RAPD and rep-PCR fingerprinting for characterization of Bifidobacterium species.

DNA fingerprinting methods, RAPD with 7 random primers, and rep-PCR using both BOXA1R and (GTG)(5) ones, were used for the discrimination of 16 type and collection Bifidobacterium strains of 9 species of human origin, B. animalis ssp. animalis and B. animalis ssp. lactis and 7 Bifidobacterium strains collected in the Culture Collection of Dairy Microorganisms (CCDM). Both RAPD and rep-PCR methods provided similar results. The strains were identified as B. animalis ssp. lactis (6 strains) and B. adolescentis (1 strain). The reclassification of the collection strain CCM 3761 as B. pseudocatenulatum species (previously classified as B. adolescentis) was confirmed.

Isolation of microbial DNA by newly designed magnetic particles.

Carboxyl group-containing magnetic nonporous poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) (P(HEMA-co-GMA)) microspheres and cobalt ferrite nanoparticles modified with alginic acid (natural carboxylic polysaccharide) were used for isolation of microbial DNA of lactic acid bacteria (LAB) from dairy products, lyophilised cell cultures, and bacterial colonies grown on hard media, and Trichophyton fungi DNA from lyophilised cells. DNA from the samples with lysed cells was reversibly adsorbed to the particles in the presence of high poly(ethylene glycol) (PEG 6000) and sodium chloride concentrations. The optimal final PEG and NaCl concentrations were 9.1 wt.% and 2.0 M, respectively. The adsorbed DNA was released from the particles in low ionic strength TE buffer. The quality of isolated DNA was checked by PCR amplification. Moreover, PCR amplicons were isolated on cobalt ferrite nanoparticles modified with alginic acid and checked by restriction analysis.

Application of PCR, rep-PCR and RAPD techniques for typing of Lactococcus lactis strains.

A collection of 34 lactococcal strains were characterized using the polymerase chain reaction (PCR) for the acmA gene, and for the 16S rDNA gene, and DNA fingerprinting methods for randomly amplified polymorphic DNA (RAPD) and repetitive extragenic palindrome-PCR (rep-PCR). PCR experiments corroborated the genotypic identification of Lactococcus lactis strains by RAPD; rep-PCR did not distinguish between L. lactis subspecies. In some cases, phenotypic classification of L. lactis subspecies did not correlate with genotypic characterization.

Apoptotic DNA alterations in pig leukocytes after phagocytosis of bacteria are linked to maturation of the immune system.

The effect of phagocytosis of living bacteria on apoptotic DNA changes was examined in pig leukocytes in relation to immune system maturation. Blood samples of pigs (aged 6, 12 and 18 weeks) were cultivated with a suspension of bacterial cells Salmonella typhimurium LB 5000 at 37 (o)C. In the experimental groups, killed bacteria and microspheric particles were used to detect the influence of the phagocytic process. Phagocytic activity and index were determined in each sample by means of microspheric particles. The ability to kill engulfed microbes (bactericidal capacity) was estimated from the decrease in bacterial colony-forming units (CFU). Samples of cultured cells were taken for DNA analysis at given intervals. DNA ladder assay was used for qualitative apoptotic DNA break detection and the TUNEL AP test was employed for quantification of apoptosis. In 18-week-old animals, spontaneous DNA degradation was observed in the control group without phagocytosis after 8 h. In contrast, cells cultivated with microspheric particles or killed bacteria became apoptotic after 4 h. The rate of apoptotic DNA degradation was decreased in the group exposed to living bacteria. This prolonged survival of phagocytes was also detected in 12-week-old animals, but not at 6 weeks of age. These findings were supported by the ability of phagocytes in 6-week-old animals to engulf microbes, but their killing (bactericidal) ability was significantly decreased in comparison with other stages of immune system maturation. These results suggest that the process of phagocytosis itself is accompanied by activation of the apoptotic program in phagocytic cells of the pig immune system, but the presence of phagocyted living bacteria can delay this activation. The prolonged survival of short-lived cells was only observed in later phases of immune system maturation.

Properties of RNase A immobilized on magnetic Poly(2-hydroxyethyl methacrylate) microspheres.

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.

Ultrastructure of nuclei of cisplatin-treated C6 glioma cells undergoing apoptosis.

