Biomedical subjects

J Matousek

Publications and source records attributed to J Matousek.

At least 19 recordsLinked to original sources

PubMed2006

Some biological actions of PEG-conjugated RNase A oligomers.

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.

Animals↗

PubMed2006

Mung bean sprout (Phaseolus aureus) nuclease and its biological and antitumor effects.

Bovine seminal ribonuclease (BS RNase), a dimeric homolog of bovine pancreatic ribonuclease (RNase A), is known to display special biological activities namely cytotoxicity for human tumor cells. Because some plant ribonucleases have a similar mass weight and structure as the animal ribonuclease, effects of a commercial product of Mung bean (Phaseolus aureus) nuclease (PhA) were studied on proliferation of ML-2 human tumor cells, as well as it's aspermatogenic, embryotoxic, immunogenic, and immunosuppressive activity, and therapeutic efficiency in athymic mice bearing human melanoma tumor. Concerning the antiproliferative activity, PhA nuclease was almost non-effective in vitro on ML-2 cells and also immunosuppressive activity on human lymphocyte in mixed culture was very low compared to that of BS RNase. However, significant antitumor activity was detected on human melanoma tumor after intratumoral or intraperitoneal administration into the mice. Furthermore conjugate of PhA nuclease with polyethylene glycol (PEG) injected seven times at the dose of 10 microg intraperitoneally showed identical antitumor activity as that of bovine seminal ribonuclease (BS RNase) injected by the same way at ten times higher dose. Both PhA and BS RNases exerted strong aspermatogenic effect on the width of spermatogenic layers while RNase A administration at ten times higher concentration was ineffective. PhA nuclease when compared by means of antibody cross reaction with RNase A, BS RNase and wheat leaf neutral RNase (WLN-RNase) was found to be immunologically similar to RNase A and WLN-RNase, meanwhile BS RNase showed much higher antigenicity in comparison with them.

Animals↗

PubMed2005

Complete nucleotide sequence and molecular probing of potato virus S genome.

Complete genomes of three isolates of Potato virus S (PVS) were cloned and sequenced. The PVS ORF-1 was characterized for the first time. It encodes a putative replication protein (RPT) that shares the highest homology (about 52%) with that of Blueberry scorch virus (BlScV). ORF-1 motifs, characteristic for carlaviruses were found for methyltransferase (MTR), helicase (HEL) and RNA-dependent RNA polymerase (RdRp). The complete sequence of PVS genome enabled to develop an immunocapture RT-PCR probing of the PVS genome. Using this system, the sequence variability of 11 genome zones was examined for 34 PVS isolates including 15 PVS-CS variants that caused a systemic infection in Chenopodium quinoa. A broad variability between PVS isolates and diverse sequence variants was found. cDNA fragments covering the coat protein (CP) leader and CP-coding region (approx. 420 bp) were pooled for PVS-O and Chenopodium-systemic PVS isolates (PVS-CS) and corresponding cDNA libraries were screened for sequence variants. Both cDNA pools differred mainly in the 5'-end of the CP gene. Methionine at the position 17 in combination with serine at the position 34 were frequently associated with the CS character of PVS. In general, hydrophobic and polar amino acids were characteristic for the positions 17 and 34, respectively in PVS-CS isolates. Genome probing and evolutionary distances suggested that the PVS-CS isolates analyzed were close to the ordinary European isolates of ordinary strain of PVS (PVS-O) but distant to the original Andean strain of PVS (PVS-A).

Carlavirus↗

PubMed2004

Biolistic inoculation of plants with viroid nucleic acids.

Parameters for biolistic transfer of viroid nucleic acids using a Helios Gene Gun device were assayed. The main achievement of this method is high efficiency of inoculation with linear monomeric viroid cDNAs and RNAs. This greatly facilitates the study of mutated sequence variants, viroid libraries and mixed populations. The lower limits for efficient inoculation of monomeric cDNA fragments with the sequence of potato spindle tuber viroid (PSTVd) and native PSTVd RNA as detected 21 days p.i. are in the range of 50 ng and 200 pg per tomato plant, respectively. At a higher dose, i.e. 2 ng of native RNA per plant, biolistic transfer causes drastic stunting compared to conventional mechanical inoculation, which points to higher PSTVd titers after the biolistic transfer. Infection is readily achieved with exact length monomeric RNA transcripts having 5'-triphosphate and 3'-OH termini in amounts ranging from 2 to 20 ng per plant, suggesting no need for any supplementary modifications of ends or RNA circularization. The biolistic transfer is efficient for viroid "thermomutants", which exhibit low or no infectivity with conventional mechanical inoculation with Carborundum. The biolistic inoculation is also efficient for two other members of the Pospiviroidae family, hop stunt and hop latent viroid.

