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Sumihiro Hase - One of the best experts on this subject based on the ideXlab platform.
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structural analysis of n linked sugar chains of human Blood Clotting Factor ix
Journal of Biochemistry, 2000Co-Authors: Yasushi Makino, Kaoru Omichi, Nahoki Kuraya, Hideyuki Ogawa, Hitoshi Nishimura, Sadaaki Iwanaga, Sumihiro HaseAbstract:The structures of N-glycans of human Blood Clotting Factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human Factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.
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structural analysis of o linked sugar chains in human Blood Clotting Factor ix
Journal of Biochemistry, 1993Co-Authors: Nahoki Kuraya, Kaoru Omichi, Hitoshi Nishimura, Sadaaki Iwanaga, Sumihiro HaseAbstract:Type and structural analysis of O-linked sugar chains in human Blood Clotting Factor IX was performed by the pyridylamination method developed for O-linked sugar chains [Kuraya, N. & Hase, S. (1992) J. Biochem. 112, 122-126]. O- and N-linked sugar chains were released with hydrazine, and then N-acetylated, followed by pyridylamination. The type of sugar chain was determined by reducing-end analysis of the pyridylaminated (PA-) sugar chains. Sugar chains with PA-GalNAc at the reducing terminal and that with PA-Fuc [Nishimura, H. et al. (1992) J. Biol. Chem. 267, 17520-17525] were obtained besides known sugar chains with PA-Glc from the Xyl-Glc-Ser type and those with PA-GlcNAc from asparagine-linked sugar chains. The sugar chains with PA-GalNAc were identified as mono- and disialyl Gal beta 1-3GalNAc by two-dimensional HPLC mapping. The structure of the sugar chain with PA-Fuc was Neu5Ac alpha 2-6Gal beta 1-4GlcNAc beta 1-3Fuc, as determined by exoglycosidase digestion, methylation analysis, and Smith degradation.
Luke Allen - One of the best experts on this subject based on the ideXlab platform.
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clinical implications of convergent procoagulant toxicity and differential antivenom efficacy in australian elapid snake venoms
Toxicology Letters, 2019Co-Authors: Christina N Zdenek, Bianca Op ,den Brouw, Daniel Dashevsky, Alexandra Gloria, Nicholas J Youngman, Ebony Watson, Patrick Green, Chris Hay, Nathan Dunstan, Luke AllenAbstract:Australian elapid snakes are some of the most venomous snakes in the world and are unique among venomous snakes in having mutated forms of the Blood Clotting Factor X in an activated form (FXa) as a key venom component. In human bite victims, an overdose of this activated Clotting enzyme results in the systemic consumption of fibrinogen due to the large amounts of endogenous thrombin generated by the conversion of prothrombin to thrombin by venom FXa. Within Australian elapids, such procoagulant venom is currently known from the tiger snake clade (Hoplocephalus, Notechis, Paroplocephalus, and Tropidechis species), brown/taipan (Oxyuranus and Pseudonaja species) clade, and the red-bellied black snake Pseudechis porphyriacus. We used a STA-R Max coagulation analyser and TEG5000 thromboelastographers to test 47 Australian elapid venoms from 19 genera against human plasma in vitro. In addition to activity being confirmed in the two clades above, FXa-driven potent procoagulant activity was found in four additional genera (Cryptophis, Demansia, Hemiaspis, and Suta). Ontogenetic changes in procoagulant function was also identified as a feature of Suta punctata venom. Phylogenetic analysis of FX sequences confirmed that snake venom FXa toxins evolved only once, that the potency of these toxins against human plasma has increased in a stepwise fashion, and that multiple convergent amplifications of procoagulant activity within Australian elapid snakes have occurred. CoFactor dependence tests revealed all procoagulant venoms in our study, except those of the tiger snake clade, to be highly calcium-dependent, whereas phospholipid dependence was less of a feature but still displayed significant variation between venoms. Antivenom testing using CSL Tiger Snake Antivenom showed broad but differential cross-reactivity against procoagulant venoms, with P. porphyriacus and S. punctata extremely well neutralised but with Cryptophis, Demansia, and Hemiaspis less well-neutralised. The relative variation was not in accordance to genetic relatedness of the species used in antivenom production (Notechis scutatus), which underscores a fundamental principle that the rapid evolution characteristic of venoms results in organismal phylogeny being a poor predictor of antivenom efficacy. Our results have direct and immediate implications for the design of clinical management plans in the event of snakebite by such lesser known Australian elapid snake species that have been revealed in this study to be as potent as the better studied, and proven lethal, species.
