Apoptosis is a natural form of cell death involved in many physiological changes in the cell. Defects in the process of apoptosis can lead to serious diseases. During some apoptotic pathways, proteins apoptosis-inducing factor (AIF) and endonuclease G (EndoG) are released from the mitochondria and they translocate into the cell nuclei, where they probably participate in chromatin degradation together with other nuclear proteins. Exact mechanism of EndoG activity in cell nucleus is still unknown. Some interacting partners like flap endonuclease 1, DNase I, and exonuclease III were already suggested, but also other interacting partners were proposed. We conducted a living-cell confocal fluorescence microscopy followed by an image analysis of fluorescence resonance energy transfer to analyze the possibility of protein interactions of EndoG with histone H2B and human DNA topoisomerase II alpha (TOPO2a). Our results show that EndoG interacts with both these proteins during apoptotic cell death. Therefore, we can conclude that EndoG and TOPO2a may actively participate in apoptotic chromatin degradation. The possible existence of a degradation complex consisting of EndoG and TOPO2a and possibly other proteins like AIF and cyclophilin A have yet to be investigated.

• MeSH
• antigeny nádorové chemie   genetika   metabolismus   MeSH
• apoptóza * MeSH
• buněčné jádro enzymologie   patologie   MeSH
• časové faktory MeSH
• DNA vazebné proteiny chemie   genetika   metabolismus   MeSH
• DNA-topoisomerasy typu II chemie   genetika   metabolismus   MeSH
• endodeoxyribonukleasy chemie   genetika   metabolismus   MeSH
• HeLa buňky MeSH
• histony chemie   genetika   metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• konfokální mikroskopie MeSH
• konformace proteinů MeSH
• lidé MeSH
• mapování interakce mezi proteiny MeSH
• molekulární modely MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• restrukturace chromatinu * MeSH
• rezonanční přenos fluorescenční energie MeSH
• transfekce MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• DNA vazebné proteiny MeSH
• DNA-topoisomerasy typu II MeSH
• endodeoxyribonukleasy MeSH
• endonuclease G MeSH Prohlížeč
• histony MeSH
• rekombinantní fúzní proteiny MeSH

We studied the cellular localization of the apoptotic proteins endonuclease G, AIF, and AMID in silico using three prediction tools and in living cells using both single-cell colocalization image analysis and nuclear translocation analysis. We confirmed the mitochondrial localization of endonuclease G and AIF by prediction analysis and by single-cell colocalization image analysis. We found the AMID protein to be cytoplasmic, most probably incorporated into the cytoplasmic side of the membranes of various organelles. The highest concentration of AMID was observed associated with the Golgi. Colocalization of AMID with lysosomes was also indirectly confirmed by analysis of AMID-rich vesicle velocity using manual tracking analysis. Bioinformatic analysis also detected nuclear localization signals in endonuclease G and AIF, but not in AMID. A novel analysis of time-lapse fluorescence image data during staurosporine-induced apoptosis revealed nuclear translocation only for endonuclease G and AIF.

• MeSH
• apoptóza fyziologie   MeSH
• buněčné jádro metabolismus   MeSH
• buněčné linie MeSH
• endodeoxyribonukleasy izolace a purifikace   metabolismus   MeSH
• faktor vyvolávající apoptózu izolace a purifikace   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• Golgiho aparát metabolismus   MeSH
• intracelulární membrány metabolismus   MeSH
• lidé MeSH
• lyzozomy metabolismus   MeSH
• mitochondriální proteiny izolace a purifikace   metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• proteiny regulující apoptózu izolace a purifikace   metabolismus   MeSH
• rekombinantní fúzní proteiny metabolismus   MeSH
• výpočetní biologie * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• AIFM1 protein, human MeSH Prohlížeč
• endodeoxyribonukleasy MeSH
• endonuclease G MeSH Prohlížeč
• faktor vyvolávající apoptózu MeSH
• ferroptosis suppressor protein 1, human MeSH Prohlížeč
• mitochondriální proteiny MeSH
• proteiny regulující apoptózu MeSH
• rekombinantní fúzní proteiny MeSH

