[Long term effectiveness of surgical cryoablation for chronic atrial fibrillation in patients undergoing surgery for severe mitral valve regurgitation]. / Dlouhodobá úcinnost chirurgické kryoablace chronické fibrilace síní provedené pri operaci pro závaznou mitrální regurgitaci.

OBJECTIVE: The objective of the study was to evaluate effectiveness of surgical cryoMAZE ablation for chronic atrial fibrillation (AF) in patients undergoing mitral valve surgical intervention. METHODOLOGY: Forty-seven patients (31 females), aged 67.3 +/- 7.3 years who underwent surgical intervention for severe mitral regurgitation were studied. Mitral valvuloplasty was performed in 21 patients, and mitral valve replacement in 26 patients. Combined procedure was employed in 35 patients; simultaneous aortocoronary bypass was performed in 16 patients, tricuspid valvuloplasty (TVP) in 5 patients, and aortic valve replacement (AVR) in 5 patients. RESULTS: The mean follow-up time was 19 +/- 10 months. After 6 or 12 months 36 or 32 patients were seen and 23 (64%) or 22 (69%) of them were in stable sinus rhythm (SR), respectively. In the subset of 24 patients with simultaneous intervention on a different valve (TVP or AVR), after 6 or 12 months, 14 (74 %) or 15 (83 %) patients had stable SR, respectively. In the follow-up period, 2 patients underwent successful catheter ablation for type I atrial flutter or for a residual left atrial atypical flutter. CONCLUSION: In the study using the method of cryoMAZE ablation for chronic AF performed during the mitral valve surgical intervention, a long-term stable SR was achieved in a high proportion of patients, particularly in patients with simultaneous intervention on two or three different valves.

Molecular evidence of Bartonella DNA in ixodid ticks in Czechia.

We report the development of a novel method for detection of Bartonella DNA in ixodid ticks. The assay is based on a specific amplification of a part of 16S rRNA gene and 16S-23S rRNA intergenic spacer region of Bartonella sp. by nested PCR and Southern blot hybridization with specific DNA probe; the method is highly sensitive and specific. The screening of 327 unfed ticks collected in different urban and suburban areas of Czechia in 2003-2005 revealed the presence of Bartonella DNA in four Ixodes ricinus individuals (1.2%), two males, one female and one nymph.

The prospects of advanced molecular methods in the detection of periodontal pathogens: hybridization in situ.

The paper summarizes the present state in the diagnostics of periodontal pathogens. Both the main advantages and drawbacks of the classic cultivation methods and those of the new DNA techniques are discussed. From the emerging methods of molecular diagnostics, the method of in situ hybridization is presented in more details. Its principle, various modifications of performance and possibilities of use are explained, including examples of its application in the detection of periodontal pathogens.

Construction and testing of a bacterial luciferase reporter gene system for in vivo measurement of nonsense suppression in Streptomyces.

A reporter gene system, based on luciferase genes from Vibrio harvei, was constructed for measurement of translation nonsense suppression in Streptomyces. Using the site-directed mutagenesis the TCA codon in position 13 of the luxB gene was replaced by all of the three stop codons individually. By cloning of luxA and luxB genes under the control of strong constitutive Streptomyces promoter ermE* in plasmid pUWL201 we created Wluxl with the wild-type sequence and pWlux2, pWlux3 and pWlux4 plasmids containing TGA-, TAG- and TAA-stop codons, respectively. Streptomyces lividans TK 24 was transformed with the plasmids and the reporter system was tested by growth of the strain in the presence of streptomycin as a translation accuracy modulator. Streptomycin increased nonsense suppression on UAA nearly 10-fold and more than 20-fold on UAG. On the other hand, UGA, the most frequent stop signal in Streptomyces, the effect was negligible.

Effect of phosphate on the expression of protein-Ser/Thr kinase pkg2 in Streptomyces granaticolor.

A time-correlated expression of eukaryotic-like protein Ser/Thr kinase Pkg2 of Streptomyces granaticolor was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) and by transcriptional fusion experiments. In a complex medium the activity of pkg2 promoter was constant during the life cycle. Direct RNA analysis proved the presence of corresponding pkg2 transcript. S1 nuclease protection analysis of the transcription initiation site showed that pkg2 gene is expressed as a leaderless mRNA. Under phosphate starvation the promoter activity was detectable merely in the early exponential phase. Under these conditions turning off of pkg2 promoter and cessation of pkg2 transcript level coincided with the start of granaticin production.

