and one mutant which lacked type 1, curli and flagella, was eliminated within 2 weeks. Analysis of the trends of elimination indicated that flagella contributed to persistence more than curli, which contributed more than type 1 fimbriae. Here was evidence for a major role in colonization, invasion and persistence played by type 1, curli and flagella.
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Martinez-Picado, Javier, Anu V. Savara, Lorraine Sutton, and Richard T. D’Aquila. "Replicative Fitness of Protease Inhibitor-Resistant Mutants of Human Immunodeficiency Virus Type 1." Journal of Virology 73, no. 5 (1999): 3744–52. http://dx.doi.org/10.1128/jvi.73.5.3744-3752.1999.
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ABSTRACT The relative replicative fitness of human immunodeficiency virus type 1 (HIV-1) mutants selected by different protease inhibitors (PIs) in vivo was determined. Each mutant was compared to wild type (WT), NL4-3, in the absence of drugs by several methods, including clonal genotyping of cultures infected with two competing viral variants, kinetics of viral antigen production, and viral infectivity/virion particle ratios. A nelfinavir-selected protease D30N substitution substantially decreased replicative capacity relative to WT, while a saquinavir-selected L90M substitution moderately decreased fitness. The D30N mutant virus was also outcompeted by the L90M mutant in the absence of drugs. A major natural polymorphism of the HIV-1 protease, L63P, compensated well for the impairment of fitness caused by L90M but only slightly improved the fitness of D30N. Multiply substituted indinavir-selected mutants M46I/L63P/V82T/I84V and L10R/M46I/L63P/V82T/I84V were just as fit as WT. These results indicate that the mutations which are usually initially selected by nelfinavir and saquinavir, D30N and L90M, respectively, impair fitness. However, additional mutations may improve the replicative capacity of these and other drug-resistant mutants. Hypotheses based on the greater fitness impairment of the nelfinavir-selected D30N mutant are suggested to explain observations that prolonged responses to delayed salvage regimens, including alternate PIs, may be relatively common after nelfinavir failure.
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Baker, Stefanie H., Debra L. Frederick, Andrew Bloecher, and Kelly Tatchell. "Alanine-Scanning Mutagenesis of Protein Phosphatase Type 1 in the Yeast Saccharomyces cerevisiae." Genetics 145, no. 3 (1997): 615–26. http://dx.doi.org/10.1093/genetics/145.3.615.
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Protein phosphatase type 1, encoded by GLC7 in Saccharomyces cerevisiae, is an essential serine/threonine phosphatase implicated in the regulation of a diverse array of physiological functions. We constructed and examined 20 mutant alleles of GLC7 in which codons encoding clusters of charged residues were changed to alanine codons. Three of 20 mutant alleles alter residues in the active site of the phosphatase and are unable to rescue the lethality of a glc7::LEU2 disruption. The 17 alleles that support growth confer a range of mutant traits including cell cycle arrest, 2-deoxyglucose resistance, altered levels of glycogen, sensitivity to high salt, and sporulation defects. For some traits, such as 2-deoxyglucose resistance and cell cycle arrest, the mutated residues map to specific regions of the protein whereas the mutated residues in glycogen-deficient mutants and sporulation-defective mutants are more widely distributed over the protein surface. Many mutants have complex phenotypes, each displaying a diverse range of defects. The wide range of phenotypes identified from the collection of mutant alleles is consistent with the hypothesis that Glc7p-binding proteins, which are thought to regulate the specificity of Glc7p, have overlapping binding sites on the surface of Glc7p. This could account for the high level of sequence conservation found among type 1 protein phosphatases from different species.
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Toppaldoddi, Katte Rao, Maira da Costa Cacemiro, Olivier Bluteau, et al. "Rare type 1-like and type 2-like calreticulin mutants induce similar myeloproliferative neoplasms as prevalent type 1 and 2 mutants in mice." Oncogene 38, no. 10 (2018): 1651–60. http://dx.doi.org/10.1038/s41388-018-0538-z.
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Bjerke, Susan L., John M. Cowan, Jelani K. Kerr, Ashley E. Reynolds, Joel D. Baines, and Richard J. Roller. "Effects of Charged Cluster Mutations on the Function of Herpes Simplex Virus Type 1 UL34 Protein." Journal of Virology 77, no. 13 (2003): 7601–10. http://dx.doi.org/10.1128/jvi.77.13.7601-7610.2003.
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ABSTRACT Herpes simplex virus type 1 (HSV-1) is a DNA virus that acquires an envelope by budding into the inner nuclear membrane of an infected cell. Recombinant HSV-1 lacking the UL34 gene cannot undergo this event. UL34 and UL31, another viral protein, colocalize in an infected cell and are necessary and sufficient to target both proteins to the inner nuclear envelope. In order to define and characterize sequences of UL34 that are necessary for primary envelopment to occur, a library of 19 UL34 charged cluster mutants and a truncation mutant lacking the putative transmembrane domain (ΔTM) were generated. Mutants in this library were analyzed in a complementation assay for their ability to function in the production of infectious virus. Seven of the mutants failed to complement a UL34-null virus. The remainder of the mutants complemented at or near wild-type UL34 levels. Failure of a mutant protein to function might be the result of incorrect subcellular localization. To address this possibility, confocal microscopy was used to determine the localization of the UL34 protein in charged cluster mutants and ΔTM. In transfection-infection experiments, all of the functional UL34 mutants and four of the six noncomplementing mutants localized to the inner nuclear envelope in a manner indistinguishable from that of wild-type UL34. All of the noncomplementing UL34 mutants mediated proper localization of UL31. Charged clusters critical for UL34 function are dispersed throughout the protein sequence and do not correlate well with highly conserved regions of the protein. These data suggest that UL34 has at least one function in addition to mediating proper localization of UL31 in infected cells and provide further support for the role of UL34 in mediating proper localization of UL31 in infected cells.
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Ying, Baoling, Kimberley Smith, and Katherine R. Spindler. "Mouse Adenovirus Type 1 Early Region 1A Is Dispensable for Growth in Cultured Fibroblasts." Journal of Virology 72, no. 8 (1998): 6325–31. http://dx.doi.org/10.1128/jvi.72.8.6325-6331.1998.
