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Molecular cloning, E.coli expression and purification of SCFV antibody fragments of diagnostic/therapeutic interest
Král, Vlastimil; Fábry, Milan; Hořejší, Magdalena; Závada, Jan; Sedláček, Juraj
2003 - anglický
We describe molecular cloning, expression, purification and properties of two single chain antibody variable fragments (scFv) of potential diagnostic use, namely scFv M75 and scFv Tu-20. The former scFv is derived from a monoclonal antibody M75 specific for a cell surface protein MN/CA IX, strongly associated with many types of human carcinomas. The latter scFv is derived from a monoclonal antibody TU-20 specific for neuronal beta-III-tubulin.
Klíčová slova:
cloning; purification; scFv
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Molecular cloning, E.coli expression and purification of SCFV antibody fragments of diagnostic/therapeutic interest
We describe molecular cloning, expression, purification and properties of two single chain antibody variable fragments (scFv) of potential diagnostic use, namely scFv M75 and scFv Tu-20. The former ...
Characterization of peptide proteinase inhibitors isolated from boar seminal plasma
Jelínková, Petra; Tichá, M.; Jonáková, Věra
2003 - anglický
Most of proteins of boar seminal plasma are present in this medium under physiological conditions in the form of aggregates and not of monomers. In the present communication we have shown that not only spermadhesins, but also proteinase inhibitors participate in the formation of associated complexes. Proteinase inhibitors were found after gel chromatographic separation mainly in the fraction with the lowest relative molecular mass. Two different types of serine proteinase inhibitors were isolated from boar seminal plasma ů (i) glycosylated inhibitor with Mr 12 kDa, (ii) non-glycosylated sperm-associated acrosin inhibitor with Mr 8 kDa, and a new type of low molecular weight protein that is probably composed of two subunits coupled by the -S-S- bridge. The proteinase inhibitor with Mr 8 kDa was proved to form a heterodimer with AQN 1 spermadhesin under physiological conditions, whereas inhibitor with Mr 12 kDa forms homodimers.
Klíčová slova:
proteinase inhibitors, aggregated forms, boar seminal plasma
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Characterization of peptide proteinase inhibitors isolated from boar seminal plasma
Most of proteins of boar seminal plasma are present in this medium under physiological conditions in the form of aggregates and not of monomers. In the present communication we have shown that not ...
Antitrophoblast cell-mediated immunity /AT-CMI/ in recurrent miscarriage and ectopic pregnacy
Madar, J.; Kučera, E.; Šůla, K.; Chaloupková, A.; Pěknicová, Jana; Tolarová, M.; Nouza, K.; Kinský, R.
2003 - anglický
Specific mechanisms operate in decidua nad trophoblast to establish and maintain immunological tolerance of the embryo in mother's uterus. due to the lack of this tolerance, disturbances of pregnancy may result. In this presentation, we have tested the hypothesis that anti-trohhoblast cell-mediated immunity (AT-CMI) is elevated in habitually aborting women and that extra-uterine localisation of the embryo leads to subsequent elevation of AT-CMI.
Klíčová slova:
trophoblast; cardiolipin
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Antitrophoblast cell-mediated immunity /AT-CMI/ in recurrent miscarriage and ectopic pregnacy
Specific mechanisms operate in decidua nad trophoblast to establish and maintain immunological tolerance of the embryo in mother's uterus. due to the lack of this tolerance, disturbances of pregnancy ...
Multigenerational study - effect of the selected compounds on the mouse fertility
Boubelík, Michael; Pěknicová, Jana; Kyselová, Vendula; Buckiová, D.
2003 - anglický
Several compounds known to be effective for their influence onto the environment were tested. Compounds tested in this study were: benzo/a/ pyrene, lindane (hexachlorocyclohexane isomer), 4-n-nonylphenol, bisphenol A and octachlorostyrene. The question was if these compounds might also function as endocrine disruptors.
Klíčová slova:
endocrine disruptors; sperm motility
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Multigenerational study - effect of the selected compounds on the mouse fertility
Several compounds known to be effective for their influence onto the environment were tested. Compounds tested in this study were: benzo/a/ pyrene, lindane (hexachlorocyclohexane isomer), ...
