Lung resistance protein (LRP) gene expression in adult acute myeloid leukemia: a critical evaluation by three techniques

Restriction fragment length polymorphism analysis of chromosomal DNA from 22 strains of Toxoplasma gondii were characterised using SalI and PstI restriction endonucleases and the TGR1E specific repetitive sequence as a probe. Two virulent strains, RH and P-CZ, had previously been isolated from humans, the remaining 20 strains were isolated from animals in the Czech Republic in 1994 and 1995. Among the 20 recently isolated strains, 19 belonged to an avirulent lineage and only one strain from the wild cat Felis silvestris belonged to a virulent lineage.     

Effect of acute ethanol administration on serum magnesium concentration in the rabbit

The size of the Lactobacillus plantarum CCM 1904 chromosome was determined by pulse-field gel electrophoresis. It was found to be 3.3-3.4 Mb using SfiI or AscI restriction endonucleases, compared to 3-4 Mb found for the other L. plantarum strains tested. L. plantarum CCM 1904 5S rDNA was clonedl by polymerase chain reaction, sequenced, and used as a probe to characterize strains. At least five rrn loci were found. The pulsed-field gel electrophoresis macrorestriction patterns were strain-specific, while the rDNA restriction hybridization patterns were species-specific.     

DNA extraction and stability for epidemiological studies

An outbreak of food poisoning involving most autonomous Spanish communities was detected in the first half of 1994. The causative food was infant formula milk contaminated by lactose-fermenting Salmonella virchow. It was not possible to isolate the causative strain from the manufacturer's facilities. During the same period of time, there was a significant increase in lactose-non-fermenting Salmonella virchow strains compared with the same period in previous years. Simultaneously, lactose-non-fermenting strains were recovered from clinical samples from children and from some milk samples that were involved in the outbreak. Therefore, it was speculated that the outbreak might be more extensive than initially thought. The following epidemiological markers were used for typing the Salmonella virchow strains involved in the outbreak: (i) phage typing: (ii) ribotyping, using a set of 20 different endonucleases: and (iii) pulsed-field gel electrophoresis, using three different endonucleases. The most useful markers for this serotype were phage typing and pulsed-field gel electrophoresis, since ribotyping was not able to distinguish all strains tested. The results obtained revealed that the outbreak was caused by at least two strains: one presenting phage type 4-4a and pulsed-field patterns A1 or A2 and L+ or L-, and another presenting phage type 2 and pulsed-field patterns A1 or A2 and L+ or L-. The results indicate that the outbreak was more extensive than initially thought and that the Virchow serotype is very clonal in Spain.     

Restriction fragment length polymorphism analysis and random amplified polymorphic DNA analysis of Campylobacter jejuni strains isolated from patients with Guillain-Barré syndrome

Campylobacter jejuni serotype O19 strains associated with the Guillain-Barré syndrome (GBS) and other strains were examined by restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction products of the flaA genes and by random amplified polymorphic DNA (RAPD) analysis. RFLP analysis showed that regardless of LIO serotype, geographic origins, or association with GBS, the O19 isolates shared an identical digestion pattern by each of four restriction endonucleases, DdeI, MboI, MseI, and AluI. In contrast, among C. jejuni O1 or O2 strains, RFLP patterns were different even among strains of the same LIO serotype. The results of the RAPD analysis were consistent with the flaA RFLP data. These data indicate that all of the O19 strains that were tested were closely related to one another whether they were or were not associated with GBS.     

Rapid characterization of four species of the Saccharomyces sensu stricto complex according to mitochondrial DNA patterns

Several strains of the four sibling species of the genus Saccharomyces (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species. S. cerevisiae and S. paradoxus presented a greater number of large fragments than S. pastorianus and S. bayanus with all the assay enzymes. With AluI and DdeI, species-specific bands clearly permitted differentiation between S. pastorianus and S. bayanus. To test the resolution of this method, wild Saccharomyces strains were analyzed. The correct assignment of these strains to a known taxon by this rapid method was confirmed by means of electrophoretic karyotyping.     