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ABSTRACT Mouse adenovirus type 1 (MAV-1) mutants with deletions of conserved regions of early region 1A (E1A) or with point mutations that eliminate translation of E1A were used to determine the role of E1A in MAV-1 replication. MAV-1 E1A mutants expressing no E1A protein grew to titers comparable to wild-type MAV-1 titers on mouse fibroblasts (3T6 fibroblasts and fibroblasts derived from Rb+/+,Rb+/−, and Rb−/− transgenic embryos). To test the hypothesis that E1A could induce a quiescent cell to reenter the cell cycle, fibroblasts were serum starved to stop DNA replication and cellular replication and then infected with the E1A mutant and wild-type viruses. All grew to equivalent titers. Steady-state levels of MAV-1 early mRNAs (E1A, E1B, E2, E3, and E4) from 3T6 cells infected with wild-type or E1A mutant virus were examined by Northern analysis. Steady-state levels of mRNAs from the mutant-infected cells were comparable to or greater than the levels found in wild-type virus infections for most of the early regions and for two late genes. The E2 mRNA levels were slightly reduced in all mutant infections relative to wild-type infections. E1A mRNA was not detected from infections with the MAV-1 E1A null mutant, pmE109, or from infections with similar MAV-1 E1A null mutants, pmE112 andpmE113. The implications for the lack of a requirement of E1A in cell culture are discussed.
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Silverman, Jessica L., Sapna Sharma, Tina M. Cairns, and Ekaterina E. Heldwein. "Fusion-Deficient Insertion Mutants of Herpes Simplex Virus Type 1 Glycoprotein B Adopt the Trimeric Postfusion Conformation." Journal of Virology 84, no. 4 (2009): 2001–12. http://dx.doi.org/10.1128/jvi.01791-09.
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ABSTRACT Glycoprotein B (gB) enables the fusion of viral and cell membranes during entry of herpesviruses. However, gB alone is insufficient for membrane fusion; the gH/gL heterodimer is also required. The crystal structure of the herpes simplex virus type 1 (HSV-1) gB ectodomain, gB730, has demonstrated similarities between gB and other viral fusion proteins, leading to the hypothesis that gB is a fusogen, presumably directly involved in bringing the membranes together by refolding from its initial or prefusion form to its final or postfusion form. The only available crystal structure likely represents the postfusion form of gB; the prefusion form has not yet been determined. Previously, a panel of HSV-1 gB mutants was generated by using random 5-amino-acid-linker insertion mutagenesis. Several mutants were unable to mediate cell-cell fusion despite being expressed on the cell surface. Mapping of the insertion sites onto the crystal structure of gB730 suggested that several insertions might not be accommodated in the postfusion form. Thus, we hypothesized that some insertion mutants were nonfunctional due to being “trapped” in a prefusion form. Here, we generated five insertion mutants as soluble ectodomains and characterized them biochemically. We show that the ectodomains of all five mutants assume conformations similar to that of the wild-type gB730. Four mutants have biochemical properties and overall structures that are indistinguishable from those of the wild-type gB730. We conclude that these mutants undergo only minor local conformational changes to relieve the steric strain resulting from the presence of 5 extra amino acids. Interestingly, one mutant, while able to adopt the overall postfusion structure, displays significant conformational differences in the vicinity of fusion loops, relative to wild-type gB730. Moreover, this mutant has a diminished ability to associate with liposomes, suggesting that the fusion loops in this mutant have decreased functional activity. We propose that these insertions cause a fusion-deficient phenotype not by preventing conversion of gB to a postfusion-like conformation but rather by interfering with other gB functions.
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Collins, Jennifer A., M. Gregory Thompson, Elijah Paintsil, Melisa Ricketts, Joanna Gedzior, and Louis Alexander. "Competitive Fitness of Nevirapine-Resistant Human Immunodeficiency Virus Type 1 Mutants." Journal of Virology 78, no. 2 (2004): 603–11. http://dx.doi.org/10.1128/jvi.78.2.603-611.2004.
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ABSTRACT Determining the fitness of drug-resistant human immunodeficiency virus type 1 (HIV-1) strains is necessary for the development of population-based studies of resistance patterns. For this purpose, we have developed a reproducible, systematic assay to determine the competitive fitness of HIV-1 drug-resistant mutants. To demonstrate the applicability of this assay, we tested the fitness of the five most common nevirapine-resistant mutants (103N, 106A, 181C, 188C, and 190A), with mutations in HIV-1 reverse transcriptase (RT), singly and in combination (for a total of 31 variants) in a defined HIV-1 background. For these experiments, the 27 RT variants that produced viable virus were cocultured with wild-type virus without nevirapine. The ratios of the viral species were determined over time by utilization of a quantitative real-time RT-PCR-based assay. These experiments revealed that all of the viable variants were less fit than the wild type and demonstrated that the order of relative fitness of the single mutants tested was as follows: 103N > 181C > 190A > 188C > 106A. This order correlated with the commonality of these mutants as a result of nevirapine monotherapy. These investigations also revealed that, on average, the double mutants were less fit than the single mutants and the triple mutants were less fit than the double mutants. However, the fitness of the single and double mutants was often not predictive of the fitness of the derivative triple mutants, suggesting the presence of complex interactions between the closely aligned residues that confer nevirapine resistance. This complexity was also evident from the observation that all three of the replication-competent quadruple mutants were fitter than most of the triple mutants, and in some cases, even the double mutants. Our data suggest that, in many cases, viral fitness is the determining factor in the evolution of nevirapine-resistant mutants in vivo, that interactions between the residues that confer nevirapine resistance are complex, and that these interactions substantially affect reverse transcriptase structure and/or function.
Dissertations / Theses on the topic "Mutants de type-1"
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Agut, Henri. "Etude biochimique et génétique de mutants thermosensibles du poliovirus type 1." Grenoble : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb375937283.
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Agut, Henri. "Etude biochimique et genetique de mutants thermosensibles du poliovirus type 1." Paris 7, 1987. http://www.theses.fr/1987PA077177.
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Addison, C. "Characterisation of herpes simplex virus type 1 ts mutants which have structural defects." Thesis, University of Glasgow, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377150.
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Toppaldoddi, Katte Rao. "Role of rare calreticulin mutants and of the endoplasmic reticulum stress in the pathogenesis of myeloproliferative neoplasms." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC322/document.
