PubMed Health⌕ Search

Biomedical subjects

J Burian

Publications and source records attributed to J Burian.

At least 19 recordsLinked to original sources
PubMed2004

Development of equipments for determination of BNCT source spectral parameters.

The knowledge of neutron and gamma ray energy spectra can strongly influence the BNCT information about delivered dose to target volume as well as to the surface healthy tissue region. This region is very often decisive to stay within the recommended healthy tissue limit. Modification of neutron Bonner spectrometer to one block i.e. Bonner spectrometer monoblock (BSM) and gamma ray Si semiconductor spectrometer are being developed and verified in real conditions of LVR-15 reactor beam. Test measurements were also carried out in conditions of known standard spectra. The accepted procedure and the first results documenting the sensitivity BSM to different spectra are presented.

Boron Neutron Capture Therapy↗
PubMed2004

[Neutron capture therapy in the treatment of glioblastoma multiforme. Initial experience in the Czech Republic].

BACKGROUND: Glioblastoma multiforme is the most frequent primary brain tumor in adults. Despite advances in surgery, radiotherapy and chemotherapy, its treatment remains unsatisfactory with very limited overall survival. In the year 2001, in cooperation with Department of Neurosurgery, Nemocnice Na Homolce and Nuclear Research Institute in Rez, we have started to treat glioblastoma patients with boron neutron capture therapy (BNCT). METHODS AND RESULTS: Cells of malignant brain tumors, especially that of glioblastomas, are able to accumulate boron compounds. If BNCT should be successful, it is necessary to reach selective accumulation of sufficient amount of 10B in the tumor and low accumulation in the normal brain tissue. After BSH administration, radiation with low energy thermal neutrons is delivered. It results in nuclear capture and fission reactions with subsequent selective damage of tumor cells. At the time of analysis 9 patients have been enrolled. Therapy was completed in 5 patients. Treatment has been very well tolerated. We observed minimal acute toxicity associated with radiation and no laboratory abnormalities after administrations of BSH. Unfortunately treatment results were quite unsatisfactory. The median time to progression and overall survival were shorter then expected with conventional treatment. CONCLUSIONS: BNCT is very well tolerated with only a modest toxicity. In contrast to standard radiation, BNCT patients receive only one dose of radiation. Nevertheless, in this small pilot study first results were inferior when compared either to outcomes of conventional therapy or to results reported from other BNCT groups. It might be explained that lower dose of radiation had been used. Further study will show whether the higher dose radiation can improve treatment results.

Adult↗
PubMed2001

An efficacious recombinant subunit vaccine against the salmonid rickettsial pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is the aetiological agent of salmonid rickettsial septicaemia, an economically devastating rickettsial disease of farmed salmonids. Infected salmonids respond poorly to antibiotic treatment and no effective vaccine is available for the control of P. salmonis. Bacterin preparations of P. salmonis were found to elicit a dose-dependent response in coho salmon (Oncorhynchus kisutch), which varied from inadequate protection to exacerbation of the disease. However, an outer surface lipoprotein of P. salmonis, OspA, recombinantly produced in Escherichia coli elicited a high level of protection in vaccinated coho salmon with a relative percent survival as high as 59% for this single antigen. In an effort to further improve the efficacy of the OspA recombinant vaccine, T cell epitopes (TCE's) from tetanus toxin and measles virus fusion protein, that are universally immunogenic in mammalian immune systems, were incorporated tandemly into an OspA fusion protein. Addition of these TCE's dramatically enhanced the efficacy of the OspA vaccine, reflected by a three-fold increase in vaccine efficacy. These results represent a highly effective monovalent recombinant subunit vaccine for a rickettsia-like pathogen, P. salmonis, and for the first time demonstrate the immunostimulatory effect of mammalian TCE's in the salmonid immune model. These results may also be particularly pertinent to salmonid aquaculture in which the various subspecies are outbred and of heterologous haplotypes.

Amino Acid Sequence↗
PubMed2001

OspA, a lipoprotein antigen of the obligate intracellular bacterial pathogen Piscirickettsia salmonis.