Nahoki Kuraya - One of the best experts on this subject based on the ideXlab platform.
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structural analysis of n linked sugar chains of human Blood Clotting Factor ix
Journal of Biochemistry, 2000Co-Authors: Yasushi Makino, Kaoru Omichi, Nahoki Kuraya, Hideyuki Ogawa, Hitoshi Nishimura, Sadaaki Iwanaga, Sumihiro HaseAbstract:The structures of N-glycans of human Blood Clotting Factor IX were studied. N-Glycans liberated by hydrazinolysis were N-acetylated and the reducing-end sugar residues were tagged with 2-aminopyridine. The pyridylamino (PA-) sugar chains thus obtained were purified by HPLC. Each PA-sugar chain was analyzed by two-dimensional sugar mapping combined with glycosidase digestion. The major structures of the N-linked sugar chains of human Factor IX were found to be sialotetraantennary and sialotriantennary chains with or without fucose residues. These highly sialylated sugar chains are located on the activation peptide of the protein.
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structural analysis of o linked sugar chains in human Blood Clotting Factor ix
Journal of Biochemistry, 1993Co-Authors: Nahoki Kuraya, Kaoru Omichi, Hitoshi Nishimura, Sadaaki Iwanaga, Sumihiro HaseAbstract:Type and structural analysis of O-linked sugar chains in human Blood Clotting Factor IX was performed by the pyridylamination method developed for O-linked sugar chains [Kuraya, N. & Hase, S. (1992) J. Biochem. 112, 122-126]. O- and N-linked sugar chains were released with hydrazine, and then N-acetylated, followed by pyridylamination. The type of sugar chain was determined by reducing-end analysis of the pyridylaminated (PA-) sugar chains. Sugar chains with PA-GalNAc at the reducing terminal and that with PA-Fuc [Nishimura, H. et al. (1992) J. Biol. Chem. 267, 17520-17525] were obtained besides known sugar chains with PA-Glc from the Xyl-Glc-Ser type and those with PA-GlcNAc from asparagine-linked sugar chains. The sugar chains with PA-GalNAc were identified as mono- and disialyl Gal beta 1-3GalNAc by two-dimensional HPLC mapping. The structure of the sugar chain with PA-Fuc was Neu5Ac alpha 2-6Gal beta 1-4GlcNAc beta 1-3Fuc, as determined by exoglycosidase digestion, methylation analysis, and Smith degradation.
Joseph Wallace Carnwath - One of the best experts on this subject based on the ideXlab platform.
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expression of active human Blood Clotting Factor viii in mammary gland of transgenic rabbits
DNA and Cell Biology, 2003Co-Authors: Laszlo Hiripi, Joseph Wallace Carnwath, R. Halter, F Makovics, Maria Baranyi, David L Paul, Zsuzsanna Bosze, Heinrich NiemannAbstract:Human Clotting Factor VIII is probably the largest protein to be expressed to date in the mammary gland of a transgenic animal, and it requires extensive posttranslational modification to achieve full biological activity. The mammary gland specific construct mWAP-hFVIII-MT-I was injected into the pronuclei of rabbit zygotes, and three transgenic offspring were obtained. Founder 385 showed germ-line transmission of a single integrated copy, and a homozygous line was established from this animal. The rhFVIII was transcribed and translated exclusively in the mammary gland. The activity of rhFVIII in the rabbit milk ranged from 5 to 8% of that found in normal human plasma. Results indicate the suitability of the transgenic rabbit mammary gland for rhFVIII production.
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Expression of human Blood Clotting Factor VIII in the mammary gland of transgenic sheep.