Biolistics↗

PubMed2004

Polymer-conjugated bovine pancreatic and seminal ribonucleases inhibit growth of human tumors in nude mice.

The hydrophilic poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) was used for RNase A or BS-RNase modification to prevent their degradation in bloodstream or fast elimination. Two PHPMA chains (classic and star-like) were synthesized and their conjugates with both enzymes were tested on the CD-1 nude mice bearing various human tumors. These RNase conjugates injected intravenously or intraperitoneally into the mice bearing melanoma, neuroblastoma or ovarian tumor caused significant reduction of transplanted tumors following ten daily doses of 2.5 and/or 1 mg/kg, respectively, while free RNase A or BS-RNase injected in doses of 10 mg/kg exerted only negligible antitumor activity. Histological examination confirmed potent cytotoxic effect of RNase A conjugates in ovarian tumor. Despite the antitumor activity observed in vivo, the in vitro cytotoxic activity of RNase A conjugates was not pronounced and did not differ from that caused by the free RNase A. The in vitro experiments with 125I-labeled preparations demonstrated that polymer conjugates were internalized by tumor cells very poorly in contrast to the dose-dependent internalization of the wild enzyme preparation. Surprisingly, mice injected with EL-4 leukemic cells, which were preincubated for 4 h with BS-RNase conjugates, exerted significantly prolonged survival compared with the control non-treated mice. It may be supposed that both BS-RNase and RNase A conjugates with PHPMA act after administration in vivo by a mechanism different from that or those occurring under in vitro conditions because in vivo they exert an antitumor action, whereas in vitro, they are ineffective. The experiments proved that RNase A, when conjugated to PHPMA, produced identical aspermatogenic and antitumor effects as BS-RNase conjugated to this polymer and that this preparation may be regarded as a potential anticancer drug.

Animals↗

PubMed2002

Analysis of transcription of plant 7SL RNA gene variants in HeLa in vitro transcription system.

We have performed an analysis of transcription of hop 7SL RNA genes in heterologous human extract from HeLa cells. Several variants of the Hl7SL-1 gene with a truncated or mutated 5' non-transcribed part revealed a crucial importance of TATA box for transcription. The USE element was found important but dispensable for transcription. Transcription of mutants in the A-like box and experiments with hop 7SL RNA pseudogenes E44 and G32 revealed the importance of internal elements. The A-like box and possibly CG doublet at position +15/+16 described by Bredow et al. (1990a) are according to our results indispensable for in vitro transcription of plant 7SL RNA genes in human extract.

Base Sequence↗

PubMed2002

Immunocapture RT-PCR probing of potato virus Y isolates.

Twenty potato virus Y (PVY) isolates were characterized. They represented two strains only, PVY(O) (three isolates) and PVY(N) (17 isolates). However, application of serological and molecular genetic methods led to a more complicated characterization. For example, five isolates induced necrotic symptoms on tobacco plants typical of PVY(N), despite reacting as PVY(O) serologically. Moreover, the PVY isolates were not identical according to molecular genetic properties. Typical PVY(NTN) PCR products were observed for 14 isolates, but five of them (Hr 220-5, Hr 387-7, Nord 242, Syn1Scot, and 41-97) did not produce potato tuber necrotic symptoms in infected cultivars. An immunocapture reverse transcription-polymerase chain reaction (RT-PCR) probing was developed using a set of 24 primer pairs derived from eight regions of the PVY genome. Using this method, five out of seven PVY(NTN) isolates including the Czech standard PVY(NTN) from the potato cv. Nicola were found to be identical. However, two PVY(NTN) isolates and all the other probed PVY samples showed unique patterns, suggesting specific differences at the nucleotide level. This method enabled specific identification of individual isolates variability even within different PVY strains.

DNA Primers↗

PubMed2001

The variability of hop latent viroid as induced upon heat treatment.