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Après la découverte des mutations de la calréticuline dans les néoplasmes classiques myéloproliferatifs négatifs pour le Ph1, les travaux se sont focalisés sur les deux mutations les plus fréquentes, c'est-à-dire la calréticuline del52 et l’ins5, mais il existe environ 20% de mutants rares de la calréticuline (une cinquantaine), qui ont été classés en type-1 « like » et type-2 « like », classification basée sur leur structure. Cependant il reste à déterminer si cette classification est pertinente du point de vue fonctionnel, ce qui pourrait avoir des conséquences pour la prise en charge des patients et leur traitement. Ici, nous démontrons que deux mutants rares de type-1 (del34 et del46) et un de type-2 (del19) se comportent de manière similaire aux deux mutations fondatrices de cette classification, del52 et ins5, respectivement. Ces résultats ont été validés par des expériences in vivo chez la souris. Tous les mutants de la calréticuline (del19, del34 et del46) nécessitent absolument le récepteur de la thrombopoïétine, appelé MPL, pour induire une transformation cellulaire en provoquant une activation indépendante de la thrombopoïétine de la voie MPL / JAK2-STAT, comme les mutants del52 et ins5. Dans les expériences de transplantation de moelle osseuse de souris, les mutants rares de type-1 sont associés à une progression fréquente de la maladie d’un tableau proche d’une thrombocytémie essentielle à une myélofibrose, tandis que le mutant rare de type 2 est associé à une légère thrombocytose. Du point de vue hématopoïétique, les mutants rares de type-1 provoquent une amplification au niveau des cellules souches hématopoïétiques donc à un stade précoce tandis que les mutants rares de type-2 provoquent une amplification tardive de la mégacaryopoïèse. Grâce à une modélisation protéique basée sur l'homologie des mutants de calréticuline, nous avons identifié des domaines oncogènes qui seraient potentiellement responsables de l'interaction pathologique de la calréticuline et de MPL pour conduire à une activation indépendante de la thrombopoïétine. Maintenant, ces résultats in silico doivent être absolument validés par des études structure fonction. Enfin, nous avons modélisé un nouveau mécanisme de signalisation dans la leucémie myéloïde chronique comprenant IRE-1alpha, un bras de la voie de réponse des protéines mal repliées (UPR), qui pourrait être responsable de la perte de la fonction de la p53 pendant la progression de la leucémie myéloïde chronique vers une leucémie aiguë. Un tel mécanisme pourrait être impliqué dans les autres MPN<br>After the discovery of calreticulin mutations in classical Ph1- Myeloproliferative Neoplasms, extensive investigation is underway on the two most frequent mutations, i.e., del52 and ins5, but it remains that the rare calreticulin mutants, which include both type-1 like and type-2 like require a similar investigation for ascertaining whether the classification of type-1 and type-2 has a functional relevance as well as for therapeutic intervention and patient management. Here we demonstrate that type-1 like (del34 and del46) and type-2 like (del19) mutants behave similarly as del52 and ins5 mutants, respectively. Moreover, we validate our findings with in vivo experiments. All the calreticulin mutants (del19, del34 and del46) absolutely require the thrombopoietin receptor, MPL, to induce cell transformation by causing ligand independent activation of the MPL/JAK2-STAT pathway. In mouse bone marrow transplantation experiments, type-1 like mutants are associated with frequent progression from an essential thrombocythemia-like phenotype to myelofibrosis whereas type-2 like mutant is associated with mild thrombocytosis. Type-1 like mutants cause clonal amplification of early hematopoetic stem cells whereas the type-2 like mutant causes late platelet amplification. Further, by homology based protein modeling of calreticulin mutants, we have identified possible oncogenic domains responsible for pathologic interaction of CALR and MPL leading to ligand independent activation of MPL. Now they must be validated by structural-functional studies Finally, we have modelled a novel signaling mechanism in chronic myeloid leukemia comprising of IRE-1alpha, an unfolded protein response (UPR) pathway arm, which may be responsible for loss of the WT p53 function during leukemic development and progression. Such a mechanism may be involved in the other MPNs
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Leung-Tack, Patricia. "L'herpès virus bovin de type 1 : caractérisation de la région Us du génome viral (souche ST) et étude de mutants délétés." Université Louis Pasteur (Strasbourg) (1971-2008), 1993. http://www.theses.fr/1993STR13260.
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La rhinotrachéite infectieuse bovine (IBR) est une affection respiratoire des bovins, provoquée par l’herpèsvirus bovin de type 1 (BHV-1). Bien que des vaccins existent, la capacité du BHV-1 à demeurer à l’état latent rend difficile le contrôle de cette maladie. Les travaux effectués dans la cadre de cette thèse ont contribué à l’acquisition des connaissances en biologie moléculaire du BHV-1, en vue d’améliorer les moyens de diagnostic de l’IBR. La séquence complète de BHV-1 (souche ST) Us a été déterminée (9745 pb). Sur cette séquence, sept cadres ouverts de lecture codent pour des produits homologues de protéines d’autres alpha-herpèsvirus. La comparaison de BHV-1 Us avec les régions Us d’autres alpha-herpèsvirus a permis de confirmer l’hypothèse de la conservation des gènes et de leurs arrangements identiques au sein des régions Us des alpha-herpèsvirus. Une souche BHV-1 délétée pour le gène équivalent de HSV-1 gE, et stable en culture de cellules, a été construite par recombinaison génétique, indiquant le caractère non essentiel de ce gène pour la réplication virale in vitro. L’utilisation d’un anticorps monoclonal spécifique a permis de mettre en évidence l’expression de la glycoprotéine gE chez le BHV-1. L’étude différentielle des comportements in vitro des deux souches BHV-1 parentales et recombinantes a montré une croissance plus lente de la souche délétée pour la protéine gE. L’ensemble des résultats a conforté l’hypothèse, émise pour les virus HSV-1 et PRV, d’une implication de la glycoprotéine, émise pour les virus HSV-1 et PRV, d’une implication de la glycoprotéine gE équivalente dans la diffusion virale<br>Bovine herpesvirus type 1 (BHV-1) causes severe respiratory tract infections, known as infectious bovine rhinotracheitis (IBR). The DNA sequence of the whole of the short unique region (Us) of the BHV-1 was determined (9745 kbp). Seven open reading frames have counterparts in the Us of other alpha-herpesvirus. Comparison analysis suggests the conservation and the same arrangement of the Us encoded genes. A gE homologue-deleted BHV-1 strain, stable in cell-culture, has been constructed, indicating that gE is not essential to in vitro replication. The use of a specific monoclonal antibody demonstrated the gE glycoprotein expression in BHV-1 virus. The in vitro study of the wild-type and the recombinant BHV-1 viruses shows that gE seems to be implicated in the viral diffusion
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Naito, Takeshi. "SC29EK, a Peptide Fusion Inhibitor with Enhanced α-Helicity, Inhibits Replication of Human Immunodeficiency Virus Type 1 Mutants Resistant to Enfuvirtide". Kyoto University, 2012. http://hdl.handle.net/2433/158065.
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Marshall, Ker Ross. "The pathogenesis, including neural latency, of equine herpesvirus type 1 in a murine infection model studied by means of lacZ, insertion mutants." Thesis, University of Cambridge, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.627614.