Detection and characterization of feline Bartonella henselae in the Czech Republic.

The aims of the study were to characterize isolates of Bartonella henselae and to determine the prevalence of bacteremic domestic cats in urban and suburban parts of Prague, Czech Republic. Five (18%) gram-negative fastidious bacterial single-cat isolates were recovered from 27 hemocultures incubated without previous freezing. Four of these isolates originated from flea infested stray cats (n=6) and one from a shelter cat without any ectoparasites (n=21). None of the 34 previously frozen specimens from flea free pet cats yielded any bacteria. All five isolates were catalase and oxidase negative. Their enzymatic activity, RFLP profile of citrate synthetase gene (gltA) and DNA-DNA hybridization results were typical of B. henselae. According to their PvuII and BglI ribotypes the isolates could be allocated to two homogeneous groups. Ribotype HindIII and RFLP of 16S-23S rRNA spacer region analysis gave unique profiles different from those of Bartonella quintana, Bartonella elizabethae and Bartonella clarridgeiae. The 16S rRNA type-specific amplification revealed an identical profile typical of B. henselae genotype II for all the cat isolates studied. Pulsed-field gel electrophoresis (PFGE) assigned a different profile to each of the isolates studied. Determination of the enzymatic activity, RFLP of gltA gene, RFLP of 16S-23S rRNA spacer region, and HindIII ribotype could be efficient tools for identification of B. henselae isolates. Ribotyping (PvuII, BglI), 16S rRNA typing and PFGE may be useful methods to prospect ecology and epidemiology of the agent.

In situ detection of Bartonella henselae cells.

A new in situ hybridization technique was developed for identification of Bartonella henselae cells in cell suspension or in tissue sections. Use of highly specific probes labeled with either fluorescein or digoxigenin allows discrimination between B. henselae and closely related B. quintana cells. No cross-hybridization with other Bartonella or non-Bartonella species was observed. Besides its specificity it showed higher sensitivity as compared to PCR based detection methods. Moreover, its application allows direct observation of B. henselae in infected tissues.

Inhibition of quorum sensing by a Pseudomonas aeruginosa dksA homologue.

The Pseudomonas aeruginosa las (lasR-lasI) and rhl (rhlR-rhlI) quorum-sensing systems regulate the expression of several virulence factors, including elastase and rhamnolipid. P. aeruginosa strain PR1-E4 is a lasR deletion mutant that contains a second, undefined mutation which allows production of elastase and rhamnolipid despite a nonfunctional las system. We have previously shown that this strain accomplishes this by increasing the expression of the autoinducer synthase gene rhlI. In this report, we show that the elastolytic phenotype of mutant PR1-E4 can be complemented with a P. aeruginosa homologue of the Escherichia coli dnaK mutation suppressor gene dksA. When supplied in trans on a multicopy plasmid, this gene completely suppressed elastase production by mutant PR1-E4. Cloning and Northern blot analysis revealed that dksA was neither mutated nor less transcribed in mutant PR1-E4. When overexpressed, dksA also reduced rhamnolipid production by both mutant PR1-E4 and the wild type, PAO1. Using Northern blot analysis and lacZ reporter fusions, we show that dksA inhibits rhlI, rhlAB, and lasB transcription. Exogenous N-butyryl-L-homoserine lactone overcame the reduced expression of rhlI and restored rhlAB and lasB expression, as well as elastase production. Our results suggest that the overproduction of the P. aeruginosa DksA homologue inhibits quorum-sensing-dependent virulence factor production by downregulating the transcription of the autoinducer synthase gene rhlI.

Evidence for phosphoprotein phosphatase in Streptomyces granaticolor.

The existence of phosphoprotein phosphatase (PPP) in aerial mycelium of S. granaticolor was demonstrated. Using inhibitors of serine and/or threonine PPP and specifically labeled substrate it was found that the PPP is of the serine and/or threonine type.

Pkg2, a novel transmembrane protein Ser/Thr kinase of Streptomyces granaticolor.

