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H Kopecka

Publications and source records attributed to H Kopecka.

52 records · Page 3Linked to original sources
PubMed1985

The stabilization of vancomycin by peptidoglycan analogs.

The glycopeptide antibiotic vancomycin is unstable in solution. It undergoes a rearrangement involving the conversion of an asparagine residue to isoaspartate to give an antibiotically inactive species, CDP-I. Peptide analogs of bacterial peptidoglycan, such as Ac-D-Ala-D-Ala and di-Ac-L-Lys-D-Ala-D-Ala bind to vancomycin and stabilize the antibiotic against degradation and consequent loss of activity. Protection by peptide is effective even under prolonged heating at 80 degrees C or steam sterilization (30 minutes, 10(4) kg/m2).

Chemical Phenomena↗
PubMed1983

Expression of poliovirus capsid polypeptide VP1 in Escherichia coli.

A recombinant plasmid (pPV1-958) carrying a poliovirus cDNA insert corresponding to nucleotides 1-5750 of the poliovirus RNA genome was constructed through in vitro recombination of two plasmids carrying overlapping poliovirus cDNA inserts (Van der Werf et al., Proc. Natl. Acad. Sci. USA 78 (1981) 5983-5987). pPV1-958 was then cleaved with PstI and the fragment carrying the sequence encoding capsid polypeptide VP1 was isolated and subcloned at the PstI site of pBR322. The VP1 sequence was successfully fused with the 5' sequence of the beta-lactamase gene by deleting 300-350 bp from each side of the PvuI site of the plasmid with nuclease BAL31. One of the resulting plasmids, pSW119, expressed an Mr 49 000 VP1-beta-lactamase-fusion protein, which was specifically immunoprecipitated with anti-VP1 immune serum but not by hyperimmune sera raised against native or heat-inactivated virus.

Capsid↗
PubMed1981

Molecular cloning of the genome of poliovirus type 1.

Poliovirus cDNA.RNA hybrids were prepared from the Mahoney strain of poliovirus type 1 by using reverse transcriptase (RNA-dependent DNA nucleotidyltransferase) and cloned in the Escherichia coli plasmid pBR322. Bacteria colonies carrying recombinant plasmids were selected by in situ hybridization with virus-specific RNase T1-resistant oligonucleotides. Analysis of the cDNA inserts by restriction mapping and electron microscopy showed that the cloned cDNAs, the longest of which was 3.2 kilobase pairs, originated from various parts of the viral RNA, covering at least 99% of the genome length. Due to overlapping of the clones, the restriction map of the poliovirus genome could be reconstructed. The complete 5' end of the genome was successfully cloned in at least one of the recombinant plasmids, pPV1-366.

Cloning, Molecular↗
PubMed1981

Restriction map of poliovirus type 2 cDNA.

Poliovirus type 1 RNA was reverse-transcribed into c-DNA and inserted at the Pst I site of the plasmid vector pBR322 of E. coli. Resulting recombinant plasmids were analyzed by hybridization with RNase T1-resistant 32P-labeled oligonucleotides, and by restriction enzyme mapping. All of the genome was cloned in a series overlapping cDNA inserts, the longest of which was 3.2 kb. The restriction map of the poliovirus cDNA is presented.

Avian Myeloblastosis Virus↗
PubMed1980

Cytotypically specific transfecting activity of DNA from C57BL/Ka mouse cells producing thymotropic in contrast to nonthymotropic retroviruses.

A comparative study has been made of the susceptibility of fibroblastic cells to transfection by DNA from C57BL/Ka mouse lines producing either fibrotropic or thymotropic retroviruses. DNA isolated from fibroblasts that release a B-ecotropic, fibrotropic virus, BL/Ka(B), was found to transfect fibroblasts of Fv-1bb genotype with release of virus similar to BL/Ka(B). Fv-1nn fibroblasts were also susceptible but expression was delayed, and xenotypic mink lung cells were refractory. In contrast, DNA prepared from a murine T-cell lymphoma line producing a B-ecotropic, thymotropic virus failed to transfect mouse fibroblasts though it transfected a nonproducer T-cell lymphoma line. The data suggest that the Fv-1 and differentiation-specific restriction mechanisms operate at different molecular levels.

Animals↗
PubMed1978

Restriction enzyme analysis of satellite DNA components from the bovine genome.

A restriction enzyme analysis was performed on satellite DNA components, isolated, as described in the preceding paper, from the bovine genome by a combination of Cs2SO4/BAMD and Cs2SO4/Ag+ density gradient centrifugation. Such an analysis has led to the unambiguous identification of eight satellite DNA components and to new information on their repeat units; this indicates that identical repeat lengths are shared by them, a fact strongly suggesting a common origin.

Animals↗
PubMed1977

The Euglena gracilis chloroplast genome: analysis by restriction enzymes.

