The reactivity of the anticancer drug picoplatin (cis-amminedichlorido(2-methylpyridine)platinum(II) complex) with the model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated by electrospray ionisation mass spectrometry (ESI MS) and X-ray crystallography. The data were compared with those previously obtained for the adducts of these proteins with cisplatin, carboplatin and oxaliplatin under the same experimental conditions. ESI-MS data show binding of Pt to both proteins, with fragments retaining the 2-methylpyridine ligand and, possibly, a chloride ion. X-ray crystallography identifies different binding sites on the two proteins, highlighting a different behaviour of picoplatin in the absence or presence of dimethyl sulfoxide (DMSO). Metal-containing fragments bind to HEWL close to the side chains of His15, Asp18, Asp119 and both Lys1 and Glu7, whereas they bind to RNase A on the side chain of His12, Met29, His48, Asp53, Met79, His105 and His119. The data suggest that the presence of DMSO favours the loss of 2-methylpyridine and alters the ability of the Pt compound to bind to the two proteins. With both proteins, picoplatin appears to behave similarly to cisplatin and carboplatin when dissolved in DMSO, whereas it behaves more like oxaliplatin in the absence of the coordinating solvent. This study provides important insights into the pharmacological profile of picoplatin and supports the conclusion that coordinating solvents should not be used to evaluate the biological activities of Pt-based drugs.

• MeSH
• dimethylsulfoxid chemie   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• karboplatina chemie   metabolismus   MeSH
• krystalografie rentgenová MeSH
• kur domácí MeSH
• molekulární modely MeSH
• muramidasa * chemie   metabolismus   MeSH
• organoplatinové sloučeniny * chemie   metabolismus   MeSH
• pankreatická ribonukleasa * chemie   metabolismus   MeSH
• skot MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• hen egg lysozyme MeSH Prohlížeč

A novel positively charged surfactant N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride was used for the dynamic coating of the inner wall of a silica capillary. This paper covers the evaluation of dynamic coating and study of the influence of the analysis conditions for the magnitude and direction of electroosmotic flow as well as for the effective and selective separation of chosen proteins (ribonuclease A, cytochrome c, lysozyme, and myoglobin). The concentration of 0.1 mM of N-dodecyl-N,N-dimethyl-(1,2-propandiol) ammonium chloride enabled the reversal of the electro-osmotic flow, however, to separate basic as well as neutral proteins the higher concentration of the studied surfactant was necessary. The final conditions for the separation of studied proteins were set at 100 mM sodium acetate pH 5.5 with 10.0 mM of the studied surfactant. The results were also compared with those of two commercially available cationic surfactants, cetyltrimethylammonium bromide and dodecyltrimethylammonium bromide. Additionally, the developed method for protein separation was applied for the determination of lysozyme in a cheese sample. The limits of detection and quantification of lysozyme were 0.9 and 3.0 mg/L, respectively. The mean concentration of lysozyme found in the cheese sample was 167.3 ± 10.3 mg/kg.

• Klíčová slova
• Capillary electrophoresis, Cationic surfactants, Dynamic coating, Lysozyme, Proteins,
• MeSH
• cytochromy c chemie   izolace a purifikace   MeSH
• elektroforéza kapilární MeSH
• kationty chemie   MeSH
• muramidasa chemie   izolace a purifikace   metabolismus   MeSH
• myoglobin chemie   izolace a purifikace   MeSH
• oxid křemičitý chemie   MeSH
• pankreatická ribonukleasa chemie   izolace a purifikace   MeSH
• povrchově aktivní látky chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytochromy c MeSH
• kationty MeSH
• muramidasa MeSH
• myoglobin MeSH
• oxid křemičitý MeSH
• pankreatická ribonukleasa MeSH
• povrchově aktivní látky MeSH