Genomic, protein homogeneity and antigenic variability of Mycoplasma agalactiae

Eleven strains of Mycoplasma agalactiae differing in pathogenicity, animal species origin and geographic localisation, showed similar chromosome restriction profiles with four endonucleases. However the international reference strain PG2 showed a unique profile. The protein and antigenic variabilities of 31 strains of M. agalactiae were investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting performed with naturally infected animal sera and purified antibodies against the 29 kDa protein. Protein profiles were similar but antigenic profiles could be separated into two main groups according to geographic origin: (i) strains isolated in south-west France and (ii) strains from north-east France. Some differences also occurred from strain to strain within each group. The antigenic profile variability found in immunoblotting, originated in two different phenomena: (i) some epitopes were expressed only in strains of one profile type and (ii) some other epitopes were common to all strains but located on several proteins which differed in number and molecular mass from one strain to another. The presence of epitopes which undergo phase variation in the same lineage of clones from a single cell is discussed.     

A strain-type clustering of potato virus Y based on the genetic distance between isolates calculated by RFLP analysis of the amplified coat protein gene

Potato virus Y (PVY) isolates have been classified into genetic strains by a host-independent criterion using a molecular typing method. The method used extracts from infected tissue, and included immunocapture-RT-PCR-RFLP analysis using 5 different restriction endonucleases (Dde I, Eco RV, Hinf I, Rsa I and Taq I). Genetic distances between the different PVY "restrictotypes" were calculated and used to define the PVY genetic strains. Three main clusters were found: PVYO, PVYN, and non-potato PVY (PVYNP), in good agreement with classical PVY strain definitions that combine different biological criteria. Our approach was incomparably quicker and more reliable and reproducible than biotyping. The potential of this approach for very quick, simple and automatable molecular epidemiological studies is discussed.     

Characterization of two restriction endonucleases, SenPT14bI and SenPT16I, in standard phage-type strains of Salmonella enteritidis

Two restriction endonucleases (ENases) were found by screening 38 standard phage strains of Salmonella (S.) Enteritidis. An isoschizomer of SacII ENase that recognizes the sequence 5'-CCGC/GG-3' was identified in S. Enteritidis PT14b, and an isoschizomer of XmaIII ENase (5'-C/GGCCG-3') was found in S. Enteritidis PT16. It is of special interest that the recognition specificities of all known ENases in Salmonella, including those of the S. Enteritidis ENases, are very similar to each other.     

Detection of kanamycin-resistant Mycobacterium tuberculosis by identifying mutations in the 16S rRNA gene

In Mycobacterium smegmatis and a limited number of Mycobacterium tuberculosis strains, the involvement of alterations of the 16S rRNA gene (rrs) in resistance to kanamycin has been shown. To investigate the extent to which mutations in a specific region of the rrs gene and the kanamycin-resistant phenotype in clinically isolated M. tuberculosis strains were correlated, 43 kanamycin-resistant strains (MICs, > or =200 microg/ml), 71 kanamycin-susceptible strains, and 4 type strains were examined. The 300-bp DNA fragments carrying the rrs gene and the intervening sequence between the rrs gene and 23S rRNA (rrl) gene fragments were amplified by PCR and were subjected to PCR-based direct sequencing. By comparing the nucleotide sequences, substitutions were found in 29 of 43 (67.4%) kanamycin-resistant clinical isolates at positions 1400, 1401, and 1483 but in none of the 71 sensitive isolates or the 4 type strains. The most frequent substitution, from A to G, occurred at position 1400. A substitution from C to T at position 1401 was found once. Two clinical isolates carried the double mutation from C to A at position 1401 and from G to T at position 1483. In addition, we found that these mutants can be distinguished from wild-type strains by digestion with the restriction endonucleases TaiI and Tsp45I. Furthermore, we found that the genotypes of kanamycin-resistant strains can be discriminated from each other by digestion with a restriction endonuclease, BstUI or DdeI.     