A 4.2-kb SphI-BamHI fragment of chromosomal DNA from Streptomyces granaticolor was cloned and shown to encode a protein with significant sequence similarity to the eukaryotic protein serine/threonine kinases. It consists of 701 amino acids and in the N-terminal part contains all conserved catalytic domains of protein kinases. The C-terminal domain of Pkg2 contains seven tandem repeats of 11 or 12 amino acids with similarity to the tryptophan-docking motif known to stabilize a symmetrical three-dimensional structure called a propeller structure. The pkg2 gene was overexpressed in Escherichia coli, and the gene product (Pkg2) has been found to be autophosphorylated at serine and threonine residues. The N- and C-terminal parts of Pkg2 are separated with a hydrophobic stretch of 21 amino acids which translocated a PhoA fusion protein into the periplasm. Thus, Pkg2 is the first transmembrane protein serine/threonine kinase described for streptomycetes. Replacement of the pkg2 gene by the spectinomycin resistance gene resulted in changes in the morphology of aerial hyphae.

Characterisation of two putative protein Ser/Thr kinases from actinomycete Streptomyces granaticolor both endowed with different properties.

The structural genes, pkg4 and pkg3, encoding two putative protein serine/threonine kinases in Streptomyces granaticolor, have been cloned and sequenced. The genes were isolated after screening genomic sublibraries with specific probes obtained by PCR amplification of chromosomal DNA using degenerate primers which correspond to amino acid sequences highly conserved in eukaryotic protein Ser/Thr kinases. The sequences of these genes predict polypeptide chains of 761 and 780 amino acids for Pkg4 and Pkg3, respectively. The genes are separated by only 2 bp and therefore probably constitute an operon. pkg4, which is positioned upstream of pkg3, contains a UUALeu codon suggesting a developmental-dependent mode of expression. The amino-terminal half of both proteins clearly shares similarities with the family of protein Ser/Thr kinases. Both proteins studied also possess a region rich in Pro and Ala residues and a repeating motif of 11 amino acid residues, the function of which is unknown, in the carboxy-terminal domain. Expression of pkg4 in Escherichia coli gave rise to two different forms: a soluble protein autophosphorylated at threonine residues and an insoluble form phosphorylated at threonine and serine residues. In contrast, when pkg3 was expressed in E. coli, no autophosphorylation was detected either in vivo or in vitro.

Antibiotic resistance gene cassettes derived from the omega interposon for use in E. coli and Streptomyces.

Three antibiotic resistance gene cassettes, derived from the omega interposon (Prentki and Krisch (1984) Gene 29, 303-313) were constructed. These cassettes carry different antibiotic resistance genes, conferring resistance to geneticin, hygromycin or viomycin, flanked by short inverted repeats containing transcription and translation termination signals and synthetic polylinkers. These cassettes were designated omega aac, omega hyg and omega vph. Resistance phenotypes conferred by these constructions are selectable in E. coli and Streptomyces. These cassettes can be used for insertional mutagenesis or for vector construction.

The genes for phosphofructokinase and pyruvate kinase of Lactobacillus delbrueckii subsp. bulgaricus constitute an operon.

In Lactobacillus delbrueckii subsp. bulgaricus, the pyk gene coding for pyruvate kinase and the pfk gene coding for phosphofructokinase formed a bicistronic operon transcribed into a 2.9-kb RNA. The nucleotide sequence of the pyk gene indicated that the encoded protein possessed an extra C-terminal domain with a potential phosphoenolpyruvate-dependent autophosphorylation site.

The role of Glu187 in the regulation of phosphofructokinase by phosphoenolpyruvate.

In bacterial phosphofructokinases, either a glutamic or an aspartic residue is present at position 187, and the mechanism of inhibition by phosphoenolpyruvate seems to be correlated to the nature of residue 187. Upon binding phosphoenolpyruvate, only the enzymes with a Glu187 would undergo a major allosteric conformational change from an active into an inactive state, whereas the enzymes with an Asp187 would only show a simple upward shift in their pH-profile of activity. The phosphofructokinase from Spiroplasma citri, which has an Asp187, has been purified and its properties follow this pattern. The behaviour of mutants of the enzyme from Escherichia coli in which Glu187 is replaced by either aspartate or leucine confirms the importance of residue 187. The major allosteric transition of E. coli phosphofructokinase is abolished by the substitution Glu187-->Asp, suggesting that a glutamate at position 187 is necessary (but not sufficient) for the protein to undergo the change from the active into the inactive state induced by phosphenolpyruvate. In addition, the presence of an acidic residue, aspartate or glutamate, at position 187 is required (but not sufficient) for the binding of ADP (or GDP). This requirement of a negative charge for ADP binding could explain the striking conservation of an aspartate residue at position 187 in all the eukaryotic phosphofructokinases.

