The roles of mid-myocardial and epicardial cells in T-wave alternans development: a simulation study.

BACKGROUND: The occurrence of T-wave alternans in electrocardiographic signals was recently linked to susceptibility to ventricular arrhythmias and sudden cardiac death. Thus, by detecting and comprehending the origins of T-wave alternans, it might be possible to prevent such events. RESULTS: Here, we simulated T-wave alternans in a computer-generated human heart model by modulating the action potential duration and amplitude during the first part of the repolarization phase. We hypothesized that changes in the intracardiac alternans patterns of action potential properties would differentially influence T-wave alternans measurements at the body surface. Specifically, changes were simulated globally in the whole left and right ventricles to simulate concordant T-wave alternans, and locally in selected regions to simulate discordant and regional discordant, hereinafter referred to as "regional", T-wave alternans. Body surface potential maps and 12-lead electrocardiographic signals were then computed. In depth discrimination, the influence of epicardial layers on T-wave alternans development was significantly higher than that of mid-myocardial cells. Meanwhile, spatial discrimination revealed that discordant and regional action potential property changes had a higher influence on T-wave alternans amplitude than concordant changes. Notably, varying T-wave alternans sources yielded distinct body surface potential map patterns for T-wave alternans amplitude, which can be used for location of regions within hearts exhibiting impaired repolarization. The highest ability for T-wave alternans detection was achieved in lead V1. Ultimately, we proposed new parameters Vector Magnitude Alternans and Vector Angle Alternans, with higher ability for T-wave alternans detection when using multi-lead electrocardiographic signals processing than for single leads. Finally, QT alternans was found to be associated with the process of T-wave alternans generation. CONCLUSIONS: The distributions of the body surface T-wave alternans amplitude have been shown to have unique patterns depending on the type of alternans (concordant, discordant or regional) and the location of the disturbance in the heart. The influence of epicardial cells on T-wave alternans development is significantly higher than that of mid-myocardial cells, among which the sub-endocardial layer exerted the highest influence. QT interval alternans is identified as a phenomenon that correlate with T-wave alternans.

Polynucleotide phosphorylase from Streptomyces aureofaciens: purification and properties.

1. Polynucleotide phosphorylase from a chlortetracycline-producing strain of Streptomyces aureofaciens was isolated by Polymin P fractionation. Using chromatography on DEAE-cellulose and Sephadex G-150 the enzyme, which appears homogeneous in gel chromatography and sedimentation analysis, was purified 2000-fole giving a final yield of 15%. 2. The sedimentation coefficient (s-o 20, w) of the native enzyme in 0.2 M NaCl is 9.15 S and its molecular weight is 210 000 plus or minus 15 000. Molecular weight estimated by sodium dodecylsulfate gel electrophoresis was about 100 000. 3. We have determined the optimal conditions for nucleoside 5'-diphosphate polymerization, their phosphate exchange and phosphorolysis of polyribonucleotides catalysed by polynucleotide phosphorylase from S. aureofaciens. 4. Chlortetracycline is a competitive inhibitor of S. aureofaciens polynucleotide phosphorylase. 5. Polynucleotide phosphorylase is activated in the polymerization reaction by ionic strength (K+, Na+, NH4+) while polyribonucleotide phosphorolysis is activated only by NH4+.

Production of multiple forms during purification of Streptomyces aureofaciens DNA polymerase: a study using nondenaturing polyacrylamide gradient gel electrophoresis.

A modified method for the detection of DNA polymerases in cell extracts and purified enzyme preparations after electrophoresis in polyacrylamide gradient cylindrical gels is described. The technique, which is based on direct assay of activity in a reaction mixture during elution of DNA polymerases from gel slices, was applied to the pursuit of enzyme forms of Streptomyces aureofaciens DNA polymerase during purification procedure. In a crude extract of S. aureofaciens mycelium many catalytically active forms of DNA polymerase ranging from 35 to 860 kDa were detected. After purification, that included mycelium homogenization, precipitation of nucleic acids by polyethylene glycol, DEAE-Sephadex, QAE-Sephadex and DNA-Sepharose chromatography, higher molecular mass forms of more than 172 kDa have not been found. The lower molecular mass forms were separated into two groups by DNA-Sepharose chromatography. On the basis of their characterization, it is assumed that the lower molecular mass forms are produced by proteolysis and the major form found after purification of S. aureofaciens DNA polymerase in the presence of suitable proteinase inhibitors should be a protein of 172 kDa.

Modification methylase M.Sau3239I from Streptomyces aureofaciens 3239.

By chromatography on phosphocellulose and Heparin-Sepharose the modification methylase M.Sau3239I was detected and partly purified from cells of Streptomyces aureofaciens 3239. Methylation by this enzyme protects DNA from cleavage by the restriction endonuclease R.Sau3239I. The enzyme catalyzes methylation of adenine to N-6-methyladenine in the 5'-CTCGmAG-3' recognition sequence.

Amino acid sequence determination of guanyl-specific ribonuclease Sa from Streptomyces aureofaciens.

Using automated Edman degradation of two nonfractionated peptide mixtures of tryptic and staphylococcal protease digests of the protein, the complete amino acid sequence of the guanyl-specific ribonuclease Sa from Streptomyces aureofaciens was established. Ribonuclease Sa contains 96 amino acid residues (Mr 10,566). A 50% sequence homology of ribonuclease Sa to the guanyl-specific ribonuclease St from S. erythreus was found.

DNA-dependent RNA polymerase from the chlorotetracycline producing strain of Streptomyces aureofaciens.

