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  1. Article ; Online: The F309S mutation increases factor VIII secretion in human cell line

    Daianne Maciely Carvalho Fantacini / Aparecida Maria Fontes / Mário Soares de Abreu Neto / Dimas Tadeu Covas / Virgínia Picanço-Castro

    Revista Brasileira de Hematologia e Hemoterapia, Vol 38, Iss 2, Pp 135-

    2016  Volume 140

    Abstract: ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate) on the secretion of ... ...

    Abstract ABSTRACT OBJECTIVES: The capacity of a human cell line to secrete recombinant factor VIII with a F309S point mutation was investigated, as was the effect of the addition of chemical chaperones (betaine and sodium-4-phenylbutyrate) on the secretion of factor VIII. METHODS: This work used a vector with a F309S mutation in the A1 domain to investigate FVIII production in the HEK 293 human cell line. Factor VIII activity was measured by chromogenic assay. Furthermore, the effects of chemical drugs on the culture were evaluated. RESULTS: The addition of the F309S mutation to a previously described FVIII variant increased FVIII secretion by 4.5 fold. Moreover, the addition of betaine or sodium-4-phenylbutyrate increased the secretion rate of FVIIIΔB proteins in HEK 293 cells, but the same effect was not seen for FVIIIΔB-F309S indicating that all the recombinant protein produced had been efficiently secreted. CONCLUSION: Bioengineering factor VIII expressed in human cells may lead to an efficient production of recombinant factor VIII and contribute toward low-cost coagulation factor replacement therapy for hemophilia A. FVIII-F309S produced in human cells can be effective in vivo.
    Keywords HEK 293 cells ; Recombinant factor VIII ; F309S mutation ; Betaine ; Sodium-4-phenylbutyrate ; Internal medicine ; RC31-1245 ; Medicine ; R ; Diseases of the blood and blood-forming organs ; RC633-647.5
    Subject code 306
    Language English
    Publishing date 2016-06-01T00:00:00Z
    Publisher Sociedade Brasileira de Hematologia e Hemoterapia
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  2. Article ; Online: Hemofilia B sob um olhar panorâmico

    Andrielle de CASTILHO-FERNANDES / Alícia Greyce Turatti PESSOLATO / Aparecida Maria FONTES

    Revista da Universidade Vale do Rio Verde, Vol 10, Iss 1, Pp 275-

    2012  Volume 289

    Abstract: Hemophilia B is a hereditary disease linked to chromosome X and consists in the deficiency of factor IXblood clotting. This hemorrhagic disease affects one in 30,000 men worldwide. The level of biologically active FactorIX in the patients’ plasma is ... ...

    Abstract Hemophilia B is a hereditary disease linked to chromosome X and consists in the deficiency of factor IXblood clotting. This hemorrhagic disease affects one in 30,000 men worldwide. The level of biologically active FactorIX in the patients’ plasma is directly related to the severity and frequency of bleeding episodes. Since the advent ofrecombinant DNA technology, it was feasible to clone the gene for factor IX, which made possible its molecularcharacterization. Knowledge of the molecular basis of hemophilia B has allowed a better understanding of therelationships between factor IX gene structure and active Factor IX protein function. The existing treatment is given bybiological replacement therapy, through intravenous infusions of Factor IX derived from human plasma or recombinantFactor IX. This article aims to summarize the knowledge about the molecular basis of hemophilia B (gene, RNA, andprotein), the cascade of blood clotting, the process of development of recombinant Factor IX and the situation inworldwide and Brazil in face of the current treatment and the future prospects of hemophilia B.
    Keywords Hemophilia B ; Factor IX ; molecular biology ; blood coagulation and therapeutics. ; General Works ; A
    Subject code 306
    Language English
    Publishing date 2012-01-01T00:00:00Z
    Publisher Universidade Vale do Rio Verde
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  3. Article ; Online: Murine leukemia virus-derived retroviral vector has differential integration patterns in human cell lines used to produce recombinant factor VIII

    Marcela Cristina Correa de Freitas / Aparecida Maria Fontes / Andrielle de Castilho Fernandes / Virginia Picanço-Castro / Elisa Maria de Sousa Russo / Dimas Tadeu Covas

    Revista Brasileira de Hematologia e Hemoterapia, Vol 36, Iss 3, Pp 213-

    2014  Volume 218

    Abstract: OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified ... ...