Transgenic research, 1999Co-Authors: Holger Niemann, E Lemme, D. Herrmann, Joseph Wallace Carnwath, R. Halter, D. PaulAbstract:By targeting the expression of sequences encoding non-milk proteins to the mammary gland of transgenic farm animals, the organ could serve as a 'bioreactor' for producing pharmacologically active proteins on a large scale. Here we report the generation of transgenic sheep bearing a fusion gene construct with the human Blood Clotting Factor VIII (hFVIII) cDNA under the transcriptional control of a 2.2 kb fragment of the mammary gland specific promoter of the ovine beta-Lactoglobulin (beta-Lac) gene. Six founder animals were generated bearing a hFVIII cDNA construct with the introns of the murine metallothionein (MtI) gene (beta-Lac/hFVIII-MtI). Founders transmitted the transgene in a Mendelian fashion and two transgenic lines were generated. Ten out of 12 transgenic F1-females expressed rhFVIII mRNA in exfoliated mammary epithelial cells isolated from the milk. But only in transgenic F1 ewes 4010 and 603 hFVIII Clotting activity estimated at 4-6 ng/ml was detected in defatted milk. Furthermore, the presence of rhFVIII-protein in ovine milk was demonstrated by a specific band at approximately 190 kD following immunoprecipitation and immunoblotting. Transgenic founder 395 expressed rhFVIII mRNA in biopsied mammary gland tissue, in exfoliated mammary cells as well as ectopically in brain, heart, spleen, kidney and salivary gland, suggesting that the employed beta-Lac promoter fragment lacks essential sequences for directing expression exclusively to the mammary gland. A rhFVIII standard preparation (rhFVIIIstd) was rapidly sequestered in a saturable fashion in ovine milk, thus rendering it largely inaccessible to immunoprecipitation although its biological activity was retained. Recovery of hFVIIIstd was dependent on milk donor, storage temperature and dilution of milk sample.
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expression of human Blood Clotting Factor viii fviii constructs in the mammary gland of transgenic mice and sheep
Journal of Animal Breeding and Genetics, 1996Co-Authors: Heiner Niemann, E Lemme, D. Herrmann, Joseph Wallace Carnwath, R. Halter, G Espanion, C Wrenzycki, D. PaulAbstract:Summary The aim of this study is to produce transgenic mice and sheep which express large amounts of human anti-hemophilic Factor VIII (FVIII) in the mammary gland. To overcome the potentially low expression of cDNA-constructs we have added heterologous introns from the murine metallothionein (MT-I) gene, resulting in gene constructs of 13 200 and 14 400 bp, respectively. Via microinjection of zygotes recovered from superovulated donors, we have produced 25 lines of transgenic mice with four different constructs of which two are under the control of the β-lactoglobulin (β-Lac) promoter and two under the control of the Whey acidic protein (WAP) promotor. One of the β-Lac- or WAP-hFVIII constructs possessed the MT-I fragment containing both introns in the 3′-untranslated region of the FVIII cDNA, respectively. Expression of FVIII cDNA was determined in mammary tissue of the transgenic mouse lines by reverse transcriptase PCR (RT-PCR). Of these 25 transgenic lines 13 (52%) expressed the integrated gene construct (WAP hFVIII 7/4, WAP hFVIII MT-I 11/5, β-Lac hFVIII 2/1, β-Lac hFVIII MT-I 5/3). By restriction enzyme analysis of the PCR products and Southern Blot analysis with FVIII probes we confirmed specificity of the expression of the transgene. Following microinjection of β-Lac hFVIII or β-Lac hFVIII MT-I constructs we have generated 8 transgenic founder sheep. One β-Lac hFVIII MT-I sheep expressed FVIII in the lactating mammary gland as detected by RT-PCR from biopsied mammary gland tissue. Two male founder animals transmitted the transgene in a Mendelian fashion to their offspring. To achieve site independent expression, new gene constructs employing matrix-attachment region elements (MAR) (MAR-β-Lac-hFVIII MT-I) were recently microinjected into pronuclei and two female and two male founder lambs were obtained. The total efficiency of microinjection into