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Gutierrez, Rodriguez Ana. "Anatomical Characterization of the Type-1 cannabinoid receptors in specific brain cell populations of mutant mice." Thesis, Bordeaux, 2016. http://www.theses.fr/2016BORD0236/document.
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Dans cette thèse l’expression du récepteur CB1 dans l’hippocampe de souris mutantes ré-exprimant spécifiquement le gène spécifiquement dans certains types cellulaires du cerveau tels que : les neurones glutamatergiques du télencéphale dorsal, et les neurones GABAergiques a été analysé. De plus, dans le but de connaître la distribution anatomique exacte des récepteurs CB1 astrogliaux par rapport aux synapses excitatrices et inhibitrices, j’ai étudié l’expression des récepteurs CB1 dans les astrocytes de souris exprimant le récepteur CB1 seulement dans les astrocytes et une souris mutante ciblée pour exprimer la protéine cytoplasmique hrGFP diffusible dans les cellules astrogliales, ce qui permet une meilleure détection des prolongements astrocytaires. Les conclusions de ce travail de thèse sont les suivantes : la distance la plus commune entre le récepteur CB1 astroglial et la synapse la plus proche est de 400 à 800 nm. La majorité des synapses entourées par des astrocytes immunopositifs pour le récepteur CB1 dans l’hippocampe, est de nature excitatrice. Les souris mutantes ré-exprimant le récepteur CB1 caractérisées dans ce travail de thèse montrent : 1) l’expression du récepteur CB1 dans différents types cellulaires, 2) la réexpression est limitée à une population neuronale particulière ou aux astrocytes, 3) les niveaux endogènes de récepteurs CB1 sont maintenus dans les différents types cellulaires ré-exprimant le récepteur CB1. De façon générale, ces résultats nous montrent que les souris mutantes ré-exprimant le récepteur CB1 sont d’excellents outils pour l’étude fonctionnelle et translationnelle sur le rôle de ce récepteur dans le cerveau sain ou pathologique<br>The Cannabinoid Type I receptor protein (CB1) expression in the hippocampus of rescue mice modified to express the gene exclusively in specific brain cell types: such as dorsal telencephalic glutamatergic neurons, or GABAergic neurons have been analyzed. Furthermore, aiming at knowing the exact anatomical distribution of the astroglial CB1 receptors with respect to the excitatory and inhibitory synapses, the CB1 receptor expression in astrocytes of mouse expressing CB1 receptor only in astrocytes and mutant mouse expressing the protein hrGFP into astrocytes (that allows for better detection of the astrocytic processes) have been also investigated. The results showed that the majority of the hippocampal synapses surrounded by CB1 receptor immunopositive astrocytes in the 400-800 nm range are of excitatory nature. Moreover, the CB1 receptor rescue mutant mice characterized in this Doctoral Thesis have proven 1) to express CB1 receptors in specific brain cell types; 2) the re-expression is limited to the particular brain cell populations; 3) the endogenous levels of CB1 receptors are maintained in the brain cell types re-expressing the receptor. Which makes this mutant mice excellent tools for functional and translational investigations on the role of the CB1 receptors in the normal and diseased brain
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Porter, Iain Malcolm. "Characterisation of the herpes simplex virus type 1 mutant, ambUL12." Thesis, University of Glasgow, 2002. http://theses.gla.ac.uk/3959/.
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The herpes simplex virus type 1 (HSV-1) UL12 gene encodes an alkaline nuclease. Although the UL12 gene is not absolutely essential for replication, UL12 null mutants produce 100-1000 fold less viable virus than wt HSV-1. It has been suggested that the alkaline nuclease functions to remove branched structures from high molecular weight concatemeric DNA prior to its cleavage into monomeric genomes that are packaged into viral capsids. Failure to remove the branches results in unstable packaging of DNA into capsids which fail to egress from the nucleus. This thesis describes detailed characterisation of the HSV-1 mutant, ambUL12 (Patel et al., 1996) which fails to express the alkaline nuclease due to the insertion of an amber stop codon into the UL12 open reading frame. The ability of ambUL12 to replicate and package both viral genomic DNA and amplicons (plasmids containing the HSV-1 origin of replication and DNA encapsidation signal) was examined. In contrast to results obtained with other alkaline nuclease mutants, which replicate and package DNA with close to wt HSV-1 efficiency (Shao et al., 1993; Martinez et al., 1996b), ambUL12 displayed a 3-5 fold drop in replication and a 15-20 fold drop in packaging of genomic DNA. Similar reductions were observed in the replication and packaging of amplicon DNA. The replication and packaging of amplicons by ambUL12 in these transient assays could be partially complemented when UL12 was supplied in trans. Close inspection of the DNA molecules synthesised during transient assays demonstrated that amplicon replication intermediates are complex high molecular weight concatemers that undergo intermolecular recombination, analogous to viral DNA replication intermediates.
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Roberts, Gethin. "Conditional expression of wild-type and mutant AtCDKA;1 in plants." Thesis, University of East Anglia, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.405910.
Full textBooks on the topic "Mutants de type-1"
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Arnold, J. Douglas. Awesome Super Nintendo Secrets. Sandwich Islands Publishing, 1992.
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Nick, Roberts. Super NES Games: Unauthorized Power Tips Book. Prima Publishing, 1993.
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Daksis, Jasmine Ilga *. Biochemical and genetic characterization of temperature-sensitive mutants of herpes simplex virus type 1 defective in the shutoff of cellular macromolecular synthesis. 1987.
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Sybert, Virginia P. Disorders of Pigmentation. Oxford University Press, 2012. http://dx.doi.org/10.1093/med/9780195397666.003.0004.
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Hyperpigmentation – Carney Complex – Dowling-Degos Disease – Dyskeratosis Congenita – Fanconi Anemia – Hemochromatosis – Incontinentia Pigmenti – LEOPARD Syndrome – Linear and Whorled Nevoid Hypermelanosis – McCune-Albright Syndrome – Naegeli Syndrome – Neurofibromatosis – Nevus Phakomatosis Pigmentovascularis – Peutz-Jeghers Syndrome – Universal Melanosis – Hypopigmentation – Albinisms – Albinism with Deafness – Hermansky-Pudlak Syndrome – Oculocutaneous Albinism Tyrosinase Negative – Oculocutaneous Albinism Tyrosinase Positive – Yellow Mutant Albinism – Cross Syndrome – Hypomelanosis of Ito – Piebaldism – Premature Canities – Vitiligo – Waardenburg Syndrome Types 1, 2, 3, and 4
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Sybert, Virginia P. Disorders of Pigmentation. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780190276478.003.0004.