We have previously shown that heat treatment of hop plants infected by hop latent viroid (HLVd) reduces viroid levels. Here we investigate whether such heat treatment leads to the accumulation of sequence variability in HLVd. We observed a negligible level of mutated variants in HLVd under standard cultivation conditions. In contrast, the heat treatment of hop led to HLVd degradation and, simultaneously, to a significant increase in sequence variations, as judged from temperature gradient-gel electrophoresis analysis and cDNA library screening by DNA heteroduplex analysis. Thirty-one cDNA clones (9.8%) were identified as deviating forms. Sequencing showed mostly the presence of quadruple and triple mutants, suggesting an accumulation of mutations in HLVd during successive replication cycles. Sixty-nine percent of base changes were localised in the left half and 31% in the right half of the secondary structure proposed for this viroid. No mutations were found in the central part of the upper conserved region. A "hot spot" region was identified in a domain known as a "pathogenicity domain" in the group representative, potato spindle tuber viroid. Most mutations are predicted to destabilise HLVd secondary structure. All mutated cDNAs, however, were infectious and evolved into complex progeny populations containing molecular variants maintained at low levels.

Base Sequence↗

PubMed2001

Ribonucleases and their antitumor activity.

The antitumor effect of ribonucleases was studied with animal ribonucleolytic enzymes, bovine pancreatic RNase A, bovine seminal RNase (BS-RNase), onconase and angiogenin. While bovine pancreatic RNase A exerts a minor antitumor effect, BS-RNase and onconase exert significant effects. Angiogenin, as RNase, works in an opposite way, it initiates vascularization of tumors and subsequent tumor growth. Ribonunclease inhibitors are not able to inhibit the antitumor effectiveness of BS-RNase or onconase. However, they do so in the case of pancreatic RNases. Conjugation of BS-RNase with antibodies against tumor antigens (preparation of immunotoxins) like the conjugation of the enzyme with polymers enhances the antitumor activity of the ribonuclease. After conjugation with polymers, the half-life of BS-RNase in blood is extended and its immunogenicity reduced. Recombinant RNases have the same functional activity as the native enzymes. The synthetic genes have also been modified, some of them with gene sequences typical for the BS-RNase parts. Recent experimental efforts are directed to the preparation of 'humanized antitumor ribonuclease' that would be structurally similar to human enzyme with minimal immunogenicity and side effects. The angiogenesis of tumors is attempted to be minimized by specific antibodies or anti-angiogenic substances.

Animals↗

PubMed2000

Effect of bovine seminal ribonuclease and bovine pancreatic ribonuclease A on bovine oocyte maturation.

Bovine seminal ribonuclease (BS-RNase) contains the MxM (noncovalent dimer) and M=M (free monomer) in constant ratio. The aim of this work was to evaluate the effect of BS-RNase, its monomer and dimer forms, and also various mutants of this enzyme on meiotic completion in cattle oocytes. It was found that BS-RNase has irreversible effects on the meiotic maturation of bovine oocytes in vitro, particularly on the completion of meiosis. The effect of BS-RNase is dose-dependent. In medium supplemented with 1 microg/ml, the results were comparable with those of the control (70% MII oocytes after 24 hr of culture). Whereas 5 microg/ml reduced the number of MII oocytes to 50%, 10 and 25 microg/ml arrested this process completely. The MxM form and RNase A at 5 microg/ml inhibited the maturation rate by 71 and 48%, respectively, but a less significant effect was observed for the M=M form, or the carboxymethylated monomers MCM31 and MCM32 (21%, 16%, and 42% MII oocytes, respectively, in comparison with control). These data demonstrate that bovine ribonucleases can have variable detrimental effects on the maturation of bovine oocyte. J. Exp. Zool. 287:394-399, 2000.

Animals↗

PubMed2000

Bovine seminal ribonuclease attached to nanoparticles made of polylactic acid kills leukemia and lymphoma cell lines in vitro.

Bovine seminal ribonuclease (BS-RNase) is a protein with a number of biological effects. It shows antitumoral, aspermatogenic, antiembryonic, immunosuppressive and antiviral properties. The cytotoxic effects appear to be specific for tumor cells as non-malignant cells seem to be unaffected in vitro. Unfortunately, the in vivo application of BS-RNase so far was successful only when it was administered intratumorally. Therefore, the objective of the present investigation was to improve the properties of BS-RNase by attachment to nanoparticles made of polylactic acid (PLA-NP) using an adsorption method. This preparation was tested in vitro against leukemia (MOLT-4) and lymphoma (H9) cell lines sensitive and resistant to cytarabine. No difference between the nanoparticle preparation and pure BS-RNase was found in these tests. To examine the in vivo effects, the preparations were tested for their aspermatogenic and antiembryonal efficacy compared to the pure BS-RNase as a rapid test for antitumoral activity. The aspermatogenic and antiembryonal effects were enhanced by the nanoparticle preparation. Consequently, BS-RNase loaded adsorptively to PLA-NP holds promise for the in vivo use as an antitumoral agent. Further research will investigate the efficacy of this preparations in an in vivo tumor model.

