TY  - CONF
AU  - Dunjić Manevski, Sofija
AU  - Cumbo, Marija
AU  - Gvozdenov, Maja
AU  - Tomić, Branko
AU  - Ušjak, Dušan
AU  - Đorđević
PY  - 2023
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/2126
AB  - Introduction: The prothrombin Belgrade variant (c.1787G>A, p.Arg596Gln) is a rare mutation found in
Serbia, Japan, China, America, India and leads to antithrombin resistance. Prothrombin Belgrade mutation influencesthrombin-antithrombin interactions and leadsto impaired inactivation of mutated thrombin. Also, it affectssodium binding site in thrombin, which isimportant forswitching from fast thrombin
configuration (coagulant properties) to slow configuration (anticoagulant properties). It has only been
found in a heterozygous state, which could mean that homozygous carriers are incompatible with life.
By using prothrombin (FII) deficient plasma, we could reconstitute plasma of wild type, heterozygous and
homozygous carrier, which could give more insight into the mechanism of this mutation.
Methods: Recombinant wild type and mutated prothrombin were generated by transient transfection
in HEK293T cell line. Western blot analysis was performed to test the efficiency of transfection. Human
Prothrombin ELISA (Nordic BioSite, Sweden) was used in order to measure recombinant prothrombin
concentration. Overall Hemostasis Potential (OHP) assay was performed to assess recombinant protein
activity. Recombinant wild type and mutated prothrombin were added to FII deficient plasma (Siemens,
Germany) in order to create reconstituted plasma, in the final concentration of 0.1 mg/mL, as it is approximately the level of prothrombin in human plasma.
Results: Reconstituted plasma samples that correspond to non-carrier, heterozygous carrier, and homozygous mutation carrier plasma were reconstructed. Recombinant proteinstested by OHP assay were
functional.
Conclusion: Reconstituted plasma samples allow us to examine the mechanism of prothrombin Belgrade mutation in various assays and in homozygous form as well.
PB  - Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade
C3  - CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
T1  - Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins
EP  - 71
SP  - 71
UR  - https://hdl.handle.net/21.15107/rcub_imagine_2126
ER  - 

@conference{
author = "Dunjić Manevski, Sofija and Cumbo, Marija and Gvozdenov, Maja and Tomić, Branko and Ušjak, Dušan and Đorđević",
year = "2023",
abstract = "Introduction: The prothrombin Belgrade variant (c.1787G>A, p.Arg596Gln) is a rare mutation found in
Serbia, Japan, China, America, India and leads to antithrombin resistance. Prothrombin Belgrade mutation influencesthrombin-antithrombin interactions and leadsto impaired inactivation of mutated thrombin. Also, it affectssodium binding site in thrombin, which isimportant forswitching from fast thrombin
configuration (coagulant properties) to slow configuration (anticoagulant properties). It has only been
found in a heterozygous state, which could mean that homozygous carriers are incompatible with life.
By using prothrombin (FII) deficient plasma, we could reconstitute plasma of wild type, heterozygous and
homozygous carrier, which could give more insight into the mechanism of this mutation.
Methods: Recombinant wild type and mutated prothrombin were generated by transient transfection
in HEK293T cell line. Western blot analysis was performed to test the efficiency of transfection. Human
Prothrombin ELISA (Nordic BioSite, Sweden) was used in order to measure recombinant prothrombin
concentration. Overall Hemostasis Potential (OHP) assay was performed to assess recombinant protein
activity. Recombinant wild type and mutated prothrombin were added to FII deficient plasma (Siemens,
Germany) in order to create reconstituted plasma, in the final concentration of 0.1 mg/mL, as it is approximately the level of prothrombin in human plasma.
Results: Reconstituted plasma samples that correspond to non-carrier, heterozygous carrier, and homozygous mutation carrier plasma were reconstructed. Recombinant proteinstested by OHP assay were
functional.
Conclusion: Reconstituted plasma samples allow us to examine the mechanism of prothrombin Belgrade mutation in various assays and in homozygous form as well.",
publisher = "Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade",
journal = "CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia",
title = "Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins",
pages = "71-71",
url = "https://hdl.handle.net/21.15107/rcub_imagine_2126"
}