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Chapter 4 covers Hyperpigmentation (including Carney Complex, Dowling-Degos Disease, Dyskeratosis Congenita, Fanconi Anemia, H Syndrome, Hemochromatosis, Incontinentia Pigmenti, LEOPARD Syndrome, Linear and Whorled Nevoid Hypermelanosis, McCune-Albright Syndrome, Naegeli Syndrome, Neurofibromatosis, Nevus Phakomatosis Pigmentovascularis, Peutz-Jeghers Syndrome, and Universal Melanosis) and Hypopigmentation ( Albinisms, Albinism with Deafness, Hermansky-Pudlak Syndrome, Oculocutaneous Albinism Tyrosinase Negative, Oculocutaneous Albinism Tyrosinase Positive, Yellow Mutant Albinism, Cross Syndrome, Hypomelanosis of Ito, Piebaldism, Premature Canities, Vitiligo, and Waardenburg Syndrome Types 1, 2, 3, and 4). Each condition is discussed in detail, including dermatologic features, associated anomalies, histopathology, basic defect, treatment, mode of inheritance, prenatal diagnosis, and differential diagnosis.
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Desroches, Julie. Peripheral analgesia involves cannabinoid receptors. Edited by Paul Farquhar-Smith, Pierre Beaulieu, and Sian Jagger. Oxford University Press, 2018. http://dx.doi.org/10.1093/med/9780198834359.003.0034.
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This landmark paper by Agarwal and colleagues was published in 2007, when the exact contribution of the activation of the cannabinoid type 1 receptor (CB1) receptors expressed on the peripheral terminals of nociceptors in pain modulation was still uncertain. At that time, while it was clearly demonstrated that the central nervous system (CNS) was involved in the antinociceptive effects induced by the activation of the CB1 receptor, many strains of mice in which the gene encoding the CB1 receptor was deleted by conditional mutagenesis were used to study the specific role of these receptors in pain. Creating an ingenious model of genetically modified mice with a conditional deletion of the CB1 receptor gene exclusively in the peripheral nociceptors, Agarwal and colleagues were the first to unequivocally demonstrate the major role of this receptor in the control of pain at the peripheral level. In fact, these mutant mice lacking CB1 receptors only in sensory neurons (those expressing the sodium channel Nav1.8) have been designed to highlight that CB1 receptors on nociceptors, and not those within the CNS, constitute an important target for mediating local or systemic (but not intrathecal) cannabinoid analgesia. Overall, they have clarified the anatomical locus of cannabinoid-induced analgesia, highlighted the potential significance of peripheral CB1-mediated cannabinoid analgesia, and revealed important insights into how the peripheral endocannabinoid system works in controlling both inflammatory pain and neuropathic pain.
Book chapters on the topic "Mutants de type-1"
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Levine, Myron, David J. Fink, Ramesh Ramakrishnan, Prashant Desai, William F. Goins, and Joseph C. Glorioso. "Neurovirulence of Herpes Simplex Virus Type 1 Accessory Gene Mutants." In Pathogenicity of Human Herpesviruses due to Specific Pathogenicity Genes. Springer Berlin Heidelberg, 1994. http://dx.doi.org/10.1007/978-3-642-85004-2_13.
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Havekes, L., E. Klasen, and G. Utermann. "Apoprotein E3-Leiden: A Variant of Human Apolipoprotein E3 Associated with Familial Type III Hyperlipoproteinemia." In Human Apolipoprotein Mutants. Springer US, 1986. http://dx.doi.org/10.1007/978-1-4615-9474-1_26.
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Gabelli, C., G. Baggio, A. Pagnan, et al. "Identification of an Italian Kindred with a Variant Apolipoprotein E (E1) Associated with Type III Hyperlipoproteinemia." In Human Apolipoprotein Mutants 2. Springer US, 1989. http://dx.doi.org/10.1007/978-1-4615-9549-6_23.
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Seedorf, U., K. N. Sastry, and S. K. Karanthanasis. "Cis-Acting Elements and Trans-Acting Factors Involved in Cell Type Specific Expression of the Human Apoliprotein AI Gene." In Human Apolipoprotein Mutants 2. Springer US, 1989. http://dx.doi.org/10.1007/978-1-4615-9549-6_4.
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Martin, J. P., F. Koehren, A. Bingen, and A. Kirn. "Characterization of Temperature-Sensitive Mutants Mouse Hepatitis Virus Type 3." In Coronaviruses. Springer US, 1987. http://dx.doi.org/10.1007/978-1-4684-1280-2_33.
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Barman, Bandana, and Anirban Mukhopadhyay. "Detection of Differentially Expressed Genes in Wild Type HIV-1 Vpr and Two HIV-1 Mutant Vprs." In Advances in Intelligent Systems and Computing. Springer International Publishing, 2015. http://dx.doi.org/10.1007/978-3-319-11933-5_67.
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Osei-Owusu, Patrick, Matthew L. Nilles, David S. Bradley, and Travis D. Alvine. "A Method for Characterizing the Type III Secretion System’s Contribution to Pathogenesis: Homologous Recombination to Generate Yersinia pestis Type III Secretion System Mutants." In Methods in Molecular Biology. Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-6649-3_13.
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Ishikawa, K., T. Suzuki, T. Funai, K. Nishiyama, C. Uchida, and A. Ichiyama. "A liver enzyme, serine:pyruvate/alanine:glyoxylate aminotransferase and its mutant in a primary hyperoxaluria type 1 case." In Biochemistry of Vitamin B6 and PQQ. Birkhäuser Basel, 1994. http://dx.doi.org/10.1007/978-3-0348-7393-2_53.
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Suzuki, Toshiaki, Kozo Nishiyama, Tsuneyoshi Funai, Keiji Tanaka, Akira Ichihara, and Arata Ichiyama. "Energy-Dependent Degration of a Mutant Serine:Pyruvate/Alanin: Glyoxylate Aminotransferase in a Primary Hyperoxaluria Type 1 C." In Intracellular Protein Catabolism. Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0335-0_16.
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Toutant, Jean-Pierre, and Martine Arpagaus. "Quaternary Structure and Hydrophobic Interactions of Drosophila Acetylcholinesterase in Wild Type Flies and in Mutants of the Ace Locus." In Insect Neurochemistry and Neurophysiology · 1989 ·. Humana Press, 1990. http://dx.doi.org/10.1007/978-1-4612-4512-4_15.
Full textConference papers on the topic "Mutants de type-1"
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Rånby, M., A. Brändstrôm, L. Hansen, K. Henson, and G. Larsen. "REC. t-PA GENETICALLY MODIFIED AT THE CLEAVAGE SITE OF ONE-CHAIN TO TWO-CHAIN CONVERSION: ENZYMOLOGY AND DIAGNOSTIC APPLICATIONS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644410.