Differential cleavage of oligonucleotides containing the benzene-derived adduct, 1,N6-benzetheno-dA, by the major human AP endonuclease HAP1 and Escherichia coli exonuclease III and endonuclease IV

We report here that the newly synthesized DNA adduct, 1,N6-benzetheno-dA (pBQ-dA), in defined oligonucleotides [Chenna and Singer, Chem. Res. Toxicol., 8, 865-874], is a substrate for the major human AP endonuclease, HAP1, and the Escherichia coli AP endonucleases, exonuclease III and endonuclease IV. The mechanism of cleavage is identical to that reported previously for 3,N4-benzetheno-dC (pBQ-dC) and leads to a phosphodiester bond cleavage 5' to the adduct. There are, however, significant differences in the rate of cleavage of this adduct by these enzymes. The two bacterial AP endonucleases are both much more efficient than the human repair enzyme. In addition, using two random oligodeoxynucleotide sequences containing a single pBQ-dA, exonuclease III and endonuclease IV are similarly active, while HAP1 shows a distinct sequence preference of approximately 10-fold in efficiency of cleavage. The repair of this adduct by the three recombinant enzymes is further confirmed by using both active site mutant HAP1 proteins and by E.coli mutant strains lacking exonuclease III and/ or endonuclease IV. This sequence-dependent repair of pBQ-dA by HAP1 may play an important role in modulating benzene-induced carcinogenesis.     

Ribonuclease inhibitors from mouse ascites cells

Ribonuclease inhibitors were found to be present attached to mouse ascites ribosomes and in the post-ribosomal supernatant. Both inhibitors inhibited pancreatic RNAses A and B and two endonucleases prepared from ascites cells but did not inhibit RNAses T1 or N1. Both inhibitors had the same sedimentation coefficient and this taken with the results above suggest that they are identical. The inhibitor was shown to interact directly with the RNAse itself.     

Site-specific endonucleases RspLKI and RspLKII from Rhodococcus species LK2 are isoschizomers of SphI and BamHI

Two site-specific endonucleases, RspLKI and RspLKII, have been isolated and purified to functional homogeneity from the soil bacterium Rhodococcus species LK2. RspLKI recognizes the 5'-GCATG decreases C-3' DNA sequence and RspLKII recognizes the 5'-G decreases GATCC-3' sequence (arrows indicate DNA cleavage sites). The isolated enzymes are class II site specific endonucleases and are isoschizomers of endonucleases SphI and BamHI, respectively.     

Species identification and virulence attributes of Saccharomyces boulardii (nom. inval.)

Saccharomyces boulardii (nom. inval.) has been used for the treatment of several types of diarrhea. Recent studies have confirmed that S. boulardii is effective in the treatment of diarrhea, in particular chronic or recurrent diarrhea, and furthermore that it is a safe and well-tolerated treatment. The aim of the present study was to identify strains of S. boulardii to the species level and assess their virulence in established murine models. Three strains of S. boulardii were obtained from commercially available products in France and Italy. The three S. boulardii strains did not form spores upon repeated testing. Therefore, classical methods used for the identification of Saccharomyces spp. could not be undertaken. Typing by using the restriction fragment length polymorphisms (RFLPs) of the PCR-amplified intergenic transcribed spacer regions (including the 5. 8S ribosomal DNA) showed that the three isolates of S. boulardii were not separable from authentic isolates of Saccharomyces cerevisiae with any of the 10 restriction endonucleases assessed, whereas 9 of the 10 recognized species of Saccharomyces could be differentiated. RFLP analysis of cellular DNA with EcoRI showed that all three strains of S. boulardii had identical patterns and were similar to other authentic S. cerevisiae isolates tested. Therefore, the commercial strains of S. boulardii available to us cannot be genotypically distinguished from S. cerevisiae. Two S. boulardii strains were tested in CD-1 and DBA/2N mouse models of systemic disease and showed intermediate virulence compared with virulent and avirulent strains of S. cerevisiae. The results of the present study show that these S. boulardii strains are asporogenous strains of the species S. cerevisiae, not representatives of a distinct and separate species, and possess moderate virulence in murine models of systemic infection. Therefore, caution should be advised in the clinical use of these strains in immunocompromised patients until further study is undertaken.     