Two activated matrices have been developed to determine whether immobilization chemistry can be used to orient proteins on a support. Restriction endonuclease EcoRI from Escherichia coli RY13 (E.C.3.1.23.13) was used as a model in these studies. Thiol-activated Sephadex G-10 was used to couple the EcoRI endonuclease through its free sulfhydryl, while amino-activated Sephadex G-10 was used to couple it randomly through its free carboxyl groups. To determine whether the enzyme was immobilized randomly or specifically, both lower and higher molecular weight substrates were used. The polymerase chain reaction amplified multiplied cloning site region of pBluescript KS obtained using T3 and T7 primers was considered as the small substrate. The plasmid SP64 containing firefly luciferase gene was the large substrate. Immobilized EcoRI preparations were characterized with respect to repeated usage and storage stability. The EcoRI immobilized on thiopropyl-Sepharose 4B could be stored for over 14 days at 4 degrees C without observable loss of activity. In an independent experiment the same gel was used thrice repeatedly without any discernible loss of activity.

• MeSH
• butylenglykoly chemie   MeSH
• deoxyribonukleasa EcoRI chemie   metabolismus   MeSH
• dextrany chemie   MeSH
• DNA metabolismus   MeSH
• enzymy imobilizované * chemie   MeSH
• gely chemie   MeSH
• konformace proteinů MeSH
• rozpustnost MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, U.S. Gov't, Non-P.H.S. MeSH
• Názvy látek
• 1,4-bis(2,3-epoxypropoxy)butane MeSH Prohlížeč
• butylenglykoly MeSH
• deoxyribonukleasa EcoRI MeSH
• dextrany MeSH
• DNA MeSH
• enzymy imobilizované * MeSH
• gely MeSH
• sephadex MeSH Prohlížeč

DNA molecules of pUC19, pBR322 and PhiX174 were irradiated by various doses of UV light and the irradiated molecules were cleaved by about two dozen type II restrictases. The irradiation generally blocked the cleavage in a dose-dependent way. In accordance with previous studies, the (A + T)-richness and the (PyPy) dimer content of the restriction site belongs among the factors that on average, cause an increase in the resistance of UV damaged DNA to the restrictase cleavage. However, we observed strong effects of UV irradiation even with (G + C)-rich and (PyPy)-poor sites. In addition, sequences flanking the restriction site influenced the protection in some cases (e.g. HindIII), but not in others (e.g. SalI), whereas neoschizomer couples SmaI and AvaI, or SacI and Ecl136II, cleaved the UV-irradiated DNA similarly. Hence the intrastrand thymine dimers located in the recognition site are not the only photoproduct blocking the restrictases. UV irradiation of the A-form generally made the irradiated DNA less resistant to restrictase cleavage than irradiation in the B-form and in some cases, the A-form completely protected the UV-irradiated DNA against the damage recognized by the restrictases. The present results also demonstrate that the UV irradiation approach used to generate partial digests in genomic DNA studies, can be extended to the (G + C)-rich and (PyPy)-poor restriction sites. The present extensive and quantitative data can be used in genomic applications of UV damage probing by restrictases.

• MeSH
• DNA bakterií chemie   MeSH
• plazmidy chemie   MeSH
• poškození DNA účinky záření   MeSH
• pyrimidinové dimery chemie   MeSH
• restrikční endonukleasy typu II chemie   MeSH
• ultrafialové záření * MeSH
• zastoupení bazí MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• pyrimidinové dimery MeSH
• restrikční endonukleasy typu II MeSH

Structural distortions on the boundary between right-handed B and left-handed Z DNA segments in plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of chemical probes. Samples of supercoiled DNA were treated with the respective chemical probe, linearized with EcoRI and inhibition of BamHI (whose recognition sequence GGATCC lies on the boundary between the (dC-dG)n segments and the pBR322 nucleotide sequence) cleavage was tested. Treatment with osmium tetroxide in the presence of pyridine or 2,2'-bipyridine, respectively, resulted in a strong inhibition of the BamHI cleavage at both restriction sites, provided the (dC-dG)n segments were in the left-handed form. In the presence of 2,2'-bipyridine submillimolar concentrations of OsO4 (at 26 degrees C) were sufficient to induce the inhibition of BamHI. Chloroacetaldehyde was used as a probe reacting selectively with atoms involved in the Watson-Crick hydrogen bonding. Similarly as in the case of osmium tetroxide treatment of pRW751 with this agent resulted in the inhibition of BamHI cleavage. It was concluded that the B-Z junction regions in pRW751 contain few solitary bases with disturbed hydrogen bonding or non-Watson-Crick base pairs.