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Native one-chain t-PA is cleaved by plasmin or by trypsin after the Arg in the sequence -Gln-Phe-Arg-Ile-Lys-. Variants of one-chain t-PA where the -Arg- was replaced by a His (Arg to His) or by a Lys (Arg to Lys) or by a Thr (Arg to Thr) were made through genetic modification. The three mutants and the wild type were expressed in animal cells and purified in the one-chain form by affinity chromatography as was t-PA from Bowes melanoma cells. In contrast to wild type and melanoma t-PA the mutants reacted poorly with polyclonal antibodies raised against the peptide -Gln-Pro-Gln-Phe-Arg-Ile-Lys--Gly-Gly- indicating mutation in the sequence. Of these proteins only the Arg to Thr mutant was resistant to plasmin cleavage as evidenced by SDS-PAGE. t-PA antigen values (ELISA) and fibrinolytic activity values (fibrin clot lysis assay) yielded the following specific activities expressed in IU/|μg: 810 (Arg to His), 640 (Arg to Lys), 290 (Arg to Thr), 810 (wild type) and 660 (melanoma t-PA). The amidolytic activities for the one-chain proteins against D-Ile-Pro-Arg-pNA at pH 9.0 and 37°C, expressed in mOD per minute at 1 M-g/mL of enzyme were: 15.8 (Arg to His), 13.6 (Arg to Lys), 8.3 (Arg to Thr), 10.0 (wild type), 9.6 (melanoma t-PA) as compared to 55.2 for two-chain melanoma t-PA.All mutants including the uncleavable Arg to Thr mutant could be used in determination of PAI activity in plasma samples. Only one-chain t-PA reacts selectively with PAI 1. Thus, use of the Arg to Thr mutant represents a theoretical advantage in PAI 1 activity determination since preparations of this mutant most likely is free of contaminating two-chain t-PA.The plasminogen activation rate as measured in a coupled assay in the presence and absence of fibrin at 0.5 jiM plasminogen and 37°C was measured and the stimulation factor calculated. This was about 950 fold for the Arg to Thr mutant wich was considerably higher than that of melanoma one chain t-PA and the other mutants wich all were about 550 fold. The stimulation factor for melanoma two-chain t-PA was in the same experiment about 120 fold. The extra fibrin sensitivity of the Arg to Thr mutant resulted in improved soluble fibrin assay according to Wiman and Renby Thromb. Haemostas, (1986) 55:189-193.In conclusion: the use of a plasmin insensitive protein-engineered mutant of t-PA gives advantages in assays for PAI 1 and soluble fibrin.
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Pannekok, H., A. J. Van Zonneveid, C. J. M. de vries, M. E. MacDonald, H. Veerman, and F. Blasi. "FUNCTIONAL PROPERTIES OF DELETION-MUTANTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643724.
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Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (trp) operons,and the detailed establishment of the structure and function of the repressor (lacl) of the lac operon. Recombinant DNA techniques and the development of appropriate expression systems have provided the means both to study structure and functionof eukaryotic (glyco-) proteins and to create defined mutations with a predestinedposition. The rationale for the construction of mutant genes should preferentiallyrely on detailed knowledge of the three-dimensional structure of the gene product.Elegant examples are the application of in vitro mutagenesis techniques to substitute amino-acid residues near the catalytic centre of subtilisin, a serine proteasefrom Bacillus species and to substituteanamino acid in the reactive site (i.e. Pi residue; methionine) of α-antitrypsin, a serine protease inhibitor. Such substitutions have resulted into mutant proteins which are less susceptible to oxidation and, in some cases, into mutant proteins with a higher specific activity than the wild-type protein.If no data are available on the ternary structure of a protein, other strategies have to be developed to construct intelligent mutants to study the relation between the structure and the function of a eukaryotic protein. At least for a number of gene families, the gene structure is thought to be created by "exon shuffling", an evolutionary recombinational process to insert an exon or a set of exons which specify an additional structural and/or functional domain into a pre-existing gene. Both the structure of the tissue-type plasminogen activator protein(t-PA) and the t-PA gene suggest that this gene has evolved as a result of exon shuffling. As put forward by Gilbert (Science 228 (1985) 823), the "acid test"to prove the validity of the exon shuffling theory is either to delete, insert or to substitute exon(s) (i.e. in the corresponding cDNA) and toassay the properties of the mutant proteins to demonstrate that an exon or a set of adjacent exons encode (s) an autonomousfunction. Indeed, by the construction of specific deletions in full-length t-PA cDNA and expression of mutant proteins intissue-culture cells, we have shown by this approach that exon 2 of thet-PA gene encodes the function required forsecretion, exon 4 encodes the "finger" domain involved in fibrin binding(presumably on undegraded fibrin) and the set of exons 8 and 9 specifies kringle 2, containing a lysine-binding sit(LBS) which interacts with carboxy-terminal lysines, generated in fibrin after plasmic digestion. Exons 10 through 14 encode the carboxy-ter-minal light chain of t-PA and harbor the catalytic centre of the molecule and represents the predominant "target site" for the fast-acting endothelial plasminogen activator inhibitor (PAI-1).As a follow-up of this genetic approach to construct deletion mutants of t-PA, we also created substitution mutants of t-PA. Different mutants were constructed to substitute cDNA encoding thelight chain of t-PA by cDNA encoding the B-chain of urokinase (u-PA), in order to demonstrate that autonomous structural and functional domains of eitherone of the separate molecules are able toexert their intrinsic properties in a different context (C.J.M. de Vries et al., this volume). The possibilities and the limitations of this approach to study the structure and the function of t-PA and of other components of the fibrinolytic process will be outlined.
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Nelles, L., H. R. Linjnen, E. Demarsin, D. Collen, and W. E. Holmes. "CHARACTERIZATION OF SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR MUTANTS PRODUCED BY SITE-SPECIFIC MUTAGENESIS OF LYS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642909.
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A cDNA encoding full length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced. The recombinant scu-PA (rscu-PA) cDNA as well as the cDNA of two mutants constructed by deoxyoligonucleotide directed mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158 ) or to Glu158 (rscu-PA-Glu158 ) were inserted into SV40 early promoter/enhancer based expression vectors, which were used to transfect Chinese Hamster Ovary (CHO) cells. The expression products were purified from serum-free conditioned media by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration.The amidolytic activity of the three rscu-PAs was low (< 500 IU/mg). The mutant rscu-PAs, in contrast to the rscu-PA and nscu-PA, could not be converted into an amidolytically active two-chain form (tcu-PA) by plasmin. The mutant scu-PAs had a very low specific activity (< 1,000 IU/mg) on fibrin plates, whereas wild type rscu-PA had a specific activity < 1000 IU/mg. The mutant scu-PAs did not cause lysis of a I-fibrin labeled plasma clot immersed in citrated human plasma. Serum-free medium from a control transfected CHO cell line showed no significant plasminogen activating activity.In a purified system, both rscu-PA-Gly and rscu-PA-Glu activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (K = 60-80 yM) but with a higher catalytic rate constant (k2 = B.01 s-1) as compared to the wild type rscu-PA (K =1.0 yM, k = 0.002 s-1).It is concluded thaz conversion of scu-PA to tcu-PA is prerequisite for the activation of plasminogen. However, Lys158 seems to be important for the stability of the Michaelis complex between scu-PA and plasminogen.