Staphylococcal utilization of siderophores produced by bacilli of the genus Bacillus

The ability of utilization by 24 staphylococcal strains of the siderophores of B. megaterium PCM 2010 and B. subtilis S was investigated. B. megaterium PCM 2010 produced hydroxamate class siderophores and B. subtilis S-catecholate class. The majority of staphylococcal strains--thirteen--could utilize these siderophores: eight strains from both these species and three strains did not possess any receptors for these chelators. The staphylococcal strains producing catecholate siderophores utilized catecholate chelators from B. subtilis S.     

Intergenic transcribed spacer PCR ribotyping for differentiation of Saccharomyces species and interspecific hybrids

The taxonomy of the genus Saccharomyces has undergone significant changes recently with the use of genotypic rather than phenotypic methods for the identification of strains to the species level. The sequence of rRNA genes has been utilized for the identification of a variety of fungi to the species level. This methodology, applied to species of Saccharomyces, allows unknown Saccharomyces isolates to be assigned to the type strains. It was the aim of the present study to assess whether typing of the intergenic spacer region by using restriction fragment length polymorphisms of PCR products (intergenic transcribed spacer PCR [ITS-PCR] ribotyping) could distinguish among type strains of the 10 accepted species of Saccharomyces and further to assess if this method could distinguish strains that were interspecific hybrids. Cellular DNA, isolated after the lysis of protoplasts, was amplified by PCR using ITS1 and ITS4 primers, purified by liquid chromatography, and digested with restriction endonucleases. Ribotyping patterns using the restriction enzymes MaeI and HaeIII could distinguish all species of Saccharomyces from each other, as well as from Candida glabrata, Candida albicans, and Blastomyces dermatitidis. The only exception to this was the inability to distinguish between Saccharomyces bayanus and S. pastorianus (S. carlsbergensis). Furthermore, interspecific hybrids resulting from the mating of sibling species of Saccharomyces were shown to share the ITS-PCR ribotyping patterns of both parental species. It should now be possible, by this simple PCR-based technique, to accurately identify these strains to the species level, thereby allowing an increase in our understanding of the characteristics required by these interspecific hybrids for their particular ecological niches.     

Novel site-specific endonucleases from Brevibacterium species

New site-specific endonucleases BecAI and BecAII have been detected in Brevibacterium species A. Endonuclease BecAII free from contaminating nonspecific endonucleases, exonucleases, and phosphatases was isolated by column chromatography on phosphocellulose, heparin sepharose, and DNA cellulose. It recognizes and cleaves the 5'-GG decreases CC-3' sequence and is a true isoschizomer of HaeIII restriction enzyme. The other restriction endonuclease, BecAI, cleaves Ad2 DNA at least by 2 sites but not the DNA of phage lambda, T7, SV40, phiX174, and plasmides pBR322 and pUC19. The substrate specificity of BecAI indicates its appurtenance to the super rare restriction endonucleases.     