Microsatellite region in the murine oncogene src - its primary structure and variability during evolution
Fučík, Vladimír; Šindelka, Radek; Beran, Jaroslav; Ferjentsik, Zoltán; Jonák, Jiří
2003 - anglický
In the fifth intron of the Mus musculus oncogene src a complex microsatellite was discovered that consists of a CA run and 31 bp apart followed by track of TG dinucleotides interrupted by extra Gs in specific intervals. As the 31 bp interlink can assume a hairpin structure, the whole region can be viewed as an imperfect inverted repeat. The microsatellite was sequenced in imbred lines of M. m. musculus and M. m. domesticus, as well as in mice of both subspecies trapped near the hybrid zone. Differences in the length of repeat regions, insertion/deletions and simple base changes characterize the strains. Hybrids between the two species could be identified by sequencing of cloned PCR products. The structure of the microsatellite in more distant species was found to be conspicuously different. Instead of frequent (CA)18-25, we can see only (CA)8 in M. spretus, CATGCGCCCCCCA in Mus pahari and CACCCCCC in Mus caroli. No similar structure is present in the corresponding human src intron.
Klíčová slova:
Mus musculus, c-src introns, microsatellite
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Microsatellite region in the murine oncogene src - its primary structure and variability during evolution
In the fifth intron of the Mus musculus oncogene src a complex microsatellite was discovered that consists of a CA run and 31 bp apart followed by track of TG dinucleotides interrupted by extra Gs in ...
A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation and Subsite-Pocket Interactions Analyzed at Atomic Resolution
Brynda, Jiří; Řezáčová, Pavlína; Fábry, Milan; Hořejší, Magdalena; Štouračová, Renata; Sedláček, Juraj; Souček, M.; Hradílek, M.; Lepšík, M.; Konvalinka, J.
2003 - anglický
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibiot shows subnanomolar Ki values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance developoment. The structure displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. The high resolution permits to assess the donor/acceptor realtions of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. Structural mechanism for the unimpaired ihnibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.
Klíčová slova:
inhibitor; HIV protease; atomic resolution
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
A Phenylnorstatine Inhibitor Binding to HIV-1 Protease: Geometry, Protonation and Subsite-Pocket Interactions Analyzed at Atomic Resolution
The x-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH2 has been determined at 1.03 A, the highest resolution so far reported for any HIV PR ...
Metody experimentálního určení fází (MIR, MAD,SAD) v krystalografické analýze biologických makromolekul
Brynda, Jiří
2003 - český
Důležitost metod poskytujících nepředpojaté řešení fázového problému se stává vstupem molekulární biologie a biochemie do tzv. post-genomické éry stále významnější. V článku jsou popsány klasické metody experimentálního určení fází:1) Metoda isomorfních derivátů s těžkými atomy 2) Metoda Anomálního rozptylu při různých vlnových délkách 3) Metoda Anomálního rozptylu při jedné vlnové délce Čím lepší jsou vaše první, experimentální mapy, tím více modelu můžete spolehlivě vystavět do nepředpojaté (modelem nezatížené) mapy před sečtením fází (původní + model). MAD fáze pocházejí z perfektně izomorfních dat a tak netrpí disordrem jako je to běžné u MIR map na malém rozlišení. MIR fáze zase na druhou stranu poskytují výtečnou fázovací sílu na nízkém rozlišení a výsledkem je lepší propojení mapy v prostoru. Není proto překvapivé, že nejlepší mapy jsou buď MAD mapy nebo kombinované MIR-MAD mapy. Importance of unbiased phases in post genomic era is emphasized. Article describes the classical methods of phase determination: 1) Multiple Isomorphous replacement, heavy-atom derivative (MIR) 2) Multiple Anomalous Dispersion (MAD) Phasing 3) Single Anomalous Dispersion (SAD) Phasing The better your initial, experimental map is, the more model you will be able to build confidently into this unbiased map before proceeding to phase combination. MAD phases result from perfectly isomorphous data, and do not deteriorate at low resolution as do MIR phases. MIR phases, on the other hand, provide superior phasing power at low resolution resulting in better connectivity. Not surprising, the best maps are either MAD or combined MIR-MAD maps.