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Haigwood, N., E.-P. Pâques, G. Mullenbach, G. Moore, L. DesJardin, and A. Tabrizi. "IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643841.
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The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.
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Maisch, Tim, Pouriya Faraj Tabrizi, and Sara Wennige. "Mutants of E. coli, which lack antioxidant enzymes, are more susceptible towards type-1 mechanism of action of photoantimicrobials compared to type-2 photoantimicrobials (Conference Presentation)." In 17th International Photodynamic Association World Congress, edited by Tayyaba Hasan. SPIE, 2019. http://dx.doi.org/10.1117/12.2525799.
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Furis, B. C., M. J. Jorgensem, M. J. Rabiet, et al. "RECOGNITION SITE DIRECTING GAMMA-CARBOXYLATION RESIDES ON THE PROPEPTIDES OF FACTOR IX AND PROTRROMBIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643992.
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Factor IX and prothrombin vitamin K-dependent proteins that participate in blood coagulation undergo post-translationalmodification in which glutamic acid residues in the amino terminus of the protein are converted to gamma-carboxyglutamic acid residues. This modification confers divalent metal ion binding ability upon the proteins.As a consequence of binding divalent metal ions these proteins undergoconformational changes necessary for biological function.The vitamin K-dependent proteins are synthesized with an NH2-terminal extension. The region distal to the NH2-terminus of the mature protein is a prototypic signal sequence while the proximal region is a propeptide with homology among the vitamin K-dependent proteins. The boundary between the pre and pro sequences has been established for factor IX by analysis of three naturally occurring factor IX mutants factor IX Cambridge factor IX Oxford-3 and factor IX San Dimas, in which processing is incomplete.For human factor IX the propeptide extends from residue -18 to -1. The homology among the propeptides of vitamin K-dependent proteins suggests that the propeptide may designate adjacent gamma-carboxyglutamic acids for carboxylation. To test this hypothesis alterations in sequence were introduced into the propeptide region of human factor IX cDNA by oligonucleotide directed site specific mutagenesis.Mutated genes were expressed in Chinese hamster ovary cells. Rapid and efficient isolationof the mutant proteins by immunoaffinity chromatography permitted detailed analysis of the mutants on quantities of protein easily obtainable at low expression levels. The extent of gamma-carboxylation was assessed by the ability of the mutant proteins to interact with conformation specific antibodies directed against the gamma-carboxyglutamic acid-dependent metal stabilized native structure of factor IX as well as by direct amino acid analysis. Unmodified recombinant factor IX contained, on average, 9 gamma-carboxyglutamic acid residues, as compared to 12 for plasma factor IX. About 70% of the recombinant wild type factor IX bound to the conformation specific antibodies. Deletion of the propiece or point mutations at residues -10 or -16 led to secretion of uncarboxylated factor IX unreaotive with antibodies specific for the native structure but with the NH2-terminus of mature factor IX. In order to assess the universality of these observations we have recently cloned human prothrombin cDNA and expressed the gene in the same Chinese hamster ovary cell system used for factor IX. In contrast to factor IX, at low levels ofexpressionof the prothrombin gene, the prothrombin is fully carboxylated relative to a plasma prothrombin standard.The recombinant prothrombin exhibits the same specific clotting activity as plasma derivedprothrombin and is fully native as evaluated by conformation specific antibodies. At high levels of expression the capacityof the cells to carboxylate prothrombin can be exceeded leading to secretion of under carboxylated prothrombin. However, the absolute amount of fully carboxylated prothrombin that can be produced in this system appears to be a least fivefold greater that the absolute amount of highly carboxylated factor IX that can be synthesized.The elimination of carboxylation observed upon mutation of the propiece of factor IX suggest that the propiece contains a recognition element required for carboxylation of the protein. Assignment of a functional role to the propiece of factor IX represents the first determination of function for any pro sequence. It is anticipated that extension of these studies to prothrombin will demonstrate that this recognition signal is used by all the members of this class of proteins. In order to determine if the propiece is sufficient to designate a protein for gamma-carboxylation we are currently constructing chimeric proteins incorporating the propieceof prothrombin into the cDNA of normally uncarboxylated proteins.
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Higgins, Deborah L., and William E. Holmes. "CHARACTERIZATION OF RECOMBINANT HUMAN TISSUE-TYPE PLASMINOGEN ACTIVATOR MISSING THE FINGER DOMAIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643842.
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Site-specific mutagenesis was used to produce a mutant form of tissue-type plasminogen activator (t-PA) which was missing the first 44 amino acids. This domain has sequence homology with the type 1 regions in proteins such as fibronectin and is commonly called the finger domain. The mutant protein (des 1-44 t-PA) was expressed in Chinese Hampster Ovary cells, and was purified using chromatography on Zn-chelate sepharose and lysine-sepharose. Sequence analysis indicated that the resulting protein was homogeneous and started at amino acid 45 in the sequence of the normal protein. The two-chain forms of both des 1-44 t-PA and normal sequence t-PA exhibited similar kinetic constants with a small synthetic substrate (H-D-Isoleucyl-L- prolyl-L-arginyl-p-nitroani 1 ide). The ability of des 1-44 t-PA to activate plasminogen was decreased to 70% of the rate of normal t-PA. The rate of plasminogen activation by normal t-PA was stimulated 51-fold in the presence of fibrin, whereas with des 1-44 t-PA it was stimulated only 40-fold. Although des 1-44 t-PA bound to lysine-agarose, little (if any) binding was observed to either intact or degraded fibrin indicating that fibrin stimulation is due in part to the ability of t-PA to recognize plasminogen bound to fibrin as a preferable substrate. The mutant t-PA was capable of forming complexes in vitro with all of the inhibitors in blood which react with normal sequence t-PA. The rate of reaction with α2-macroglobulin, however, was slower with des 1-44 t-PA than with normal sequence t-PA. The similar resistance of des 1-44 t-PA and normal sequence t-PA to proteolysis and the ability to react with a battery of monoclonal antibodies suggests that the deletion did not cause perturbed folding, but rather that alterations in function of des 1-44 t-PA were due to the lack of the finger domain.