No effective recombinant vaccines are currently available for any rickettsial diseases. In this regard the first non-ribosomal DNA sequences from the obligate intracellular pathogen Piscirickettsia salmonis are presented. Genomic DNA isolated from Percoll density gradient purified P. salmonis, was used to construct an expression library in lambda ZAP II. In the absence of preexisting DNA sequence, rabbit polyclonal antiserum raised against P. salmonis, with a bias toward P. salmonis surface antigens, was used to identify immunoreactive clones. Catabolite repression of the lac promoter was required to obtain a stable clone of a 4,983 bp insert in Escherichia coli due to insert toxicity exerted by the accompanying radA open reading frame (ORF). DNA sequence analysis of the insert revealed 1 partial and 4 intact predicted ORF's. A 486 bp ORF, ospA, encoded a 17 kDa antigenic outer surface protein (OspA) with 62% amino acid sequence homology to the genus common 17 kDa outer membrane lipoprotein of Rickettsia prowazekii, previously thought confined to members of the genus Rickettsia. Palmitate incorporation demonstrated that OspA is posttranslationally lipidated in E. coli, albeit poorly expressed as a lipoprotein even after replacement of the signal sequence with the signal sequence from lpp (Braun lipoprotein) or the rickettsial 17 kDa homologue. To enhance expression, ospA was optimized for codon usage in E. coli by PCR synthesis. Expression of ospA was ultimately improved (approximately 13% of total protein) with a truncated variant lacking a signal sequence. High level expression (approximately 42% tot. prot.) was attained as an N-terminal fusion protein with the fusion product recovered as inclusion bodies in E. coli BL21. Expression of OspA in P. salmonis was confirmed by immunoblot analysis using polyclonal antibodies generated against a synthetic peptide of OspA (110-129) and a strong antibody response against OspA was detected in convalescent sera from coho salmon (Oncorhynchus kisutch).

Amino Acid Sequence↗
PubMed1999

Replication control of a small cryptic plasmid of Escherichia coli.

The role of the RepA initiator protein in replication and copy-number control of pKL1, a small cryptic plasmid of Escherichia coli, was elucidated. The identified ori region encompasses a copy-number control element (cop) and an active single-strand initiation signal (ssi), n'-pasH, which were essential for efficient plasmid replication. The cop region also harbors a region of plasmid incompatibility, inc, encompassing a stem-loop structure, the repA promoter, Prep, as well as two distinct RepA binding sites, BD-1 and BD-2. RepA was shown to bind to these sites quite differently, binding primarily as a monomer or dimer to BD-1 to initiate RepA transcription and plasmid replication, and as higher oligomers to BD-2 to autoregulate repA transcription, the balance being reflected in plasmid copy number. An active integration host factor (IHF) binding sequence was located in the cop region and plasmid replication was shown to be dependent on host IHF encoding genes himA and himD. Low concentrations of IHF predisposed the cop region to RepA binding, although when highly expressed in trans RepA effectively displaced bound IHF and it overcame IHF dependency. Incompatibility was shown to be due to the titration of RepA at the cop locus but could be easily overridden by excess RepA. Both RepA binding sites were required to maintain incompatibility and effective pKL1 replication. Neither antisense RNA nor iterons were found to be involved in pKL1 regulation, thus pKL1 is a novel example of autoregulation of DNA replication. When produced in excess from a helper plasmid, RepA induced pKL1 replication to unusually high levels (>2500 copies/cell). In addition, pKL1 replication could be artificially modulated and a wide range of copy numbers maintained.

Bacterial Proteins↗
PubMed1999

High efficiency gene replacement in Salmonella enteritidis: chimeric fimbrins containing a T-cell epitope from Leishmania major.

A simple, high frequency chromosomal gene replacement method of general utility was developed for Salmonella enteritidis. This system uses an unstable, imperfectly segregating, temperature-sensitive replicon, pHSG415, as a carrier of the recombinant gene of interest. It also allows for site-specific replacement of chromosomal genes without the need for antibiotic resistance markers in the recombinant genes or the use of specific bacterial strains. This strategy was used to replace the chromosomal sefA and agfA fimbrin genes of S. enteritidis 3b with recombinant genes containing a 48 bp DNA fragment encoding PT3, an immunoprotective T-cell epitope from GP63 of Leishmania major. The fidelity of chimeric fimbrial replacements were confirmed by DNA sequence analysis. Nearly 30% of the S. enteritidis clones selected in the final stage of sefA mutagenesis contained the sefA::PT3 recombinant gene, whereas for agfA the efficiency was as high as 10%. To our knowledge, this is the first report of fimbrial epitope replacement in the Salmonellae and the first chimeric fimbrin genes that have been reconstituted into a wild-type genetic background for any organism. As such, this model represents a promising 'organelle' expression system for epitope display in vaccinology.