pronuclei of ovine zygotes has been 2.9% transgenic lambs (12/413). Analysis of mouse and sheep milk using two different Clotting assays and a sandwich ELISA did not reliably indicate the presence of active FVIII in milk. Currently, identification of FVIII in the milk of transgenic founder and F1 females with the aid of a sensitive antibody is under investigation. Zusammenfassung Expession von Genkonstruktion mit dem menschlichen Blutgerinnungsfaktor VIII in der Milchdruse transgener Mause und Schafe Zielsetzung unserer Untersuchungen ist die Erstellung transgener Mause und Schafe, die grosere Mengen an humanem Gerinnungsfaktor VIII (FVIII) in der Milchdruse exprimieren. Um die potentiell niedrige Expression von cDNA-Konstrukten zu uberwinden, haben wir heterologe Introns vom Maus-Metallothionein-Gen (MT-I Gen) hinzugefugt, was in Genkonstrukten von 13 200 bzw. 14 400 bp resultierte. Durch Mikroinjektion in Zygoten, die von superovulierten Spendertieren gewonnen waren, haben wir 25 transgene Mauselinien mit vier verschiedenen FVIII-Konstrukten erstellt. Von diesen Konstrukten waren jeweils zwei unter der Kontrolle des β-Lactoglobulin-Promotors (β-Lac) und zwei unter der Kontrolle des Whey-Acidic-Protein (WAP) Promotorelements. Jeweils eines der β-Lac und eines der WAP hFVIII-Konstrukte besas das MT-I Fragment mit den beiden Introns in der 3-untranslatierten Region der FVIII cDNA. Die Expression der FVIII cDNA im Milchdrusengewebe der transgenen Mauselinien wurde uber Reverse Transkriptase-PCR (RT-PCR) bestimmt. Von den 25 transgenen Linien exprimierten 13 (52%) das integrierte Genkonstrukt (WAP-hFVIII 7/4, WAP-hFVIII Mt-I 11/5, β-Lac-hFVIII 2/1, β-Lac-hFVIII MT-I 5/3). Durch Restriktionsenzymanalyse der PCR-Produkte und Southern Blot Analyse der FVIII-Proben haben wir die Speziftat der Expression der Transgene bestatigt. Nach Mikroinjektion von β-Lac-hFVIII oder β-Lac-hFVll-MT-I Konstrukten haben wir auch 8 transgene Founderschafe erhalten. Ein β-Lac-hFVIII-MT-I Schaf exprimierte FVIII in der laktierenden Milchdruse, was uber RT-PCR in biopsiertem Milchdrusengewebe festgestellt wurde. Zwei mannliche Foundertiere gaben das Transgen entsprechend der Mendelschen Regeln an die Nachkommen weiter. Um eine vom umliegenden Genom unabhangige Expression zu erreichen, haben wir neuartige Gen-konstrukte mit Matrix Attachment Region Elements (MAR) (MAR-β-Lac-hFVIII-MT-I) in die Vorkerne von Schafzygoten injiziert und zwei weibliche und zwei mannliche Founderlammer erhalten. Insgesamt betrug die Effizienz der Mikroinjektion 2,9% (12 von 413 Lammern waren transgen). Die Analyse von Maus- und Schafmilch mit zwei verschiedenen Gerinnungsassays sowie einem Sandwich ELISA ergab keine zuverlassigen und wiederholbaren Hinweise auf die Anwesenheit von aktivem FVIII in der Milch. Zur Zeit wird die Milch mittels transgener Founder- und F1-Schafe mit Hilfe eines empfindlichen Antikorpers und Immunprazipitation auf Anwesenheit des FVIII-Proteins untersucht.
R. Halter - One of the best experts on this subject based on the ideXlab platform.
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expression of active human Blood Clotting Factor viii in mammary gland of transgenic rabbits
DNA and Cell Biology, 2003Co-Authors: Laszlo Hiripi, Joseph Wallace Carnwath, R. Halter, F Makovics, Maria Baranyi, David L Paul, Zsuzsanna Bosze, Heinrich NiemannAbstract:Human Clotting Factor VIII is probably the largest protein to be expressed to date in the mammary gland of a transgenic animal, and it requires extensive posttranslational modification to achieve full biological activity. The mammary gland specific construct mWAP-hFVIII-MT-I was injected into the pronuclei of rabbit zygotes, and three transgenic offspring were obtained. Founder 385 showed germ-line transmission of a single integrated copy, and a homozygous line was established from this animal. The rhFVIII was transcribed and translated exclusively in the mammary gland. The activity of rhFVIII in the rabbit milk ranged from 5 to 8% of that found in normal human plasma. Results indicate the suitability of the transgenic rabbit mammary gland for rhFVIII production.