• MeSH
• acetaldehyd analogy a deriváty   MeSH
• elektroforéza v agarovém gelu MeSH
• konformace nukleové kyseliny * MeSH
• plazmidy * MeSH
• restrikční enzymy MeSH
• sekvence nukleotidů MeSH
• superhelikální DNA * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetaldehyd MeSH
• chloroacetaldehyde MeSH Prohlížeč
• restrikční enzymy MeSH
• superhelikální DNA * MeSH

The oxidative mechanisms of injury-induced damage of neurons within the spinal cord are not very well understood. We used a model of T8-T9 spinal cord injury (SCI) in the rat to induce neuronal degeneration. In this spinal cord injury model, unilateral avulsion of the spinal cord causes oxidative stress of neurons. We tested the hypothesis that apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1) regulates this neuronal oxidation mechanism in the spinal cord region caudal to the lesion, and that DNA damage is an early upstream signal. The embryonic neural stem cell therapy significantly decreased DNA-damage levels in both study groups - acutely (followed up to 7 days after SCI), and chronically (followed up to 28 days after SCI) injured animals. Meanwhile, mRNA levels of APE/Ref-1 significantly increased after embryonic neural stem cell therapy in acutely and chronically injured animals when compared to acute and chronic sham groups. Our data has demonstrated that an increase of APE/Ref-1 mRNA levels in the caudal region of spinal cord strongly correlated with DNA damage after traumatic spinal cord injury. We suggest that DNA damage can be observed both in lesional and caudal regions of the acutely and chronically injured groups, but DNA damage is reduced with embryonic neural stem cell therapy.

• MeSH
• akutní nemoc MeSH
• cauda equina enzymologie   patologie   chirurgie   MeSH
• chronická nemoc MeSH
• degenerace nervu enzymologie   genetika   patologie   chirurgie   MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) genetika   metabolismus   MeSH
• embryonální kmenové buňky enzymologie   patologie   transplantace   MeSH
• kometový test MeSH
• krysa rodu Rattus MeSH
• kultivované buňky MeSH
• lokomoce MeSH
• messenger RNA metabolismus   MeSH
• modely nemocí na zvířatech MeSH
• neurony enzymologie   patologie   transplantace   MeSH
• oxidační stres MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• poranění míchy enzymologie   genetika   patologie   chirurgie   MeSH
• poškození DNA * MeSH
• potkani Sprague-Dawley MeSH
• upregulace MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• Apex1 protein, rat MeSH Prohlížeč
• DNA-lyasa (apurinová nebo apyrimidinová) MeSH
• messenger RNA MeSH

Structural distortions on the boundary between right-handed and left-handed DNA segments in negatively supercoiled plasmid pRW751 (a derivative of pBR322 containing (dC-dG)13 and (dC-dG)16 segments) were studied by means of osmium tetroxide, pyridine and glyoxal. These two probes react preferentially with single-stranded DNA, but only the latter requires non-paired bases for the reaction. Nuclease S1 and testing of the inhibition of BamHI cleavage (whose recognition sequences GGATCC lie on the "outer" boundaries between the (dC-dG)n and the pBR322 nucleotide sequence) were used to detect the site-specific chemical modification in pRW751. As a result of glyoxal treatment BamHI cleavage was strongly inhibited in topoisomeric samples whose superhelical density was sufficiently negative to stabilize the (dC-dG)n segments in the left-handed form. Osmium tetroxide, pyridine modification resulted in a similar inhibition of BamHI cleavage and in a formation of nuclease S1 sensitive sites. The results suggest that the "outer" B-Z junctions in pRW751 contain one or few non-paired bases or non-Watson-Crick base pairs.