Polyspermine-ribonuclease A (PS-RNase A) and polyspermine-dimeric ribonuclease A (PS-dimeric RNase A) were prepared by cross-linking ribonuclease A or its covalently linked dimer to polyspermine (PS) using dimethyl suberimidate. The two RNase A derivatives were tested for a possible antitumor action. The in vitro and in vivo cytotoxic activity of PS-RNase A, although strong, is not higher than that known for free polyspermine. PS-dimeric RNase A, which was characterized by mass spectroscopy, titration of free amine groups, and enzymatic assays, proved instead to be a definitely more efficient antitumor agent, both in vitro and in vivo. This result could tentatively be explained in view of the importance of positive charges for ribonuclease activity, considering the higher basicity of PS-dimeric RNase A compared to that of PS-(monomeric)RNase A. It must be also taken into account that the dimeric RNase A moiety of PS-dimeric RNase A could evade the cytoplasmic ribonuclease inhibitor, which instead could trap the monomeric RNase A moiety of the other derivative. The two RNase A derivatives degrade poly(A).poly(U) under conditions where native RNase A is inactive. The results of this work demonstrate once again the importance of positive charges for the functions of mammalian pancreatic type ribonucleases in general, in particular for RNase A derivatives, and the potential therapeutic use of the ribonuclease A derivatives.

• MeSH
• dimerizace MeSH
• gelová chromatografie MeSH
• kultivované buňky MeSH
• lidé MeSH
• molekulární struktura MeSH
• pankreatická ribonukleasa chemie   izolace a purifikace   metabolismus   toxicita   MeSH
• proliferace buněk účinky léků   MeSH
• reagencia zkříženě vázaná chemie   MeSH
• RNA metabolismus   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• spermin chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pankreatická ribonukleasa MeSH
• reagencia zkříženě vázaná MeSH
• RNA MeSH
• spermin MeSH

Previously we have shown that monomeric RNase A has no significant biological activity, whereas its oligomers (dimer to tetramer) prepared by lyophilizing from 50% acetic acid solutions, show remarkable aspermatogenic and antitumor activities. Furthermore, conjugates prepared by chemical binding of native RNase A to polyethylene glycol (PEG) have shown a significant aspermatogenic and antitumor activities. In this work we show that the chemical conjugation of PEG to the RNase A C-dimer, and to the two RNase A trimers (NC-trimer and C- trimer) decreases the aspermatogenic activity of the oligomers while increasing their inhibitory activity on the growth of the human UB900518 amelanotic melanoma transplanted in athymic nude mice. Moreover, the PEG-conjugated RNaseA oligomers are devoid, like the free oligomers, of any embryotoxic activity.

• MeSH
• antispermatogenní látky farmakologie   MeSH
• dimerizace MeSH
• embryo savčí účinky léků   MeSH
• lidé MeSH
• melanom experimentální farmakoterapie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• pankreatická ribonukleasa chemie   farmakologie   MeSH
• peptidové fragmenty chemie   farmakologie   MeSH
• polyethylenglykoly chemie   farmakologie   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• spermatogeneze účinky léků   MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antispermatogenní látky MeSH
• pankreatická ribonukleasa MeSH
• peptidové fragmenty MeSH
• polyethylenglykoly MeSH
• protinádorové látky MeSH

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.

• MeSH
• buněčné dělení účinky léků   MeSH
• dimerizace MeSH
• embryo savčí účinky léků   MeSH
• konformace proteinů MeSH
• lidé MeSH
• melanom farmakoterapie   MeSH
• myši MeSH
• nádorové buňky kultivované MeSH
• pankreatická ribonukleasa chemie   izolace a purifikace   farmakologie   MeSH
• peptidové fragmenty farmakologie   MeSH
• protinádorové látky farmakologie   MeSH
• skot MeSH
• spermatogeneze účinky léků   MeSH
• transplantace heterologní MeSH
• vztahy mezi strukturou a aktivitou MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• pankreatická ribonukleasa MeSH
• peptidové fragmenty MeSH
• protinádorové látky MeSH