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Expression of human Blood Clotting Factor VIII in the mammary gland of transgenic sheep.
Transgenic research, 1999Co-Authors: Holger Niemann, E Lemme, D. Herrmann, Joseph Wallace Carnwath, R. Halter, D. PaulAbstract:By targeting the expression of sequences encoding non-milk proteins to the mammary gland of transgenic farm animals, the organ could serve as a 'bioreactor' for producing pharmacologically active proteins on a large scale. Here we report the generation of transgenic sheep bearing a fusion gene construct with the human Blood Clotting Factor VIII (hFVIII) cDNA under the transcriptional control of a 2.2 kb fragment of the mammary gland specific promoter of the ovine beta-Lactoglobulin (beta-Lac) gene. Six founder animals were generated bearing a hFVIII cDNA construct with the introns of the murine metallothionein (MtI) gene (beta-Lac/hFVIII-MtI). Founders transmitted the transgene in a Mendelian fashion and two transgenic lines were generated. Ten out of 12 transgenic F1-females expressed rhFVIII mRNA in exfoliated mammary epithelial cells isolated from the milk. But only in transgenic F1 ewes 4010 and 603 hFVIII Clotting activity estimated at 4-6 ng/ml was detected in defatted milk. Furthermore, the presence of rhFVIII-protein in ovine milk was demonstrated by a specific band at approximately 190 kD following immunoprecipitation and immunoblotting. Transgenic founder 395 expressed rhFVIII mRNA in biopsied mammary gland tissue, in exfoliated mammary cells as well as ectopically in brain, heart, spleen, kidney and salivary gland, suggesting that the employed beta-Lac promoter fragment lacks essential sequences for directing expression exclusively to the mammary gland. A rhFVIII standard preparation (rhFVIIIstd) was rapidly sequestered in a saturable fashion in ovine milk, thus rendering it largely inaccessible to immunoprecipitation although its biological activity was retained. Recovery of hFVIIIstd was dependent on milk donor, storage temperature and dilution of milk sample.
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expression of human Blood Clotting Factor viii fviii constructs in the mammary gland of transgenic mice and sheep
Journal of Animal Breeding and Genetics, 1996Co-Authors: Heiner Niemann, E Lemme, D. Herrmann, Joseph Wallace Carnwath, R. Halter, G Espanion, C Wrenzycki, D. PaulAbstract:Summary The aim of this study is to produce transgenic mice and sheep which express large amounts of human anti-hemophilic Factor VIII (FVIII) in the mammary gland. To overcome the potentially low expression of cDNA-constructs we have added heterologous introns from the murine metallothionein (MT-I) gene, resulting in gene constructs of 13 200 and 14 400 bp, respectively. Via microinjection of zygotes recovered from superovulated donors, we have produced 25 lines of transgenic mice with four different constructs of which two are under the control of the β-lactoglobulin (β-Lac) promoter and two under the control of the Whey acidic protein (WAP) promotor. One of the β-Lac- or WAP-hFVIII constructs possessed the MT-I fragment containing both introns in the 3′-untranslated region of the FVIII cDNA, respectively. Expression of FVIII cDNA was determined in mammary tissue of the transgenic mouse lines by reverse transcriptase PCR (RT-PCR). Of these 25 transgenic lines 13 (52%) expressed the integrated gene construct (WAP hFVIII 7/4, WAP hFVIII MT-I 11/5, β-Lac hFVIII 2/1, β-Lac hFVIII MT-I 5/3). By restriction enzyme analysis of the PCR products and Southern Blot analysis with FVIII probes we confirmed specificity of