Amino Acid Sequence↗
PubMed1998

Determinants of recombinant production of antimicrobial cationic peptides and creation of peptide variants in bacteria.

Cationic peptides possessing antibacterial activity are virtually ubiquitous in nature, and offer exciting prospects as new therapeutic agents. We had previously demonstrated that such peptides could be produced by fusion protein technology in bacteria and several carrier proteins had been tested as fusion partners including glutathione-S-transferase, S. aureus protein A, IgG binding protein and P. aeruginosa outer membrane protein OprF. However these fusion partners, while successfully employed in peptide expression, were not optimized for high level production of cationic peptides (Piers, K., Brow, M. L., and Hancock, R. E. W. 1993, Gene 137, 7-13). In this paper we took advantage of a small replication protein RepA from E. coli and used its truncated version to construct fusion partners. The minimal elements required for high level expression of cationic peptide were defined as a DNA sequence encoding a fusion protein comprising, from the N-terminus, a 68 amino acid carrier region, an anionic prepro domain, a single methionine and the peptide of interest. The 68 amino acid carrier region was a block of three polypeptides consisting of a truncated RepA, a synthetic cellulose binding domain and a hexa histidine domain. The improved system showed high level expression and simplified downstream purification. The active peptide could be yielded by CNBr cleavage of the fusion protein. This novel vector was used to express three classes of cationic peptides including the alpha-helical peptide CEMA, the looped peptide bactenecin and the extended peptide indolicidin. In addition, mutagenesis of the peptide gene to produce peptide variants of CEMA and indolicidin using the improved vector system was shown to be successful.

Amino Acid Sequence↗
PubMed1998

In vivo and in vitro cloning and phenotype characterization of tellurite resistance determinant conferred by plasmid pTE53 of a clinical isolate of Escherichia coli.

A determinant encoding resistance against potassium tellurite (Te(r)) was discovered in a clinical isolate of Escherichia coli strain KL53. The strain formed typical black colonies on solid LB medium with tellurite. The determinant was located on a large conjugative plasmid designated pTE53. Electron-dense particles were observed in cells harboring pTE53 by electron microscopy. X-Ray identification analysis identified these deposits as elemental tellurium and X-ray diffraction analysis showed patterns typical of crystalline structures. Comparison with JCPDS 4-0554 (Joint Committee on Powder Diffraction Standards) reference data confirmed that these crystals were pure tellurium crystals. In common with other characterized Te(r) determinants, accumulation studies with radioactively labeled tellurite showed that reduced uptake of tellurite did not contribute to the resistance mechanism. Tellurite accumulation rates for E. coli strain AB1157 harboring pTE53 were twice higher than for the plasmid-free host strain. In addition, no efflux mechanism was detected. The potassium tellurite resistance determinant of plasmid pTE53 was cloned using both in vitro and in vivo techniques in low-copy-number vectors pACYC184 and mini-Mu derivative pPR46. Cloning of the functional Te(r) determinant into high-copy cloning vectors pTZ19R and mini-Mu derivatives pBEf and pJT2 was not successful. During in vivo cloning experiments, clones with unusual "white colony" phenotypes were found on solid LB with tellurite. All these clones were Mucts62 lysogens. Their tellurite resistance levels were in the same order as the wild type strains. Clones with the "white" phenotype had a 3.6 times lower content of tellurium than the tellurite-reducing strain. Transformation of a "white" mutant with a recombinant pACYC184 based Te(r) plasmid did not change the phenotype. However, when one clone was cured from Mucts62 the "white" phenotype reverted to the wild-type "black" phenotype. It was suggested that the "white" phenotype was the result of an insertional inactivation of an unknown chromosomal gene by Mucts62, which reduced the tellurite uptake.

Cloning, Molecular↗
PubMed1997

Small cryptic plasmids of multiplasmid, clinical Escherichia coli.

