Cryoablation of junctional tachycardia at high risk of atrio-ventricular block.

INTRODUCTION: Transcatheter cryoablation is an alternative option for the treatment of supraventricular tachycardia, due to its very low risk of permanent atrio-ventricular block. However, the overcost of cryocatheter and the high recurrence rate of this emerging technology braked its large use. This study reports the results of an approach using cryoablation for the treatment of junctional tachycardia (JT) in selected patients at high risk of atrio-ventricular (AV) block. PATIENTS AND METHODS: Out of a series of 199 patients with JT treated by catheter ablation, 26 benefited from cryoablation (mean age 32.8+/-15 years, 15 males). The indications were the presence of an accessory pathway with a high risk of atrio-ventricular block (n=7), a slow pathway difficult to ablate, with a risk of atrio-ventricular block (n=7), a recurrence after a RF procedure, during which a transient atrio-ventricular block has occurred (n=4), and finally patients at young age (n=8). RESULTS: The primary success rate was 92%. No permanent AV block has been reported, neither with RF nor with cryoablation. The recurrence rate at 9+/-10 months was at 29% after cryoablation and 8.6% after RF. In case of AV nodal reentrant tachycardia, the additional cost of cryotherapy catheter has been avoided in 76.85% of cases. The use of a cryotherapy catheter and RF catheter has been necessary for the remaining cases. CONCLUSION: This study demonstrates that an approach, reserving cryoablation in selected patients at high risk of AV block is an alternative strategy to "the systematic use" of cryotherapy in the ablation of JT with a high efficacy, an excellent safety and a reduced cost.

Fluorescent advanced glycation end products in the detection of factual stages of cartilage degeneration.

Patients treated for knee disorders were included in this study. They were examined clinically (Lequesne and Tegner scores) and by standard X-ray investigation. Patients underwent a surgical procedure, either arthroscopy or knee replacement. At the initial phase of surgery, a sample of cartilage was taken for laboratory examination. Progression of the disorder and the clinical examination was correlated with the actual state of the cartilage using a novel fluorescence approach. The intrinsic fluorescence of cartilages was shown as a suitable and sensitive method for detection of the actual state of cartilages because the correlation with X-ray examination and clinical status was found. Intrinsic fluorescence properties of cartilages from patients with chondropathy and osteoarthritis were described and found to be age-dependent. We also observed a higher concentration of advanced glycation end products due to inflammatory and/or degenerative processes in the cartilage. In addition, acute pathological changes due to diseases such as meniscal lesions or anterior cruciate ligament rupture caused a significant increase of formation of advanced glycation end products even in the group of young patients. In fact, such an observation could be crucial and important for the detection of knee conditions suspected of early meniscal and/or ACL lesions especially among young patients.

Protein structure and dynamics determined by protein modeling combined with spectroscopic techniques.

Beside of the protein crystals, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy or Raman spectroscopy, and with molecular biology techniques. This method could achieve reliable results comparable with resolution obtained from crystal structures. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we proved that a hydrogen bond between Arg423 and Glu472 supported the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth-strands, which both contain residues that take part in the ATP-binding (Ref. 34).

Protein modeling combined with spectroscopic techniques: an attractive quick alternative to obtain structural information.

Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys501, Gly502 and Arg544, also Glu446, Phe475 and Gln482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth beta-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.

Fluorescence competition assay for the assessment of ATP binding to an isolated domain of Na+, K(+)-ATPase.

An equation allowing estimation of the dissociation constant for binding of a non-fluorescent ligand to the enzyme is presented that is based on the competitive replacement of the ligand by its fluorescent analog. We derived an explicit formula for the probe fluorescence intensity, which is suitable for nonlinear least-squares analysis. We used this formula to evaluate the binding of ATP to the large cytoplasmic loop of Na+,K(+)-ATPase. The estimated value of KD (6.2+/- 0.7 mM) is comparable with the results from other laboratories for similar constructs obtained by a different method.

Frequency-domain lifetime fluorometry of double-labeled creatine kinase.

Myofibril-bound creatine kinase EC 2.7.3.2 (CK), a key enzyme of muscle energy metabolism, has been selected for studies of conformational changes that underlie the cellular control of enzyme activity. For fluorescence spectroscopy measurements, the CK molecule was double-labeled with IAF (5-iodoacetamidofluorescein) and ErITC (erythrosin 5'-isothiocyanate). Measurement of fluorescence resonance energy transfer (FRET) from fluorescein to erythrosin was used to obtain information about the donor-acceptor pair distance. Frequency-domain lifetime measurements evaluate the donor-acceptor distance in the native CK molecule as 7.8 nm. The Förster radius equals 5.3 nm with the resolution range from 0.2 to 1.0 nm. Erythrosin-fluorescein labeling (EFL) was tested for artificial conformational changes of the CK molecule with high-salt concentration treatment. The transition distance, defined by His-97 and Cys-283 and derived from a 3D model equals 0.766 nm for the open (inactive) form and 0.277 nm for the closed (reactive) form of the CK molecule. In this way, the resolution range of the used spectroscopy method is significant, concerning the difference of 0.489 nm. Nevertheless, the CK enzyme activity, assessed by the hexokinase-coupled assay, was diminished down to 1 % of the activity of the native enzyme. EFL is suitable for description of conformational behavior implied from the regulation of creatine kinase. However, the observed inhibition restricts EFL to studies of conformational changes during natural catalytic activity.

Limitations in linearized analyses of binding equilibria: binding of TNP-ATP to the H4-H5 loop of Na/K-ATPase.

Binding of TNP-ATP [2',3'- O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, a fluorescent analogue of ATP] to the K605 protein was studied. This is an isolated N-domain in the cytoplasmic loop of the Na/K-ATPase alpha-subunit, lying between membrane-spanning segments 4 and 5 (sequence L(354)-I(604)). A titration equation is derived that explicitly makes it possible to relate the fluorescence of TNP-ATP and K605 solutions to total probe concentration in the sample. Using this, it is possible to obtain the value of the dissociation constant from the titration experiment without resorting to the Scatchard plot, which is far from optimal from the statistical point of view. Using the new formula with non-linear regression analysis, a value of the dissociation constant K(D) for TNP-ATP binding to the K605 protein of 3.03 +/- 0.28 microM at 22 degrees C was obtained. A series of fits to simulated data with added noise demonstrated clearly the advantage of non-linear regression using the new formula over the commonly employed linear regression using the Scatchard plot. The procedure presented is generally applicable to binding assays using changes in the fluorescence of ligands on binding.