Animals↗

PubMed2000

Analysis of variability of P1 gene region of N strain of potato virus Y using temperature-gradient gel electrophoresis and DNA heteroduplex analysis.

Reaction conditions specific for reverse taranscription-polymerase chain reaction (RT-PCR) of potato virus Y strain NTN (PVYNTN) were used to amplify a 394 bp fragment of the P1 gene from selected PVY isolates with the aim to study the PVY variability within this genomic region. The P1 gene fragment from the Nicola isolate (Nicola P1/1 clone) was sequenced and characterized by temperature-gradient gel electrophoresis (TGGE). The Nicola P1/1 clone differed from that from the Hungarian isolate by double point mutation resulting in two changes at the deduced amino acid level. The clone showed simple transition from double-stranded to single-stranded form with two characteristic melting end points of about 41 degrees C and 48 degrees C. A more complicated TGGE pattern was, however, found for the whole P1 cDNA library of the Nicola isolate, suggesting accumulation of some minor sequence variants of PVY in this isolate. Based on the TGGE pattern, 46 degrees C was selected as the standard temperature for electrophoretic analysis of heteroduplex DNAs formed with the Nicola P1/1 DNA as reference. More than 40 other PVY isolates from PVYN group were analysed using this method. In most cases only minor fractions of electrophoretically distinguishable DNA heteroduplexes were found, however, in most isolates of PVYN-Wilga type, mixtures of the major sequence variants were observed. Two of these variants from the hybrid 220-5 (Czech Republic) were sequenced. Both of them differed from the Nicola P1/1 clone by 6 point mutations, which led to several changes at the amino acid level.

Amino Acid Sequence↗

PubMed1999

Molecular characterization and genome organization of 7SL RNA genes from hop (Humulus lupulus L.).

A wide spectrum of hop 7SL RNA-encoding sequences was detected by temperature gradient-gel electrophoresis. Four hop 7SL RNA genes were cloned and characterized. A new subvariant of the upstream sequence element (USE) 5'TCCCACATGG 3' and two distinct variants of TATA signal were found at positions characteristic for RNA polymerase III-driven transcription in plants. In addition, a more distant conserved sequence element 5' CATGTATAAACTTTCTGC 3' was present in all cloned genes, about 160 bp upstream of the 7SL RNA coding sequence. Consensus secondary structures calculated for hop 7SL RNAs revealed characteristic features, although some structure differences from formerly published models were predicted. Specific in-vitro transcription of plant 7SL RNA genes was observed in a heterologous system (HeLa extract). This in-vitro transcription assay showed significant differences among individual clones in transcription rates, suggesting the requirement of complexity of 7SL RNA sequence for its efficient transcription in HeLa extract. Southern blot analysis of hop DNA revealed 12 7SL-specific signals corresponding to HindIII fragments ranging from 0.45 to 7.8 kb. Several 7SL RNA-encoding sequences and various intergenic spacers were amplified from the individual HindIII fragments of about 1.3 and 2.8 kb. These facts suggest that at least some of the hop 7SL RNA genes are organized in genomic clusters.

Base Sequence↗

PubMed1999

A dimeric mutant of human pancreatic ribonuclease with selective cytotoxicity toward malignant cells.

Monomeric human pancreatic RNase, devoid of any biological activity other than its RNA degrading ability, was engineered into a dimeric protein with a cytotoxic action on mouse and human tumor cells, but lacking any appreciable toxicity on mouse and human normal cells. This dimeric variant of human pancreas RNase selectively sensitizes to apoptotic death cells derived from a human thyroid tumor. Because of its selectivity for tumor cells, and because of its human origin, this protein represents a potentially very attractive, novel tool for anticancer therapy.