    Abstract OBJECTIVE: Nowadays recombinant factor VIII is produced in murine cells including in Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK). Previous studies, using the murine leukemia virus-derived retroviral vector pMFG-FVIII-P140K, modified two recombinant human cell lines, HepG2 and Hek293 to produce recombinant factor VIII. In order to characterize these cells, the present study aimed to analyze the integration pattern of retroviral vector pMFG-FVIII-P140K.METHODS: This study used ligation-mediated polymerase chain reaction to locate the site of viral vector integration by sequencing polymerase chain reaction products. The sequences were compared to genomic databases to characterize respective clones.RESULTS: The retroviral vector presented different and non-random profiles of integration between cells lines. A preference of integration for chromosomes 19, 17 and 11 was observed for HepG2FVIIIdB/P140K and chromosome 9 for Hek293FVIIIdB/P140K. In genomic regions such as CpG islands and transcription factor binding sites, there was no difference in the integration profiles for both cell lines. Integration in intronic regions of encoding protein genes (RefSeq genes) was also observed in both cell lines. Twenty percent of integrations occurred at fragile sites in the genome of the HepG2 cell line and 17% in Hek293.CONCLUSION: The results suggest that the cell type can affect the profile of chromosomal integration of the retroviral vector used; these differences may interfere in the level of expression of recombinant proteins.
    Keywords Factor VIII ; Virus integration ; Hemophilia A ; Internal medicine ; RC31-1245 ; Medicine ; R
    Language English
    Publishing date 2014-06-01T00:00:00Z
    Publisher Sociedade Brasileira de Hematologia e Hemoterapia
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  4. Article: Efficient recovery of undifferentiated human embryonic stem cell cryopreserved with hydroxyethyl starch, dimethyl sulphoxide and serum replacement

    Orellana, Maristela Delgado / Gil Cunha De Santis / Kuruvilla Joseph Abraham / Aparecida Maria Fontes / Danielle Aparecida Rosa Magalhães / Viviane de Cássia Oliveira / Everton de Brito Oliveira Costa / Patrícia Vianna Bonini Palma / Dimas Tadeu Covas

    Cryobiology. 2015 Aug., v. 71, no. 1

    2015  

    Abstract: The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing ... ...

    Abstract The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application.Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated.We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05).We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies.
    Keywords blood serum ; cryopreservation ; dimethyl sulfoxide ; embryonic stem cells ; freeze-thaw cycles ; freezing ; genetic stability ; humans ; starch ; storage ; thawing ; therapeutics
    Language English
    Dates of publication 2015-08
    Size p. 151-160.
    Publishing place Elsevier Inc.
    Document type Article
    ZDB-ID 80098-3
    ISSN 1090-2392 ; 0011-2240
    ISSN (online) 1090-2392
    ISSN 0011-2240
    DOI 10.1016/j.cryobiol.2015.01.005
    Database NAL-Catalogue (AGRICOLA)

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  5. Article ; Online: Association between Knops blood group polymorphisms and susceptibility to malaria in an endemic area of the Brazilian Amazon

    Aparecida Maria Fontes / Simone Kashima / Ricardo Bonfim-Silva / Rochele Azevedo / Kuruvilla Joseph Abraham / Sérgio Roberto Lopes Albuquerque / José Orlando Bordin / Dante Mário Langhi Júnior / Dimas Tadeu Covas

    Genetics and Molecular Biology, Vol 34, Iss 4, Pp 539-

    2011  Volume 545

    Abstract: Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the ... ...