the expression of the transgene. Following microinjection of β-Lac hFVIII or β-Lac hFVIII MT-I constructs we have generated 8 transgenic founder sheep. One β-Lac hFVIII MT-I sheep expressed FVIII in the lactating mammary gland as detected by RT-PCR from biopsied mammary gland tissue. Two male founder animals transmitted the transgene in a Mendelian fashion to their offspring. To achieve site independent expression, new gene constructs employing matrix-attachment region elements (MAR) (MAR-β-Lac-hFVIII MT-I) were recently microinjected into pronuclei and two female and two male founder lambs were obtained. The total efficiency of microinjection into pronuclei of ovine zygotes has been 2.9% transgenic lambs (12/413). Analysis of mouse and sheep milk using two different Clotting assays and a sandwich ELISA did not reliably indicate the presence of active FVIII in milk. Currently, identification of FVIII in the milk of transgenic founder and F1 females with the aid of a sensitive antibody is under investigation. Zusammenfassung Expession von Genkonstruktion mit dem menschlichen Blutgerinnungsfaktor VIII in der Milchdruse transgener Mause und Schafe Zielsetzung unserer Untersuchungen ist die Erstellung transgener Mause und Schafe, die grosere Mengen an humanem Gerinnungsfaktor VIII (FVIII) in der Milchdruse exprimieren. Um die potentiell niedrige Expression von cDNA-Konstrukten zu uberwinden, haben wir heterologe Introns vom Maus-Metallothionein-Gen (MT-I Gen) hinzugefugt, was in Genkonstrukten von 13 200 bzw. 14 400 bp resultierte. Durch Mikroinjektion in Zygoten, die von superovulierten Spendertieren gewonnen waren, haben wir 25 transgene Mauselinien mit vier verschiedenen FVIII-Konstrukten erstellt. Von diesen Konstrukten waren jeweils zwei unter der Kontrolle des β-Lactoglobulin-Promotors (β-Lac) und zwei unter der Kontrolle des Whey-Acidic-Protein (WAP) Promotorelements. Jeweils eines der β-Lac und eines der WAP hFVIII-Konstrukte besas das MT-I Fragment mit den beiden Introns in der 3-untranslatierten Region der FVIII cDNA. Die Expression der FVIII cDNA im Milchdrusengewebe der transgenen Mauselinien wurde uber Reverse Transkriptase-PCR (RT-PCR) bestimmt. Von den 25 transgenen Linien exprimierten 13 (52%) das integrierte Genkonstrukt (WAP-hFVIII 7/4, WAP-hFVIII Mt-I 11/5, β-Lac-hFVIII 2/1, β-Lac-hFVIII MT-I 5/3). Durch Restriktionsenzymanalyse der PCR-Produkte und Southern Blot Analyse der FVIII-Proben haben wir die Speziftat der Expression der Transgene bestatigt. Nach Mikroinjektion von β-Lac-hFVIII oder β-Lac-hFVll-MT-I Konstrukten haben wir auch 8 transgene Founderschafe erhalten. Ein β-Lac-hFVIII-MT-I Schaf exprimierte FVIII in der laktierenden Milchdruse, was uber RT-PCR in biopsiertem Milchdrusengewebe festgestellt wurde. Zwei mannliche Foundertiere gaben das Transgen entsprechend der Mendelschen Regeln an die Nachkommen weiter. Um eine vom umliegenden Genom unabhangige Expression zu erreichen, haben wir neuartige Gen-konstrukte mit Matrix Attachment Region Elements (MAR) (MAR-β-Lac-hFVIII-MT-I) in die Vorkerne von Schafzygoten injiziert und zwei weibliche und zwei mannliche Founderlammer erhalten. Insgesamt betrug die Effizienz der Mikroinjektion 2,9% (12 von 413 Lammern waren transgen). Die Analyse von Maus- und Schafmilch mit zwei verschiedenen Gerinnungsassays sowie einem Sandwich ELISA ergab keine zuverlassigen und wiederholbaren Hinweise auf die Anwesenheit von aktivem FVIII in der Milch. Zur Zeit wird die Milch mittels transgener Founder- und F1-Schafe mit Hilfe eines empfindlichen Antikorpers und Immunprazipitation auf Anwesenheit des FVIII-Proteins untersucht.