3T3 Cells↗

PubMed1999

Bovine seminal ribonuclease selectively kills human multidrug-resistant neuroblastoma cells via induction of apoptosis.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with specific antitumor activity. The cytotoxic action of this agent was examined in human neuroblastoma (NB) cell lines (SK-N-SH and UKF-NB-4) possessing the multidrug resistance (MDR) phenotype and NB cell lines (IMR-32, UKF-NB-1, UKF-NB-2 and UKF-NB-3) without MDR. Although MDR cells expressed large amounts of mdr-1 mRNA, contained functional P-glycoprotein and had 20- to 105-fold lower sensitivities to doxorubicin and vincristine than cells with non-MDR phenotypes, BS-RNase was equally toxic to all NB cells at concentrations employed (0.2 to 100 microg/ml). BS-RNase showed high selectivity for NB cells and was non-toxic to normal fibroblasts and epithelial cells. Ultrastructural investigation and annexin V assay showed that BS-RNase is a powerful inductor of apoptosis. The antitumoral effects of BS-RNase were also demonstrated in vivo using established subcutaneous xenografts in athymic (nude) mice of the MDR-1-bearing UKF-NB-4 cell line. Intratumoral injections (12.5 mg/kg) of BS-RNase over four weeks resulted in complete tumor regression and absence of tumor regrowth over a two-week observation period after cessation of treatment. The results show that BS-RNase selectively kills NB cells by inducing apoptosis and that this agent is active against mdr-1 expressing cells both in vitro and in vivo. BS-RNase fulfills important criteria for a candidate antitumor agent in NB patients with advanced disease.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Qualitative and quantitative analyses of seminal ribonuclease in reproductive tract fluids of bulls.

Bovine seminal ribonuclease (BS RNase) is synthetized in the ampulary gland and seminal vesicles of the bull as shown by indirect immunofluorescence and by using a sensitive sandwich-linked immunosorbent assay (ELISA). The ampullary and seminal vesicle BS RNase concentrations in samples of 15 bulls were 656 micrograms/ml and 1285 micrograms/ml, respectively. Seminal plasma BS RNase levels in 22 breeding bulls were slightly lower than in the seminal vesicles--1132 micrograms/ml. There were no significant differences in the concentration of this enzyme in seminal plasma of 16 bulls ranked as having high and low fertility. Even with its immunosuppressive activity this enzyme does not seem important for the protection of sperm cells in the female reproductive tract.

Animals↗

PubMed1998

Antitumor action of bovine seminal ribonuclease.

Unlike the bovine pancreatic ribonuclease (RNAase A), bovine seminal ribonuclease (BS RNAase) displays various biological activities including antitumor cytotoxicity. To learn more about its antitumor activity, we investigated BS RNAase effect on athymic nude mice bearing various tumors. BS RNAase (250 micrograms per mouse per day) was administered to the mice with prostate carcinoma for three weeks by three different routes (intraperitoneally--i.p., subcutaneously--s.c., and intratumorally-i.t.). Administration i.p. was ineffective, while s.c. administration reduced significantly size of tumors and i.t. administration abolished half of the tumors in treated mice. The i.t. administration of BS RNase to nude mice bearing melanoma showed even better results. Eighty % of mice were without tumors and in the other mice the tumors were significantly diminished. The best antitumor effect was obtained in case of seminoma. All mice bearing this tumor were cured after ten doses of BS RNAase.

Animals↗

PubMed1998

Antitumor action of bovine seminal ribonuclease. Cytostatic effect on human melanoma and mouse seminoma.

This paper reports on the antitumor activity of BS RNase on human melanoma and mouse seminoma. Human melanoma cells established in culture were extremely susceptible to BS RNase, administered in concentrations ranging from 1-100 microg/ml. Concentrations of BS RNase over 10 microg/ml caused complete inhibition of cell growth. Bovine pancreatic ribonuclease (RNase A), a prototype of the ribonuclease superfamily, did not exert any effect under these conditions. Based on our previous results, athymic mice bearing human melanoma or mouse seminoma were treated with intratumoral administration of BS RNase (12.5 mg/kg b.w.). This dose was injected for five consecutive days excluding weekends. The intratumoral administration of BS RNase to nude mice bearing human melanoma showed a significant antitumor effect. There were no tumors seen in eighty percent of mice treated for three weeks, and tumors in the other mice diminished significantly. After some delay the tumors started to regrow. Prolonging of the treatment to five weeks had a similar effect. The effect of BS RNase on mouse seminoma was well pronounced. Five to seven doses of BS RNase were sufficient to eliminate tumors in all treated mice. However, as in the previous experiment, the growth of tumor tissue later reappeared.

Animals↗