RNA polymerase from Streptomyces aureofaciens has been purified by polyethyleneimine precipitation followed by chromatography first on DEAE-cellulose, then heparin-Sepharose and finally on an aminooxybutylcellulose matrix containing immobilised S. aureofaciens DNA. The enzyme is composed of three subunits of approximately 145, 136 and 44 kDa that are in a ratio of approx. 1:1:2. In many isolations two additional subunits of approximately 68 and 39 kDa and some minor protein bands of approximately 110, 85 and 61 kDa are also present. Thus, the structure of this enzyme is very similar to other bacterial RNA polymerases, exhibiting an alpha 2 beta beta' core and the additional proteins rho and sigma.

Ribosomal ribonucleic acids from Streptomyces griseus.

Nucleic acids from Streptomyces griseus 178 were isolated during cultivation. After their fractionation on a column of methylated serum albumin adsorbed on Kieselguhr, the 16 S and 23 S RNA were isolated. To characterize RNAs their sedimentation coefficients, Tm and nucleotide composition were determined. During cultivation of S. griseus 178 rRNA level reaches two maximum peaks and the production of streptomycin influences nucleic aicds of the producer organism.

The synthesis of highly phosphorylated nucleotides, RNA and protein by Streptomyces aureofaciens.

During the sudden decrease in RNA synthesis in Streptomyces aureofaciens, i.e. around the 6th hour of cultivation, synthesis of adenosine and guanosine tetraphosphates and pentaphosphates begins. The synthesis of these nucleotides is highest during the onset of chlortetracycline production, around the 20th hour of cultivation and continues. During this phase of growth of S. aureofaciens, RNA and protein synthesis are reduced by about one order of magnitude as compared to the rate which can be observed at the beginning of cultivation, but the synthesis is not inhibited by exogenous CTC.

NMR studies on interactions of ribonuclease Sa with Guo-3'-P.

Some features of the interaction of guanyloribonuclease Sa from Streptomyces aureofaciens with its competitive inhibitor Guo-3'-P were investigated by 1H and 31P NMR spectroscopy. The pH dependence of chemical shifts of C(2)-H protons of the histidine residue of the enzyme were analysed, in the absence and presence of Guo-3'-P. This analysis showed that only one of the two histidines of ribonuclease Sa is located in the active site of the enzyme. 31P NMR resonances of the nucleotide and of its complex with the enzyme indicated that this histidine interacts with the phosphate group of the substrate. The possible relationship between the observed perturbation of the NMR titration curve of the active site of histidine and a conformational change in the enzyme molecule at a pH of approximately 7.5 is also discussed.

The number and role of histidine residues in the active site of guanyloribonuclease Sa.

The number and role of histidine residues in the active site of extracellular guanyloribonuclease Sa produced by Streptomyces aureofaciens (RNAase Sa) were studied via chemical modification by ethoxyformic anhydride by means of circular dichroism measurements. It was shown that only one of two histidines of RNAase Sa is situated in the active site of the enzyme. Ethoxyformylation of RNAase Sa in the presence of Guo-3'-P, Guo-5'-P and dGuo-5-P, all of them being competitive inhibitors of the enzyme, supported the assumption that an essential histidine residue is bound to the phosphate group in the position 3' of the ribose ring. The circular dichroism measurements of native and modified RNAase Sa and of its complex with Guo-3'-P showed that the modification of the essential histidine residue resulted in alteration of binding of RNAase Sa to Guo-3'-P; histidine thus may play a key role in the formation of such a complex.

Specific endonucleases in Streptomyces aureofaciens.

Two strains of Streptomyces aureofaciens were found to contain restriction endodeoxynucleases; S. aureofaciens IKA 18/4 contains Sau I which splits lambda DNA into three fragments, S. aureofaciens IKA 22201 Sau Ii which splits lambda DNA into more than 15 fragments.

[Extracellular guanyl-specific ribonuclease Sa from the actinomycete Streptomyces aureofaciens. Primary structure and homology with ribonucleases from bacteria and fungi]. / Vnekletochnaia guanilspetsifichnaia ribinukleaza Sa aktinomitseta Streptomyces aureofaciens. Pervichnaia struktura i gomologiia s ribonukleazami bakterii i gribov.

The complete amino acid sequence of a guanyl-specific RNAse from Streptomyces aureofaciens has been established using a rapid method of primary structure analysis which eliminates the peptide fractionation. The automated Edman degradation of the carboxymethylated RNAse Sa and of non-fractionated peptide mixtures produced by tryptic and staphylococcal protease digests of the modified protein were used. The RNAse contains 96 amino acid residues, Mr 10,566. The secondary structures of RNAse Sa and microbial RNAses have been calculated using a modified Chou--Fasman procedure. A comparison of the primary and secondary structures of the RNAses revealed different degrees of sequence homology and a similar distribution of predicted structural regions (alpha-helices, beta-structure and beta-turn). The predicted secondary structure patterns are discussed in the light of the RNAse X-ray analysis date.

Temporal variability of radon in the atmosphere of Domica and Vazecká Karst caves (Slovakia).

Continual monitoring of radon activity concentration was performed in two caves: Domica and Vazecká. Radon in the air of the Domica cave was monitored from June 2010 to July 2011. Radon research in the Vazecká cave started in June 2012 and it is still being carried out. Radon concentration in cave atmosphere exhibited seasonal, short-term and daily variations. Daily average of radon in Domica varied from 0.5 to 2.7 kBq m(-3). Seasonal trend was characterised by the highest concentration in September and the lowest from February to March. Radon concentration in the Vazecká cave was significantly higher, and the daily average ranged from 1.0 to 5.3 kBq m(-3). The highest values were registered from June to September and in January. The seasonal and daily variations of 222Rn activity concentration in the atmosphere of both caves are assumed to be associated with the atmospheric temperature. No effect of atmospheric pressure on radon short-term variation was found.