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Collen, D. "SYNERGISM, MUTANTS AND HYBRIDS OF TISSUE-TYPE PLASMINOGEN ACTIVATO(t-PA) AND SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR(scu-PA):POTENTIAL FORTHROMBOLYTIC THERAPY." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643725.
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With the development and clinical investigation of the fibrin-specific thrombolytic agents t-PA and scu-PA, many questions relating to their optimal use remain to be answered. It is, however, becoming apparent that these agents, in addition to several advantages,suffer some shortcomings, e.g. their therapeutic dose is large and their fibrin-specificity is limited.Therefore,the quest for better thrombolytic agents remains open.We will report results of four main lines of research which we are pursuing to obtain better agents or regimens for fibrin-specific thrombolytic therapy.1. Synergism between t-PA and scu-PA. t—PA and scu—PA in molar ratios between 4:1 and 1:4 show no synergism for thrombolysis of a plasma clot immersed in plasma in vitro(Thromb. Haemost. 56, 35, 1986) but display significant in vivo synergismin a rabbit model (Circulation 74, 838, 19867and in man (Am. Heart J. 112, 1083,1986).Recently we have confirmed synergism for thrombolysis between t-PA and scu-PA in a coronary thrombosis model in the dog(Zuskind et al., unpubl.) and in the baboon (Collen et al., unpubl.). Sequential infusion of t-PA followed by scu-PA butnot of scu-PA followed by t-PA is syneristic(Collen et al., this meeting).2. Mutants of t-PA. In collaboration with Larssen et al.,deletion mutants of t-PA, obtained by in vitro mutagenesis are characterized with respect to pharmacokinetics and thrombolytic properties.Mutants lacking the finger—like domain and/or the growth factor domainand/or one or all of the glycosylation sites have a much slower clearance (Larssenet al., this meeting) but unaltered specific thrombolytic properties and fibrin-specificity (Collen et al., this meeting).3. Mutants of scu-PA. A truncated form of scu-PA, lacking the 143 NH2~terminal amino acids was shown to be pharmacologically and thrombolytically indistinguishable from intact scu—PA (Stump et al.).Mutants of scu—PA in which Lys 158 is replaced,whereby they can no longer be converted to urokinase, still haveintrinsic plasminogen activating properties and act synergistically with t-PA on thrombolysis in vivo (Nelles et al., this meeting).4. Hybrids of t-PA and scu-PA. In collaboration with Pierard et al. (this meeting) hybrids of NH2~terminal regions of t-PA and COOH-terminal regions of u-PA were constructed which, after translation in transient expression systems, showed apparent specific activities comparable to that of natural two-chain u-PA. One hybrid, composed of the finger domain of t-PA and the B-chain of u-PA, was scaled up, purified and characterized (Gheysen et al., this meeting). This hybrid had theenzymatic properties typical of single chain u-PA, but had not acquired the fibrinaffinity of t-PA.Based on the finding that the isolated A-chain of t-PA retains the intact fibrin-affinity of the native molecule (Holvoet et al.,Eur.J. Biochem. 158, 173, 1986) andthat a low Mr form of scu-PA retains the functional properties of the intact moleule (J. Biol. Chem. 261, 17120, 1986), we have constructed and expressed a hybrid consisting of the NH -terminal region of t-PA (amino acids 1 to 263) and the COOH-terminal region of scu-PA (amino acids 144to 411) (Lijnen et al., this meeting). This hybrid has both fibrin affinity of t-PA (although less pronounced) and the enzymatic properties of scu-PA. The activation of plasminogen by the hybrid is apparently stimulated by fibrin.We believe that continued research along these lines will yield thrombolytic agents or therapeutic schemes, which may be superior to t-PA and/or scu-PA in terms of specific thrombolytic activity and fibrin-specificity.
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Chen, Chia-Yuan, Michael J. Patrick, Paola Corti, David Frakes, Beth L. Roman, and Kerem Pekkan. "In Vivo Hemodynamic Performance of Wild-Type vs. Mutant Zebrafish Embryos Using High-Speed Confocal Micro-PIV." In ASME 2010 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2010. http://dx.doi.org/10.1115/sbc2010-19317.
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In developing cardiovascular systems, definite performance comparison between disease and healthy hemodynamics requires quantitative tools to support advanced microscopy. Mutations in the activin receptor-like kinase 1 (ALK1) gene are responsible for the autosomal dominant vascular disease, hereditary hemorrhagic telangiectasia type 2 (HHT2), characterized by high flow arteriovenous malformations (AVMs) [1]. Recent studies show that the zebrafish mutant violet beauregrade (vbg), which harbors a mutation in alk1, develops an abnormal circulation with dilated cranial vessels and AVMs [2]. Quantitative understanding of mechanical influences on the alk1 mutant phenotype will aid treatment of HHT2 patients. Inspired by earlier studies that demonstrate the capability of using confocal micro-PIV technique to quantify biofluid dynamics in vivo [3], primarily in major vessels (dorsal aorta, vitelline veins), the present study focused on secondary branching great vessels of zebrafish embryos where microcirculation flow regimes are different. Furthermore, confocal microscopy, essentially being an imaging modality, requires rigorous validation efforts with respect to the gold standard measurement protocols (such as PIV) and synthetic scan data. Another objective of this work was to document the intra-species differences of wall shear stress (WSS) and flow physics during embryonic development in aortic arch systems of zebrafish [4].
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Savichenko, D. L., and S. Z. Guchetl. "THE PROBLEM OF DETECTION OF HETEROZYGOUS GENOTYPES BY MUTATION OF THE FAD 2-1 GENE OF SUNFLOWER (HELIANTHUS ANNUUS L.) USING DNA MARKERS." In 11-я Всероссийская конференция молодых учёных и специалистов «Актуальные вопросы биологии, селекции, технологии возделывания и переработки сельскохозяйственных культур». V.S. Pustovoit All-Russian Research Institute of Oil Crops, 2021. http://dx.doi.org/10.25230/conf11-2021-102-106.
Full textAbstract:
The development of marker-associated breeding requires the development of DNA marker systems that meet the needs of the breeding process. We are looking for the opportunities to improve the efficiency of breeding for high oleic acid content in sunflower oil. One of the directions is the improvement of the existing marker system for detection the heterozygous status of the alleles of the FAD 2-1 gene. We carried out the search for information published in databases on this DNA locus. We studied the structure of the nucleotide sequences of the mutant allele and the wild-type allele. We proposed the way of improving the existing marker system.