Dunjić Manevski, S., Cumbo, M., Gvozdenov, M., Tomić, B., Ušjak, D.,& Đorđević. (2023). Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia
Institute of Molecular Genetics and Genetic Engineering (IMGGE), University of Belgrade., 71-71.
https://hdl.handle.net/21.15107/rcub_imagine_2126

Dunjić Manevski S, Cumbo M, Gvozdenov M, Tomić B, Ušjak D, Đorđević. Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins. in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia. 2023;:71-71.
https://hdl.handle.net/21.15107/rcub_imagine_2126 .

Dunjić Manevski, Sofija, Cumbo, Marija, Gvozdenov, Maja, Tomić, Branko, Ušjak, Dušan, Đorđević, "Reconstitution of non-carrier, heterozygous and homozygous prothrombin belgrade mutation carrier plasma using recombinant proteins" in CoMBoS2 – the Second Congress of Molecular Biologists of Serbia, Abstract Book – Trends in Molecular Biology, Special issue 06-08 October 2023, Belgrade, Serbia (2023):71-71,
https://hdl.handle.net/21.15107/rcub_imagine_2126 .

TY  - JOUR
AU  - Jovanović, Živko
AU  - Stanisavljević, Nemanja
AU  - Nikolić, Aleksandra
AU  - Medović, Aleksandar
AU  - Mikić, Aleksandar
AU  - Radović, Svetlana
AU  - Đorđević, Vuk
PY  - 2011
UR  - https://imagine.imgge.bg.ac.rs/handle/123456789/504
AB  - Primenom dve različite procedure ekstrahovana je drevna DNK iz ugljenisanih semena graška i urova starih 3200 godina sa lokaliteta Hisar kod Leskovca. Korišćena je modifikovana CTAB metoda i dobijena je relativno mala količina drevne DNK u poređenju sa drugom primenjenom metodom - komercijalno dostupnim kitom za izolaciju DNK. Nakon ekstrakcije primenjeno je umnožavanje celog genoma pomoću Phi29 DNK polimeraze. Umnožena DNK je korišćena za PCR reakciju primenom prajmera za 26S rDNK gen, koji je lociran u jedarnom genomu. Dobijen je fragment iste veličine kao i PCR fragment 26S rDNK savremenih srodnika graška i urova. Može se zaključiti da je moguće ekstrahovati drevnu DNK iz ugljenisanih semena graška i urova i koristiti je za dalje arheobotaničke analize na molekularnom nivou.
AB  - The extracts were prepared from the samples of 3,200-year-old charred pea and bitter vetch seeds from the site of Hissar near Leskovac, South Serbia, using two different DNA extraction procedures. We used CTAB method with some modification and obtained low quantity of ancient DNA in comparison with the second method used - commercial available kit. After the extraction, a whole genome amplification using Phi29 DNA polymerase was performed. The amplified DNAs were used for PCR reaction using primers for 26S rDNA gene, which is located on the nuclear genome. The single band corresponding to 26S rDNA fragment from modern relatives was obtained. We conclude that DNA from charred pea and vetch seed can be extracted and used for further archaeobotanical analysis at the molecular level.
PB  - Institut za ratarstvo i povrtarstvo, Novi Sad
T2  - Ratarstvo i povrtarstvo
T1  - Grašak i urov Tetovac - made in ranogvozdenodobni Leskovac, Deo drugi - ekstrakcija drevne DNK iz ugljenisanih semena sa nalazišta Hisar u južnoj Srbiji
T1  - Pisum & Ervilia Tetovac: Made in Early Iron Age Leskovac, Part two: Extraction of the ancient DNA from charred seeds from the site of Hissar in South Serbia
EP  - 232
IS  - 1
SP  - 227
VL  - 48
DO  - 10.5937/ratpov1101227J
ER  - 