RNase A (bovine pancreatic ribonuclease) and BS-RNase (bovine seminal ribonuclease) are monomeric and dimeric enzymes, respectively, with aspermatogenic and antitumor activities. While the aspermatogenic and, in some experimental situations, the antitumor effects of the RNase A are only minor, the activity of BS-RNase in these phenomena is very significant. These differences can be annulled by means of conjugation of the enzymes with PEG (polyethylene glycol) chains. Aspermatogenic activity was studied histologically following subcutaneous injections of RNase A and BS-RNase conjugates in ICR mice, and the antitumor activity in athymic nude mice with growing human melanoma with i.p. injection of these conjugated ribonucleases. The experiments proved that RNase A, when conjugated to PEG, produced identical aspermatogenic and antitumour effects as BS-RNase conjugated to this polymer. Immunogenicity of RNase A and BS-RNase did not change substantially after the conjugation with PEG polymers. Binding of produced antibodies to both ribonucleases attached to PEG, however, was substantially reduced.

• MeSH
• antispermatogenní látky chemie   farmakologie   MeSH
• endoribonukleasy chemie   farmakologie   MeSH
• injekce intraperitoneální MeSH
• injekce subkutánní MeSH
• lidé MeSH
• melanom experimentální farmakoterapie   MeSH
• myši inbrední ICR MeSH
• myši nahé MeSH
• myši MeSH
• pankreatická ribonukleasa chemie   farmakologie   MeSH
• polyethylenglykoly chemie   MeSH
• protinádorové látky chemie   farmakologie   MeSH
• screeningové testy protinádorových léčiv MeSH
• skot MeSH
• sperma chemie   MeSH
• spermatogeneze účinky léků   MeSH
• testis účinky léků   MeSH
• tvorba protilátek účinky léků   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antispermatogenní látky MeSH
• endoribonukleasy MeSH
• pankreatická ribonukleasa MeSH
• polyethylenglykoly MeSH
• protinádorové látky MeSH
• ribonuclease SPL MeSH Prohlížeč

Recently hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for BS-RNase modification to prevent its degradation in bloodstream or fast elimination. Polymer-conjugated BS-RNase preparations proved to be cytotoxic after intravenous or intraperitoneal application, whereas native BS-RNase was ineffective. Here RNase A unimer was conjugated with two HPMA polymers (classic and star) and their antitumor effects both in vitro and in vivo were compared with those of BS-RNase polymers. Surprisingly, the antitumor effect of RNase A conjugates was also pronounced. The RNase A conjugates (classic and star) injected intravenously to mice bearing melanoma tumor caused a significant reduction in tumor volume following ten doses of 5 and 1 mg/kg, respectively. Despite the antitumor activity observed in vivo, the in vitro tested cytotoxic activity of RNase A did not differ from that caused by native RNase A while native BS-RNase (50 microg/ml) totally inhibited DNA synthesis in treated cells. The experiments with 125I-labeled preparations demonstrated concentration-dependent internalization of native BS-RNase by tumor cells within an hour, whereas the polymer conjugate (S-BS) was not internalized. On the contrary, the in vivo experiments showed that whereas 40% of S-BS conjugate persisted in bloodstream for 24h after administration, 98% of the native BS-RNase was already eliminated. Improved antitumor activities of PHPMA-modified RNases in vivo might be ascribed to their prolonged retention in bloodstream, better proteolytic stability and resistance to the action of the ribonuclease inhibitor.

• MeSH
• endoribonukleasy aplikace a dávkování   chemie   terapeutické užití   MeSH
• injekce intraperitoneální MeSH
• injekce intravenózní MeSH
• konformace proteinů MeSH
• kyseliny polymethakrylové aplikace a dávkování   chemie   MeSH
• lidé MeSH
• lymfocyty metabolismus   MeSH
• melanom experimentální farmakoterapie   patologie   MeSH
• molekulární struktura MeSH
• myši nahé MeSH
• myši MeSH
• nádorové buňky kultivované metabolismus   MeSH
• nosiče léků MeSH
• pankreatická ribonukleasa aplikace a dávkování   chemie   terapeutické užití   MeSH
• protinádorové látky aplikace a dávkování   chemie   terapeutické užití   MeSH
• radioizotopy jodu MeSH
• skot MeSH
• vazebná místa fyziologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• xenogenní modely - testy protinádorové aktivity MeSH
• způsoby aplikace léků MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• skot MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Duxon MeSH Prohlížeč
• endoribonukleasy MeSH
• kyseliny polymethakrylové MeSH
• nosiče léků MeSH
• pankreatická ribonukleasa MeSH
• protinádorové látky MeSH
• radioizotopy jodu MeSH
• ribonuclease SPL MeSH Prohlížeč