1. Chloroplast DNA was isolated from autotrophically and mixotrophically grown Euglena gracilis cells. 2. Aliquots of chloroplast DNA were mechanically degraded to an average molecular weight of 4-7 X 10(6) and G+C-rich DNA fragments (density 1.701 g/cm3) were separated from the bulk DNA (density 1.685 g/cm3) using preparative CsCl density gradients. 3. Total chloroplast DNA and its DNA subfractions, which first were characterized with respect to average G+C content and hybridization capacity for chloroplast rRNA, were hydrolysed with restriction endonucleases (endo R-EcoRI, end R-HindII, endoR-HindIII, endo R-HindII+III, endoR-Hpal, endo R-HpaII and endoR-HaeIII). The fragments were separated on gels under a variety of electrophoretic conditions. 4. With each enzyme tested, a rather large number of bands was obtained. In all cases, different banding patterns were obtained for total DNA, and the DNA subfractions. 5. Chloroplast DNA from autotrophically and mixotrophically grown cells gave identical banding patterns. 6. Digestion of total DNA with the endoR-HaeIII yielded 51-52 fragments separated in the gels in a total of 36 bands of which 11-12 bands were composed of 2-3 fragments as estimated by densitometry. The molecular weights of all fragments combined was 87 X 10(6) or 95% of the genome (92 X 10(6)). 7. Chloroplast RNA hybridized to 5.1% with total chloroplast DNA, equal to three RNA cistrons per genome (Mr92 X 10(6)). These cistrons are located on seven different types of endo R-HaeIII fragments. The hybridising fragments are preferentially found in the G+C-rich subfraction and in bands which are composed of 2-3 fragments.

Chloroplasts↗
PubMed1975

A rapid purification method of restriction endonucleases from Haemophilus strains.

A simple and rapid method of purification of restriction endonucleases from different Haemophilus strains is presented. By this method highly purified and stable enzymes can be obtained. Separation of different restriction activities present in the same strain is possible. This method was so far successfully used with Haemophilus influenzae, Haemophilus parainfluenzae and Haemophilus aegyptius strains. The main advantages over previously published procedures reside in the simplication of certain purification steps (for instance the BioGel A 0.5 M filtration is replaced by a hydroxyapatite batch step), elimination of exonuclease activity by fractionation with (NH4) 2SO4, separation of different restriction activities by phosphocellulose chromatography, application of this method to various strains and high purification degree of enzymes.

Chromatography↗
PubMed1975

Mapping the transcription site of the SV40-specific late 16 S mRNA.

This paper describes the purification of polysomal RNA from SV40-lytically infected CV1 (monkey) cells and separation of the two distinct classes of SV40-specific mRNA sedimenting at 16 S and 19 S. These classes have been hybridized with the whole SV40 DNA genome as well as with the SV40 Hind fragments. The results have permitted the mapping of SV40-specific late 16 S mRNA from approximately 0.945 to 0.175 map units.

Base Sequence↗
PubMedDate not supplied

[cRNA probes (riboprobes) synthesized in vitro for detecting enterovirus by molecular hybridization].

Radioactively labelled RNA transcripts made in vitro of various fragments from cDNA clones of poliovirus type 1 and of hepatitis A virus under the control of bacteriophage T7 or SP6 promoters have been evaluated for diagnostic purposes. The RNA transcripts were 2 orders of magnitude more sensitive as hybridization probes than corresponding cDNA preparations labelled by the nick translation procedure. A combination of hybridization analysis and sequence comparison showed that some regions of the genome of a number of enteroviruses are highly conserved, while others show very little homology; the general order of conservation is: 5'-non-coding greater than 3'-terminal greater than central (2C) greater than VP3 greater than VP1. The 350 bases of the poliovirus VP1 region were highly specific for that virus, while the 450-bases of the 5'NC region showed extensive cross-reaction with other enteroviruses. However, these probes did not hybridize with HAV, which was detected only by HAV-specific riboprobes. The transcripts have been successfully applied as hybridization probes in diagnostic tests on supernatants of infected cell culture lysates and in clinical samples, mainly in stool extracts.

Animals↗
PubMedDate not supplied

Detection of enteroviruses in cases of neurological disorders in the State of Pará, Brazil.

Eighty-one cerebrospinal fluid (CSF) samples mainly from cases of aseptic meningitis and motor deficiency syndrome were sent to the Virology Section of Evandro Chagas Institute, Belém Pará, in the period of January 1995 to January 1996 in order to isolate viruses. All samples were inoculated onto HEp-2 cell culture and newborn mice, with negative results. The probability of isolating viruses by these methods is reduced because of the low concentration of viral particles in these specimens. In order to obtain more information about the etiology of these cases, a group of 23 samples were selected to be tested by a more sensitive technique than the virus isolation - the reverse transcription polymerase chain reaction (RT-PCR). Specific primers directed to conserved regions in the enterovirus genome were used, considering that this group of viruses is frequently associated with these neurological disorder. The age of the patients ranged from 1 to 55 years and nearly all of them lived in Belém, State of Pará, North of Brazil. Of 15 samples analyzed by RT PCR nine (60%) were positive; of these, 6 (66.6%) had motor deficiency and 3 (33.3%) developed aseptic meningitis. These results show that it is important to investigate enterovirus as cause of these syndromes.

Adolescent↗
PubMedDate not supplied

Detection of enteroviral sequence in endomyocardial tissues from patients with cardiac diseases in northern Brazil.

In the present report we describe the results from a pilot study aimed at detecting enterovirus sequence in cardiac tissues, obtained through endomyocardial biopsies, from patients suffering from cardiac diseases in the Amazon region. Six samples that were collected from three patients were analysed by RT-PCR showing 3 positive and 3 negative results. These preliminary findings suggest the participation of enteroviruses in the etiology of cardiac diseases, mainly myocarditis, and warrant further and broader local studies on this subject.

Adult↗