Clinical isolates of Escherichia coli were found to host a multiplicity of plasmids. These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants of E. coli DH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities. The majority of these were unusually small, cryptic plasmids (SCPs). From one strain, KL4, 13 electrophoretic bands were resolved to five plasmids, three of which were SCPs. SCPs were phenotypically barren, and the smallest of these, pKL1, contained barely enough information for self-replication. A derivative of pKL1, pKL1Km, in which the transposon was restricted to a small 350-bp region, was stably maintained in Shigella, Salmonella, Serratia, and Citrobacter species and its replication was polA independent. pKL1 encoded only a single protein, RepA (Mr 17960), which specifically bound to pKL1 DNA. No apparent homologies with other RepA protein sequences could be detected. Thus the SCP, pKL1, is a novel minimal plasmid replicon encoding only enough information to ensure perpetuation. A hypothesis is presented describing SCPs as a class of selfish DNA that persists simply due to its ability to replicate and to its stability based on high copy number.

Conjugation, Genetic↗
PubMed1997

Monitoring of the boron neutron-capture reaction by a simple animal model.

Sodium borocaptate (BSH, Na2Bl2HllSH), a slow neutron-capture compound, was injected into the left forebrain ventricle of 1-week-old rats (150 microg BSH/3 microl phosphate buffered saline). After 90 min, the animals were irradiated by epithermal neutrons (LVR-15 nuclear reactor in Rez near Prague, flux density 8.8 x 10(7) neutrons cm-2 s-1, 8 MW reactor power, 8.2 cGy/min) for 5, 10 or 20 min. The brains were examined histologically 8 h after irradiation. In animals irradiated for 5 to 10 min (41 and 82 cGy-Eq, respectively) lethal damage of cells was found in the external granular layer of the cerebellum and the subependymal layer of the forebrain. Irradiation for 20 min (164 cGy-Eq) caused more extensive destruction of cell populations in these regions and, in addition, dead cells appeared also in the more differentiated postmitotic compartments, namely the deeper layers of the cerebellum, layers II/III of the cerebral cortex and corpus callosum. In the forebrain periventricular layer, the extent of cell damage was declining towards the olfactory bulbs. In intact animals, as well as in those injected only with the 150 microl phosphate buffered saline, the radiation damage was low and limited only to the most sensitive dividing populations of the cerebellum and the forebrain. The study demonstrates a differentiation-dependent damage of the rat brain cells by alpha particles and presents a simple model for evaluation of the biological effectiveness of slow neutron beams constructed for neutron-capture therapy of tumors.

Animals↗
PubMed1996

Diagnostic potential of sefA DNA probes to Salmonella enteritidis and certain other O-serogroup D1 Salmonella serovars.

Salmonella enteritidis thin fimbriae, SEF14, were found to be restricted to S. dublin and the predominantly poultry-associated members of the Salmonella O-serogroup D1, S. enteritidis, S. berta, S. gallinarum and S. pullorum, when tested by Western and ELISA analysis from among 90 Salmonella isolates of 42 serovars, as well as from members of several related genera of the Enterobacteriaceae. These five serovars and a single isolate of S. typhi (D1) were also detected by hybridization of genomic DNA from 732 Salmonella isolates of 117 serogroups to gene probes derived from the S. enteritidis sefA (fimbrin gene), sefB (chaperone) or sefC (outer membrane protein) genes encoding proteins involved in SEF14 biosynthesis. None of 250 Enterobacteriaceae or 27 other eubacterial isolates tested hybridized to the sef probes. The sefA, sefB and sefC genes were amplified from these six Salmonella serovars by PCR using primer pairs designed from sefA, sefB or sefC of S. enteritidis. DNA sequencing of sefA genes from these five serovars indicated limited sequence variability among sefA genes and recognition of individual base pairs which could potentially differentiate certain strains of S. enteritidis, S. dublin and S. gallinarum.

Animals↗
PubMed1994

fimA and tctC based DNA diagnostics for Salmonella.

Immunochemical analyses of 85 isolates of 17 Salmonella serovars using polyclonal antiserum to SEF21, the type 1 fimbriae of Salmonella enteritidis, demonstrated antigenic relatedness among both type 1 and type 2 fimbriae of Salmonella. However, anti-SEF21 antiserum was not entirely suitable as a Salmonella diagnostic probe due either to a variability of, or a rare deficiency of, detectable fimbriae. Partial amino acid sequence analyses of the SEF21 structural fimbrin protein revealed 99% homology to Salmonella typhimurium FimA. Therefore, oligonucleotide probes for Salmonella detection were designed following sequencing of S. enteritidis fimA and comparison to the corresponding genes of S. typhimurium, Escherichia coli, Klebsiella pneumoniae and Serratia marcescens. One oligonucleotide probe hybridized to all 612 Salmonella isolates of 89 serovars tested while two other probes detected 97.5% and 99.7% of the isolates. Three consistently weak positive reactions were obtained, therefore, inclusivity was optimized by identification of a Salmonella-specific tctC DNA probe that detected 609 of 612 Salmonella isolates. No hybridization of these Salmonella probes was detected to 250 other Enterobacteriaceae isolates or to 14 other eubacterial species. Therefore, in combination, DNA probes to fimA and tctC proved to be highly reliable diagnostics for Salmonella bacteria. Accordingly, PCR assays targeting fimA and tctC were developed.