C6 glioma cells, treated with a cytostatic dose of cisplatin (1.66 x 10(-5) M) ceased dividing by 24 h and, most of them had undergone apoptosis by 72-96 h. The reactive cells were classified into 5 types (T-I to V), according to the ultrastructure of nuclei. At 4 h, 20.4% of cells (T-I) showed minute condensation and margination of chromatin. The nuclear envelope (NE) formed slim and deep invaginations consisting of the inner or both membranes. The later kind of NE invaginations often extended to the enlarged nucleoli and contained nucleolus-like material at its cytoplasmic side. Some nuclear pores were covered with a dome-shaped "cap" formed by fine filamentous material. The number of T-I cells increased to 53.3% by 72 h. In T-II cells, which appeared at 24 h, the chromatin was condensed into dense irregular masses separated from the NE by a lucent space with filamentous structures preventing complete margination of chromatin. Nucleoli of T-II cells were small and showed partial segregation of their components. The "capped" pores were absent in these apparently more damaged cells. From 24 h, cells with large and lobulated nuclei (T-III) started to increase in number and peaked at 72 h (6.6%). Except for some small lobules, the chromatin of T-III cells was moderately aggregated and the NE was well preserved. Typical apoptotic cells with highly condensed and marginated chromatin (T-IV) peaked at 48-72 h (2.4-4.8%). They appeared in 2 varieties, including cells with wrinkled nuclei with less condensed and incompletely marginated chromatin or more lobulated forms with highly condensed marginated chromatin suggesting their origin from T-II or T-III cells. T-IV cells, as well as their fragments, underwent phagocytosis and secondary necrosis (T-V cells, 48.6% at 96 h). Two alternative routes of nuclear changes leading to cisplatin-triggered apoptosis, as represented by the sequence T-I --> T-III --> T-IV/V or T-I --> T-II --> T-IV/V, may explain the initially less or more damaged cells. These alternatives, together with progressive recruitment of reactive cells, suggest intrapopulation differences in the sensitivity of cells or in the cell cycle perturbations induced by cisplatin. Except for the T-IV and T-V cells, observed alterations of cytoplasmic organelles, including mitochondria, were fewer than reported in previous studies on cisplatin.

PCR identification of Salmonella cells in food and stool samples after immunomagnetic separation.

The purpose of the present study was to investigate the application of various sample preparation methods (cell washing before lysis, purification of DNA using phenol extraction method, immunomagnetic separation-IMS) for the final PCR identification of Salmonella cells. The presence of PCR inhibitors in processed food products (milk powder and dried eggs) can be the cause of false-negative results in PCR without IMS of target cells. It was also demonstrated that IMS-PCR was successfully used for identification and quick confirmation of untypical Salmonella strains isolated from human stool samples and rabbit meat. However, IMS cannot eliminate intracellular PCR inhibitors present in immunoseparated Salmonella cells. These inhibitors must be taken into consideration in evaluation of PCR procedure.

Apoptotic damage of DNA in human leukaemic HL-60 cells treated with C2-ceramide was detected after G1 blockade of the cell cycle.

Apoptosis was induced by treatment of HL-60 cells with C2-ceramide. Apoptotic damage of DNA was detected according to the sub-G1 peak on a flow cytometer, according to the typical morphology and according to the DNA fragmentation "ladder" after gel electrophoresis. It was shown that the apoptotic cleavage followed after G1 blockade of the cell cycle. A high correlation coefficient (rs=0.957) was found between the percentages of G1 blocked cells and apoptotic cells. This high correlation together with the appearance of the sub-G1 peak suggests that the G1 blocked HL-60 cells were subject to apoptotic death. It is deduced that the mechanisms leading to G1 blockade of the cell cycle and activation of apoptosis in HL-60 cells are interconnected.

Estimation of apoptosis in C6 glioma cells treated with antidepressants.

Gel electrophoresis of DNA was used for estimation of DNA changes caused in C6 glioma cells by treatment with psychotropic drugs (imipramine, amitryptiline and fluoxetine). Some discrete bands containing a population of short DNA fragments appeared after 1 and 5 days of cultivation. Apoptotic DNA breaks were verified at single cell level using the TUNEL test in cells treated with fluoxetine.

Structures of poly(dA-dT, ip5dU) containing various small amounts of the antiherpetic 5-isopropyl-2'-deoxyuridine.