Klíčová slova:
phase problem; MIR,MAD,SAD
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Metody experimentálního určení fází (MIR, MAD,SAD) v krystalografické analýze biologických makromolekul
Důležitost metod poskytujících nepředpojaté řešení fázového problému se stává vstupem molekulární biologie a biochemie do tzv. post-genomické éry stále významnější. V článku jsou popsány klasické ...
Crystallographic study of an anti=carbonic anhydrase IX monoclonal antibody M75
Štouračová, Renata; Závada, Jan; Závadová, Zuzana; Pastoreková, S.; Brynda, Jiří; Fábry, Milan; Král, Vlastimil; Hořejší, Magdalena; Sedláček, Juraj
2003 - anglický
Carbonic anhydrase IX (CA IX) is a cell surface protein, strongly associated with certain types of human carcinomas. Structural study of a CA IX-binding monoclonal antibody (mAb) M75, complexed with its epitope peptide may contribute toward elucidation of the role of CA IX. Monoclonal antibody M75 was obtained and proved to react excellently with native and denaturated CA IX. Using synthetic oligopeptides, the epitope of mAb M75 was localized in the proteoglycan domain of CA IX, in the region of a tandem repeat and identified as amino acids PGEEDLP. The Fab fragment was obtained by papain cleavage. We obtained crystals of free Fab M75 and Fab M75 complexed with two different epitope peptides. The data set for Fab M75 was collected and the structure solving is underway.
Klíčová slova:
anti-carbonic ahydrase antibody, cancer, crystallization
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Crystallographic study of an anti=carbonic anhydrase IX monoclonal antibody M75
Carbonic anhydrase IX (CA IX) is a cell surface protein, strongly associated with certain types of human carcinomas. Structural study of a CA IX-binding monoclonal antibody (mAb) M75, complexed with ...
Structural basis of HIV-1 and HIV-2 protease inhibition bya monoclonal antibody
Řezáčová, Pavlína; Lescar, J.; Brynda, Jiří; Fábry, Milan; Bentley, G. A.; Sedláček, Juraj
2003 - anglický
The murine monoclonal antibody 1696, produced by immunisation with the HIV-1 protease,inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates, with inhibitionconstants in the low nanomolar range. This antibody cross-reacts with peptides that include theN-terminus of the enzyme (residues 1-7), a region which is highly conserved in sequence amongdifferent viral strains and which, furthermore, is crucial for homodimerization to the activeenzymatic form.We report here two crystal structures of a recombinant single-chain Fv fragment of mAb 1696,expressed in E. coli, as a complex with a cross-reactive peptides from the HIV-1 PR and theHIV-2 PR at 2.7 ? resolution and 1.9 ? resolution respectively. On the basis of the interactionsseen in the complex three-dimensional structures, the cross-reactivity between mAb 1696 withthe HIV-1 and HIV-2 protease and their N-terminal peptides can be explained. In addition, acandidate mechanism of HIV PR inhibition by mAb 1696 is proposed which may help thedesign of alternative HIV protease inhibitors, aimed at dissociating the homodimeric viral enzyme.
Klíčová slova:
antibody fragment; crystal structure; HIV protease inhibition
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
Structural basis of HIV-1 and HIV-2 protease inhibition bya monoclonal antibody
The murine monoclonal antibody 1696, produced by immunisation with the HIV-1 protease,inhibits the catalytic activity of the enzyme of both the HIV-1 and HIV-2 isolates, with inhibitionconstants in ...
ISOLATION AND CHARACTERIZATION OF MANNAN-BINDING PROTEINS FROM BOAR SEMINAL PLASMA
Jelínková, Petra; Liberda, J.; Ryšlavá, H.; Tichá, M.; Jonáková, Věra
2003 - anglický
The mannan-binding proteins of boar seminal plasma may be involved in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both with the complex of D-mannose-containing saccharide structures and heparin may also play an important role in the sperm release from the oviductal reservoir and in the capacitation process.
Klíčová slova:
mannan-binding proteins; oviductal reservoir; boar seminal plasma
Plné texty jsou dostupné na jednotlivých ústavech Akademie věd ČR.
ISOLATION AND CHARACTERIZATION OF MANNAN-BINDING PROTEINS FROM BOAR SEMINAL PLASMA
The mannan-binding proteins of boar seminal plasma may be involved in the formation of the sperm oviductal reservoir in pig. The ability of these proteins to interact both with the complex of ...
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