    Abstract Complement receptor 1 (CR1) gene polymorphisms that are associated with Knops blood group antigens may influence the binding of Plasmodium parasites to erythrocytes, thereby affecting susceptibility to malaria. The aim of this study was to evaluate the genotype and allele and haplotype frequencies of single-nucleotide polymorphisms (SNPs) of Knops blood group antigens and examine their association with susceptibility to malaria in an endemic area of Brazil. One hundred and twenty-six individuals from the Brazilian Amazon were studied. The CR1-genomic fragment was amplified by PCR and six SNPs and haplotypes were identified after DNA sequence analysis. Allele and haplotype frequencies revealed that the Kn b allele and H8 haplotype were possibly associated with susceptibility to Plasmodium falciparum. The odds ratios were reasonably high, suggesting a potentially important association between two Knops blood antigens (Kn b and KAM+) that confer susceptibility to P. falciparum in individuals from the Brazilian Amazon.
    Keywords Brazilian Amazon population ; CR1 haplotypes ; Knops blood group polymorphism ; malaria ; Genetics ; QH426-470 ; Biology (General) ; QH301-705.5 ; Science ; Q ; DOAJ:Genetics ; DOAJ:Biology ; DOAJ:Biology and Life Sciences
    Subject code 616
    Language English
    Publishing date 2011-01-01T00:00:00Z
    Publisher Sociedade Brasileira de Genética
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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  6. Article ; Online: Clonagem e expressão da glicoproteína transmembrana do retrovírus HTLV-1 em células de mamíferos Cloning and transmembrane glycoprotein expression of the retrovirus HTLV-1 in mammals' cells

    Flora Cristina Lobo Penteado / Luciene Medeiros / Maristela Delgado Orellana / Patricia Palma / Aparecida Maria Fontes / Osvaldo Massaiti Takayanagui / Dimas Tadeu Covas

    Revista da Sociedade Brasileira de Medicina Tropical, Vol 39, Iss 2, Pp 169-

    2006  Volume 173

    Abstract: O retrovírus linfotrópico de células T humanas tipo 1 é o agente etiológico da leucemia das células T do adulto e da paraparesia espástica tropical/mielopatia associada ao HTLV-1. O genoma proviral é composto por 9.032 pares de bases, contendo genes ... ...

    Abstract O retrovírus linfotrópico de células T humanas tipo 1 é o agente etiológico da leucemia das células T do adulto e da paraparesia espástica tropical/mielopatia associada ao HTLV-1. O genoma proviral é composto por 9.032 pares de bases, contendo genes estruturais e regulatórios. A glicoproteína transmembrana gp 21 é codificada pelo gene estrutural env. O desenvolvimento de metodologias para a expressão heteróloga de proteínas, assim como a obtenção de uma linhagem celular que expresse a gp 21 recombinante constitutivamente são os principais objetivos do trabalho. O fragmento codificante da gp 21 foi amplificado por Nested-PCR e clonado no vetor pCR2.1-TOPO. Posteriormente, foi realizada a subclonagem no vetor de expressão pcDNA3.1+. A transfecção da linhagem celular de mamíferos HEK 293 foi realizada de maneira transitória e permanente. A produção da gp 21 recombinante foi confirmada por citometria de fluxo e a linhagem celular produtora será utilizada em ensaios de imunogenicidade. The retrovirus HTLV-1 is the etiological agent of the adult T-cell leukemia and HTLV-1 associated myelopathy/tropical spastic paraparesis. The proviral genome has 9,032 base pairs, showing regulatory and structural genes. The env gene encodes for the transmembrane glycoprotein gp 21. The development of methodologies for heterologous protein expression, as well as the acquisition of a cellular line that constituently expresses the recombinant, were the main goals of this work. The DNA fragment that encodes for gp 21 was amplified by nested-PCR and cloned into a pCR2.1-TOPO vector. After which, a sub-cloning was realized using the expressing vector pcDNA3.1+. The transfection of mammalian cells HEK 293 was performed transitorily and permanently. Production of the recombinant gp 21 was confirmed by flux cytometry experiments and the cell line producing protein will be used in immunogenicity assays.
    Keywords Glicoproteína transmembrana ; HTLV-1 ; Clonagem ; Expressão de proteínas ; Transmembrane glycoprotein ; Cloning ; Protein expression ; Arctic medicine. Tropical medicine ; RC955-962
    Language English
    Publishing date 2006-04-01T00:00:00Z
    Publisher Sociedade Brasileira de Medicina Tropical (SBMT)
    Document type Article ; Online
    Database BASE - Bielefeld Academic Search Engine (life sciences selection)

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