Three different concentrations of the antiherpetic agent 5-isopropyl-2'-deoxyuridine (ip5dU) were introduced into the synthetic DNA poly(dA-dT) to analyze resulting copolymers by electron microscopy, UV absorption and CD spectroscopy. The poly(dA-dT, ip5dU) containing 1.3 and 4.3% ip5dU did not much differ from the parent poly(dA-dT) but poly (dA-dT, ip5dU) with 7.1% ip5dU behaved in an unusual way. Results are explained by the notion that if bulky isopropyls occur sufficiently close to each other then stable hairpins protruding from the double helix are formed, presumably to accommodate the ip5dU-s into the loops.

Comparison of permuted region lengths in the genomes of related Salmonella typhimurium phages P22 and L.

Lengths of permuted regions in the P22 and L phage genomes were estimated from the relative yields of DNA in many electrophoretic bands obtained using several restriction endonucleases. It was found that 3.6 kb (8.7%) of P22-DNA and 7.2 kb (17.8%) of L-DNA were circularly permuted. In both phages the sequential packaging process proceeded in the same direction and four headful-size DNA molecules were, on the average, cleaved in one packaging series. The differences in circular permutation may originate from different genome lengths because their average headful portions are very similar (42.5 kb in P22 and 42.3 kb in L).

Isolation and characterization of two rumen Streptococcus bovis bacteriophages.

A method for the isolation of Streptococcus bovis bacteriophages from ruminal fluid of calves is described. Thirty to 2 x 10(3) phages per ml infecting Streptococcus bovis strains 4/1 and 47/3 were isolated directly from ruminal fluid. Two bacteriophages were characterized on the basis of plaque morphology, host ranges, electron microscopic morphology and DNA restriction endonuclease digestion patterns. The F1 and F3 phages formed clear plaques of different sizes. The plaque size of the F1 phage was about 1-1.5 mm in diameter, while the plaques of the F3 phage were larger (1.5-2.5 mm in diameter). Both phages are placed in group B of Bradley's scheme and have different host ranges. The first isolation of Streptococcus bovis phage DNA is reported. Restriction analysis of their DNAs showed that phages F1 and F3 had different numbers of cleavage sites in their genomes and that they were not identical.

Integration of transposon Tn10 into phage L (Salmonella typhimurium).

Transposon Tn10 was transposed into phage L (Salmonella typhimurium) from F'ts114lac+zzf::Tn10 plasmid of strain TT629 (Chumely et al. 1979). Phage L with the insertion Tn10 (L::Tn10-8) was isolated in the form of a prophage in the lysogenic strain S. typhimurium LT2-18 (L::Tn10-8), in which it can be induced with UV light. The phage induced in this way is defective; however, it forms plaques at a multiplicity of infection (moi) greater than one and transduces the tetracycline-resistance determinant to tetracycline-sensitive cells. Analysis of its DNA by restriction endodeoxyribonucleases revealed insertion of the intact transposon Tn10 of 9300 bp in the E fragment, formed during the action of EcoRI, at a distance of 16,800 bp from the pac site.

Genetic mapping of the G101 bacteriophage (Pseudomonas aeruginosa) by temperature-sensitive mutations.

Temperature-sensitive (ts mutations of the G101 phage were isolated after mutagesis with hydroxylamine. A complementation analysis of 61 ts mutants showed that these mutants may be divided into a least 12 complementation groups. Two ts mutants probably originated in genes which control lytic functions of the G101 phage. It was shown by three factor crosses that all of the 12 ts mutations tested are localized on that side of the "c" region where the probable cI repressor gene is positioned. Seven ts mutations is closely linked to the cI26 clear marker, three exhibit a closer linkage and two do not exhibit any linkage with cI. All mutations isolated until now can be arrange linearly. According to the present knowledge the preliminary genetic map of the G101 phage is linear.

Genetic mapping of the region c of the bacteriophage G101 (Pseudomonas aeruginosa).

Morphological mutants of the c type of the bacteriophage G101 (Pseudomonas aeruginosa) were isolated after mutagenesis with hydroxylamine. Complementation analysis of 27 c mutants showed that the c region is formed by at least two genes. Two types of c mutants were obtained. One of them (cI26) behaves analogously to a mutant in the gene controlling the synthesis of the repressor of phage lambda. The second type of the c mutants (cII1, cII18) specifies a gene having probably an auxiliary function in the "c" region. According to the low frequency of recombination between the genes cI26 and c II18 (1.37 recombination units), these genes responsible for lysogenization are localized in a short region of the chromosome.