• MeSH
• deoxyribonukleasa BamHI antagonisté a inhibitory   MeSH
• DNA ultrastruktura   MeSH
• endonukleasy specifické pro jednořetězcové nukleové kyseliny metabolismus   MeSH
• glyoxal farmakologie   MeSH
• konformace nukleové kyseliny účinky léků   MeSH
• oxid osmičelý farmakologie   MeSH
• plazmidy * MeSH
• pyridiny farmakologie   MeSH
• rekombinantní DNA MeSH
• sekvence nukleotidů MeSH
• superhelikální DNA účinky léků   ultrastruktura   MeSH
• zastoupení bazí * MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• deoxyribonukleasa BamHI MeSH
• DNA MeSH
• endonukleasy specifické pro jednořetězcové nukleové kyseliny MeSH
• glyoxal MeSH
• oxid osmičelý MeSH
• pyridine MeSH Prohlížeč
• pyridiny MeSH
• rekombinantní DNA MeSH
• superhelikální DNA MeSH

A new Type II restriction endonuclease ApaCI purified from Acetobacter pasteurianus is an isoschizomer of BamHI that cleaves at the nucleotide sequence 5'-G/GATCC-3' of double-stranded DNA. The single restriction activity present in this strain permits rapidly purified 30,000 units of cleavage activity from 10 g of freshly harvested cells. The resulting ApaCI preparation is free of contaminant nuclease activities that might interfere with in vitro manipulation of DNA.

• MeSH
• Acetobacter enzymologie   MeSH
• bakteriofág lambda genetika   metabolismus   MeSH
• deoxyribonukleasa BamHI izolace a purifikace   metabolismus   MeSH
• DNA virů genetika   metabolismus   MeSH
• sekvence nukleotidů MeSH
• substrátová specifita MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• deoxyribonukleasa BamHI MeSH
• DNA virů MeSH

Many aspects of protein function regulation require specific protein-protein interactions to carry out the exact biochemical and cellular functions. The highly conserved members of the 14-3-3 protein family mediate such interactions and through binding to hundreds of other proteins provide multitude of regulatory functions, thus playing key roles in many cellular processes. The 14-3-3 protein binding can affect the function of the target protein in many ways including the modulation of its enzyme activity, its subcellular localization, its structure and stability, or its molecular interactions. In this minireview, we focus on mechanisms of the 14-3-3 protein-dependent regulation of three important 14-3-3 binding partners: yeast neutral trehalase Nth1, regulator of G-protein signaling 3 (RGS3), and phosducin.

• MeSH
• DNA-glykosylasy chemie   ultrastruktura   MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) chemie   ultrastruktura   MeSH
• fosfoproteiny chemie   ultrastruktura   MeSH
• konformace proteinů MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• multienzymové komplexy chemie   ultrastruktura   MeSH
• oční proteiny chemie   ultrastruktura   MeSH
• proteiny 14-3-3 chemie   ultrastruktura   MeSH
• proteiny RGS chemie   ultrastruktura   MeSH
• proteiny vázající GTP - regulátory chemie   ultrastruktura   MeSH
• Schizosaccharomyces pombe - proteiny chemie   ultrastruktura   MeSH
• sekvence aminokyselin MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• DNA-glykosylasy MeSH
• DNA-lyasa (apurinová nebo apyrimidinová) MeSH
• fosfoproteiny MeSH
• multienzymové komplexy MeSH
• Nth1 protein, S pombe MeSH Prohlížeč
• oční proteiny MeSH
• phosducin MeSH Prohlížeč
• proteiny 14-3-3 MeSH
• proteiny RGS MeSH
• proteiny vázající GTP - regulátory MeSH
• RGS3 protein, human MeSH Prohlížeč
• Schizosaccharomyces pombe - proteiny MeSH

The described method is a modification of the hot alkaline method (Kado and Liu, 1981). The modification lies in replacement of SDS with Triton X- 100. The DNA purified by this method was readily digested with restriction endonucleases for a variety of applications, e. g. restriction analysis of plasmid DNA in epidemiological studies, restriction mapping and cloning of high molecular weight plasmids, and for identification of transpozon mutants.

• MeSH
• molekulární biologie metody   MeSH
• molekulová hmotnost MeSH
• plazmidy izolace a purifikace   MeSH
• restrikční enzymy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• restrikční enzymy MeSH