Occurrences of penicillin-binding protein 2B gene in clinically isolated penicillin-resistant Streptococcus pneumoniae

We analyzed 88 strains of Streptococcus pneumoniae (S. pneumoniae) isolated in Showa University Hospital from June 1995 to July 1996. The ratios of antibiotic resistance were 39% to penicillin G, 50% to erythromycin, and 2% to imipenem. No resistant to cefotaxime and ofloxacin was observed. Thirty-four strains (39%) were considered to be penicillin-resistant S. pneumoniae (PRSP) strains (MIC of penicillin G > or = 0.5 microgram/ml), according to the breakpoint determined by the Japanese Working Group for Penicillin-Resistant Streptococcus pneumoniae. The ratio of PRSP was higher in S. pneumoniae isolated from inpatients (25/47) when compared to that from outpatients. By PCR analysis, DNA regions of autolysin were amplified in all the 88 strains, confirming that the isolates were S. pneumoniae. Penicillin-binding protein 2B (PBP2B) class B region was positive in 32 strains, and PBP2B class A was in 2 strains. Twenty eight of 34 strains of PRSP contained the PBP2B class B gene. In the remaining six PRSP strains, neither the PBP2B class A nor B region was amplified. The PBP2B class B region was detected as a 180-kb fragment of SmaI digestion of S. pneumoniae DNA by Southern blot analysis, confirming that the detection of PBP2B class B gene by PCR is reliable. We concluded that the PBP2B class B gene is considered to be a major gene responsible for phenotypic resistance of PRSP. We performed genotyping by SmaI digestion pattern using pulsed field gel electrophoresis. No identical pattern was observed in isolates from inpatients, suggesting that apparent nosocomial infection of S. pneumoniae was negligible.     

Structural resolution of the folding pathway of a protein by correlation of phi-values with inter-residue contacts

The phenotypic identification of the classical propionibacteria is essentially still problematic and alternative techniques for the identification of the various species are required. A rapid and sensitive technique for the routine identification of the classical propionibacteria, based on the amplification of 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products, was previously described. Although this technique enabled differentiation between the various classical species examined it was only evaluated on a limited number of type and reference strains. During this study, the taxonomic relationship between 135 Propionibacterium strains from diverse ecological niches, representing four classical species was investigated using this PCR/RFLP technique. Visual differentiation between the classical Propionibacterium was possible after restriction endonuclease digestion of the PCR products obtained using primers 16sP1-16sP4 and 16sP3-16sP4 with the restriction endonucleases HaeIII, AluI and HpaIII, respectively. With the exception of strains independently identified as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the "old" species into the various classical species as they currently exist. It was therefore concluded that the PCR/RFLP protocol is suitable for the rapid and routine identification of the classical propionibacteria.     

Interferonogenic capacity of strains influenza B virus and their sensitivity to exogenous interferon

This study is a continuation of the investgation of the influence of endogenous and exogenous interferon on influenza infection. Influenza B virus strains, both laboratory and fresh isolates, were found to be poor interferon inducers in contrast to influenza A virus strains. The study also showed that influenza B virus strains not only induced endogenous interferon poorly but were also resistant to exogenous interferon. This evidence points to marked differences of the investigated influenza B virus strains not only induced endogenous interferon poorly but were also resistant to exogenous interferon. This evidence points to marked differences of the investigated influenza B virus strains from influenza A virus strains which further indicates their peculiar nature.     

Serodiagnosis of Lyme borreliosis by Borrelia burgdorferi sensu stricto, B. garinii, and B. afzelii western blots (immunoblots)

The performance of Western blots (immunoblots) prepared with eight strains of Borrelia burgdorferi representing B. burgdorferi sensu stricto, B. garinii, and B. afzelii genospecies was tested with a panel of sera with various clinical presentations collected from eight geographic regions. European sera were generally more reactive to blots prepared with B. garinii or B. afzelii strain antigens, in particular B. garinii 20047 and B. afzelii VS461. North American sera were more reactive with B. burgdorferi sensu stricto strains. Our observation of significant differences in the levels of reactivity of some sera on Western blots of certain strains is potentially important for the development and implementation of generic interpretive criteria. Preferential reactivity of sera from patients with nerve and/or palsy symptoms to B. garinii strains and with cutaneous disease to B. afzelii strains was observed. On the basis of our results, we have concluded that strain 20047 is the best strain to use for the development of a generic Lyme borreliosis Western blot for Europe.