Cloning, sequencing, and expression in Escherichia coli of the gene coding for phosphofructokinase in Lactobacillus bulgaricus.

A fragment of 1,185 bp containing the gene coding for phosphofructokinase (ATP:D-fructose-6-phosphate-1-phosphotransferase; EC 2.7.1.11) in Lactobacillus bulgaricus has been cloned, sequenced, and expressed in Escherichia coli. The amino acid sequence of this enzyme was homologous to those of the ATP-dependent phosphofructokinases from E. coli, Thermus thermophilus, Spiroplasma citri, and Bacillus stearothermophilus, suggesting that these enzymes have closely related structures despite their different regulatory properties. The recombinant protein had the same structural and functional properties as did the original enzyme. The 3' end of the 1,185-bp fragment showed the presence of an open reading frame corresponding to the N-terminal amino acid sequence of the pyruvate kinase from L. bulgaricus. This gene organization, the same as that in S. citri (C. Chevalier, C. Saillard, and J. M. Bové, J. Bacteriol. 172:2693-2703, 1990) and B. stearothermophilus (D. Walker, W. N. Chia, and H. Muirhead, J. Mol. Biol. 228:265-276, 1992; H. Sakai and T. Ohta, Eur. J. Biochem. 311:851-859, 1993) but different from that in E. coli (H. W. Hellinga and P. R. Evans, Eur. J. Biochem. 149:363-373, 1985), indicated that the same transcription unit apparently contained the genes for phosphofructokinase and pyruvate kinase, the two key enzymes of glycolysis. The possibility that these genes could be transcribed at the same time suggested that in L. bulgaricus, the coordinated regulation of phosphofructokinase and pyruvate kinase occurs at the levels of both biosynthesis and enzymatic activity.

DNA-dependent RNA polymerase from an extremely thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum.

Extremely thermophilic bacterium Calderobacterium hydrogenophilum contains DNA-dependent RNA polymerase with unusual properties. Purified enzyme is thermoresistant (40 min at 100 degrees C) and exhibits similar subunit composition as eubacterial RNA polymerases (e.g. Escherichia coli). However, the enzyme is not susceptible to antibiotics which inhibit eubacterial RNA polymerases (rifampicin and streptolydigin). The activity of the enzyme is inhibited by actinomycin D, daunomycin and heparin.

Molecular and functional properties of protein SS1 from small ribosomal subunits of Streptomyces aureofaciens.

Small ribosomal subunits of gram-positive cells of Streptomyces aureofaciens contain an acidic protein designated SS1. Purified protein SS1 has the same mobility in sodium dodecyl sulfate/polyacrylamide gel as ribosomal protein S1 of Escherichia coli (apparent Mr 68 000). Protein SS1 was dissected under mild conditions with trypsin and generated fragments were compared with well-characterized fragments of protein S1. The protein SS1 contains a structure homologous with the C-terminal fragment of protein S1. The affinity of protein SS1 to poly(U) is virtually identical with that of E. coli protein S1. In contrast to protein S1, the addition of SS1 to partially S1-depleted ribosomes of E. coli had no stimulatory effect on poly(U)-directed synthesis of polyphenylalanine. At molar excess of SS1 over ribosomes, the protein had comparable inhibitory effect on polypeptide synthesis as had S1 of E. coli. Ribosomes of S. aureofaciens required about one order of magnitude higher concentration of poly(U) for maximum synthetic activity than did ribosomes of E. coli. The addition of proteins SS1 or S1 to ribosomes of S. aureofaciens had no stimulatory effect on translation of poly(U). Our data indicate that the high-molecular-mass acidic protein SS1 of small ribosomal subunits of S. aureofaciens exhibits only a part of the functional properties of E. coli protein S1.