Amino Acid Sequence↗
PubMed1993

DNA-based diagnostic tests for Salmonella species targeting agfA, the structural gene for thin, aggregative fimbriae.

Salmonella enteritidis 27655-3b and a few diarrheagenic Escherichia coli strains produce morphologically and antigenically related, thin, aggregative fimbriae, collectively named GVVPQ fimbriae (S. K. Collinson, L. Emödy, T. J. Trust, and W. W. Kay, J. Bacteriol. 174:4490-4495, 1992). To determine whether GVVPQ fimbriae are common to Salmonella spp. and other enteropathogenic members of the family Enterobacteriaceae, 113 isolates were phenotypically screened for Congo red binding and aggregative colony morphology. Presumptive positive and representative negative strains were examined by Western blotting (immunoblotting) by using antiserum to SEF 17, the native GVVPQ fimbria of S. enteritidis. Only four S. enteritidis strains and six E. coli isolates possessed substantial amounts of GVVPQ fimbriae after 24 h of incubation on T medium. Following 5 days of incubation, 56 of 93 Salmonella isolates (60%) and 1 of 7 additional E. coli clinical isolates possessed detectable levels of GVVPQ fimbriae. Since variable expression of GVVPQ fimbriae was observed among Salmonella isolates and some E. coli strains produced scant amounts, as revealed by immunoelectron microscopy, the ability to produce these fimbriae was evaluated by genotypic screening. The structural gene for the SEF 17 fimbrin, agfA, was amplified by the polymerase chain reaction, cloned, and sequenced to provide a characterized DNA probe. An agfA DNA fragment hybridized strongly to 603 of 604 (99.8%) Salmonella isolates but very weakly to 31 of 266 other members of the family Enterobacteriaceae including 26 of 137 E. coli strains, 3 of 14 Citrobacter spp., and single isolates of Shigella sonnei and Enterobacter cloacae. The agfA DNA probe proved to be a valuable diagnostic tool for Salmonella isolates arrayed on hydrophobic grid membrane filters. Unique agfA sequences were targeted in the development of a polymerase chain reaction assay specific for Salmonella spp.

Amino Acid Sequence↗
PubMed1991

[Classification of plasmids--approaches and problems].

An important part in human and veterinary infections is played by plasmids resistant to antibiotics. During basis investigations of plasmids and their epidemiology it was necessary to introduce typical classification groups of plasmids. Different approaches can be used when creating such groups. In the submitted work the author discusses some experimental approaches to the problem. Part of the work is devoted to phenotype fi, to the morphological, serological and phagotyping conjugation of pili. The author discusses in detail the classification of plasmids into incompatible groups, their technical and methodological problems. Special attention was focused on new approaches of classification of plasmids based on typing of replicons.

Bacteriophage Typing↗
PubMed1990

[The plasmid profile of Salmonella typhimurium strains untypable by phages].

The authors examined 250 strains of Salmonella typhimurium, untypable by phagi, for the sensitivity to antibacterial substances and lysogeny. Testing of the sensitivity to a set of 13 antibacterial substances revealed that 157 strains (63%) were polyresistent. Most frequently they were resistent to ampicillin (96% strains), sulphamethoxydine (71.2%), carbenicillin (68% strains), tetracycline (56% strains), cephalotine (56%), chloramphenicol (53%) and septrin (51% strains). The presence of a prophage was revealed in 99.2% strains. In a narrower selection of 54 strains plasmid DNA was isolated and electrophoretic analysis revealed the presence of 1-5 plasmids. Six strains were plasmid-free. The magnitude of plasmids varied between 2 and 41.4 kb. The authors indicate possibilities of using the plasmid profile in the differentiation of strains.

Bacteriophage Typing↗