@article{
author = "Jovanović, Živko and Stanisavljević, Nemanja and Nikolić, Aleksandra and Medović, Aleksandar and Mikić, Aleksandar and Radović, Svetlana and Đorđević, Vuk",
year = "2011",
abstract = "Primenom dve različite procedure ekstrahovana je drevna DNK iz ugljenisanih semena graška i urova starih 3200 godina sa lokaliteta Hisar kod Leskovca. Korišćena je modifikovana CTAB metoda i dobijena je relativno mala količina drevne DNK u poređenju sa drugom primenjenom metodom - komercijalno dostupnim kitom za izolaciju DNK. Nakon ekstrakcije primenjeno je umnožavanje celog genoma pomoću Phi29 DNK polimeraze. Umnožena DNK je korišćena za PCR reakciju primenom prajmera za 26S rDNK gen, koji je lociran u jedarnom genomu. Dobijen je fragment iste veličine kao i PCR fragment 26S rDNK savremenih srodnika graška i urova. Može se zaključiti da je moguće ekstrahovati drevnu DNK iz ugljenisanih semena graška i urova i koristiti je za dalje arheobotaničke analize na molekularnom nivou., The extracts were prepared from the samples of 3,200-year-old charred pea and bitter vetch seeds from the site of Hissar near Leskovac, South Serbia, using two different DNA extraction procedures. We used CTAB method with some modification and obtained low quantity of ancient DNA in comparison with the second method used - commercial available kit. After the extraction, a whole genome amplification using Phi29 DNA polymerase was performed. The amplified DNAs were used for PCR reaction using primers for 26S rDNA gene, which is located on the nuclear genome. The single band corresponding to 26S rDNA fragment from modern relatives was obtained. We conclude that DNA from charred pea and vetch seed can be extracted and used for further archaeobotanical analysis at the molecular level.",
publisher = "Institut za ratarstvo i povrtarstvo, Novi Sad",
journal = "Ratarstvo i povrtarstvo",
title = "Grašak i urov Tetovac - made in ranogvozdenodobni Leskovac, Deo drugi - ekstrakcija drevne DNK iz ugljenisanih semena sa nalazišta Hisar u južnoj Srbiji, Pisum & Ervilia Tetovac: Made in Early Iron Age Leskovac, Part two: Extraction of the ancient DNA from charred seeds from the site of Hissar in South Serbia",
pages = "232-227",
number = "1",
volume = "48",
doi = "10.5937/ratpov1101227J"
}

Jovanović, Ž., Stanisavljević, N., Nikolić, A., Medović, A., Mikić, A., Radović, S.,& Đorđević, V.. (2011). Grašak i urov Tetovac - made in ranogvozdenodobni Leskovac, Deo drugi - ekstrakcija drevne DNK iz ugljenisanih semena sa nalazišta Hisar u južnoj Srbiji. in Ratarstvo i povrtarstvo
Institut za ratarstvo i povrtarstvo, Novi Sad., 48(1), 227-232.
https://doi.org/10.5937/ratpov1101227J

Jovanović Ž, Stanisavljević N, Nikolić A, Medović A, Mikić A, Radović S, Đorđević V. Grašak i urov Tetovac - made in ranogvozdenodobni Leskovac, Deo drugi - ekstrakcija drevne DNK iz ugljenisanih semena sa nalazišta Hisar u južnoj Srbiji. in Ratarstvo i povrtarstvo. 2011;48(1):227-232.
doi:10.5937/ratpov1101227J .

Jovanović, Živko, Stanisavljević, Nemanja, Nikolić, Aleksandra, Medović, Aleksandar, Mikić, Aleksandar, Radović, Svetlana, Đorđević, Vuk, "Grašak i urov Tetovac - made in ranogvozdenodobni Leskovac, Deo drugi - ekstrakcija drevne DNK iz ugljenisanih semena sa nalazišta Hisar u južnoj Srbiji" in Ratarstvo i povrtarstvo, 48, no. 1 (2011):227-232,
https://doi.org/10.5937/ratpov1101227J . .