Magnetic hydrogel microspheres 1.5 microm in size were prepared by dispersion copolymerization of 2-hydroxyethyl methacrylate and ethylene dimethacrylate in the presence of magnetite, which formed the core of the particles. RNase A was coupled to the particles by the cyanuric chloride method. Gel electrophoresis of plasmid DNA pUC 19 (contaminated by bacterial RNA) confirmed RNA degradation with the immobilized enzyme. The effect of temperature and pH on the relative activity of immobilized RNase A was estimated after incubation of the samples at different temperatures (30-80 degrees C) and pH (4.0-8.0). Maximum relative activity was observed at 70 degrees C and pH 6.5. The matrices based on magnetic poly(HEMA) had a low tendency to adsorb RNA.

• MeSH
• bakteriální RNA metabolismus   MeSH
• biotechnologie metody   MeSH
• enzymy imobilizované chemie   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• magnetismus MeSH
• methakryláty chemie   MeSH
• mikrosféry * MeSH
• pankreatická ribonukleasa chemie   metabolismus   MeSH
• polyhydroxyethylmethakrylát chemie   MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální RNA MeSH
• enzymy imobilizované MeSH
• ethylene dimethacrylate MeSH Prohlížeč
• methakryláty MeSH
• pankreatická ribonukleasa MeSH
• polyhydroxyethylmethakrylát MeSH

Bovine seminal ribonuclease (BS RNase) displays immunosuppressive and antitumor activities on mammalian cells, whereas bovine pancreatic ribonuclease (RNase A) is not cytotoxic. To learn more about the mechanism of BS RNase cytotoxicity, various mutants and hybrid proteins were prepared. A series of RNase A variants substituted with amino acid residues from BS RNase were prepared. Concerning quaternary structure, a significant impact was achieved in the variant TM (Q28L K31C S32C), which forms a dimer joined covalently by two intersubunit disulfide bonds. This variant is more efficient than RNase A but less active than BS RNase. Introduction of cationic residues at positions 55, 62, and 64 or substitution at positions 111 and 113 enhanced the immunosuppressive activity of RNase A but did not confer its antitumor activity. The substitution at positions 28, 31, 32, 55, 62, 64, 111, and 113 in variant T13 exerted the best immunosuppressive and antitumor effect observed among the round of the RNase A variants. Replacement of the active-site histidine residues H12 and H119 with asparagine led to the loss of both catalytic and biological activities. Five previously prepared hybrid enzymes (SRA 1-5), synthesized by introducing 16 amino acid residues from RNase A into BS RNase, exerted the same immunosuppressive activities as did the wild-type BS RNase. However, the substitution at positions 111, 113, and 115 in variant SRA 5 caused a marked decrease in its antitumor effect, indicating that these residues play an important role in antitumor efficiency. A different mechanism of action of RNases on tumor cells and/or on blastogenic transformed lymphocytes has been assumed.

• Klíčová slova
• Non-programmatic,
• MeSH
• buněčné dělení účinky léků   MeSH
• buňky K562 MeSH
• imunosupresiva chemie   metabolismus   farmakologie   MeSH
• konformace proteinů MeSH
• lidé MeSH
• lymfocyty cytologie   účinky léků   MeSH
• mutace MeSH
• pankreatická ribonukleasa chemie   metabolismus   farmakologie   MeSH
• protinádorové látky chemie   metabolismus   farmakologie   MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• imunosupresiva MeSH
• pankreatická ribonukleasa MeSH
• protinádorové látky MeSH