Affinity enhancement of complementary peptide recognition

A peptide hydropathically complementary to Big Endothelin [Big ET] residues 16-29 has been synthesized in a multimeric form starting from an octadentate poiylysine core, essentially in a way similar to the procedure used for the production of multiple antigenic peptides [MAP's], Interaction between the multimeric complementary peptide [8δET] and the Big ET fragment 16-32 containing the target complementary region, also synthesized in a multimeric form [8ET], was evaluated by analytical high performance affinity chromatography and solid phase binding assays. While the binding interaction between the monomerics peptide pair was in the micromolar range, the recognition between the corresponding multimeric form was characterized by enhanced binding affinity of at least two orders of magnitude. In solution, complex formation between multimeric complementary peptide and target Big ET sequence in the monomeric and multimeric form was accompanied by precipitation at concentrations higher than 0.5 mg/mL and 0.1 mg/mL, respectively. Polyclonal antibodies raised against the multimeric target sequence recognized multimeric and monomeric ET target sequences with binding affinities similar to binding affinities exhibited by the multimeric complementary peptide. Multimerization of hydropathically complementary peptides could provide an improved opportunity to measure and thus probe quantitative binding properties of complementary peptides.     

Influence of Age, Lead Axis, Frequency of Arrhythmic Episodes, and Atrial Dimensions on P Wave Triggered SAECG in Patients with Lone Paroxysmal Atrial Fibrillation

Signal-averaged P wave of 42 patients with lone paroxysmal atrial fibrillation (PAF) and 29 normal subjects (N) were recorded, using three orthogonal leads and analyzed in the time and frequency (entire P wave or a 100-ms segment ranging from 75 ms before to 25 ms after the end of P wave) domains. PAFs were divided into a group of 12 having ≥ 2 attacks a month (HF) and a group of 30 having ≤ 2 attacks a year (LF). Statistically significant differences were absent with regard to ages of PAF and N; ages of HF, LF, and N at the time of signal-averaged ECG; ages of HF and LF at the time of the first arrhythmic episode; and elapsed times from the first episode. Length of P wave and some frequency-domain parameters were found to be significantly correlated with age. PAF showed a significantly longer duration of P wave in the frontal plane using the time-domain analysis. Frequency analysis was found to be useful in evaluating the influence of attack frequency. HF showed significantly higher values of some frequency-domain parameters than LF and N, while the three groups did not differ for time-domain analysis. P wave duration and frequency content of the three orthogonal leads proved to be significantly different in PAF and N. Right and left atrial echocardiographic dimensions proved to be higher (even if within normal limits) in HF than in LF and N. Results suggest that frequency analysis should be performed on the entire P wave.     

Electrocardiographic Monitoring During Coloscopy

Electrocardiographic abnormalities were observed in 15 of 23 patients (65%), five with known heart disease, who were monitored for one hour prior to, during and for one hour after coloscopy. In one patient, ischemic S-T depression, 2 mm. below the resting level, persisted during the one hour following coloscopy. In all other patients, the electrocardiographic abnormalities disappeared before the end of the monitoring period.     

Bile lipid composition and haemostatic variables in a case of high density lipoprotein deficiency (Tangier disease)

A 62-year-old man with clinical and biochemical findings consistent with homozygous Tangier disease is presented. Widespread atherosclerosis was present. Bile lipid analysis showed a low molar percentage of cholesterol with a low saturation index. The data suggest that high density lipoprotein cholesterol may act as a preferential precursor of biliary cholesterol. Coagulation and platelet studies indicated that the patient's platelets were hyper-responsive to aggregating agents and produced an increased amount of thromboxane B2. A platelet storage pool deficiency was also found.     

Time- and Dose-Dependent Effects of Chronic Wound Fluid on Human Adult Dermal Fibroblasts

BACKGROUND Wound healing is a biologic process that is altered in patients affected by chronic venous ulcers. The wound microenvironment is reflected in the chronic wound fluid (CWF), an exudate containing serum components and tissue-derived proteins.
OBJECTIVES We investigated the effects of increasing doses of CWF collected from patients suffering from chronic venous ulcers on human adult dermal fibroblasts cultured in vitro and the relationship among CWF effects and treatment length.
METHODS Fibroblasts were treated with 60, 240, and 720 μg/mL CWF for 3 and 7 days. We evaluated cell proliferation and viability by MTT and Trypan blue assay, cell morphology by light microscopy, F-actin microfilaments organization by tetramethylrhodamine B isothiocyanate-conjugated phalloidin, α-smooth muscle actin expression by immunofluorescence, and senescence-associated β-galactosidase activity.
RESULTS CWF induced an increase in cell proliferation in the first 3 days of treatment. In contrast, at 7 days, a strong decrease in cell viability was observed. These changes were related to a cytoskeletal F-actin reorganization and not to fibroblast–myofibroblast differentiation nor to changes in cellular senescence.
CONCLUSIONS This study shows a dose-dependent and biphasic effect of CWF on dermal fibroblasts, suggesting that a continuous exposure to chronic wounds microenvironment may induce late cellular dysfunctions possibly involved in the delayed wound healing.     

Optimized Measurement of Activation Rate at Left Atrial Sites with Complex Fractionated Electrograms During Atrial Fibrillation

Local Activation Rate in Atrial Fibrillation. Background: Complex fractionated atrial electrograms (CFAE) have become targets for catheter ablation of atrial fibrillation (AF). Frequency components of AF signals have also become important markers for identifying potential mechanisms of AF, yet inaccuracies exist, particularly in standard dominant frequency (SDF) calculations especially at CFAE sites. We developed new methodology to improve accuracy of AF rate determinations at such recording sites. Objective: To develop optimal methods for estimating activation rates in paroxysmal and persistent AF. Methods: Electrograms were obtained from one right atrial, coronary sinus, and 6 left atrial (LA) endocardial regions manifesting CFAEs in paroxysmal (N = 7) and persistent (N = 7) AF patients. SDF was measured from 8.4 s intervals and compared to (1) optimized DF (ODF) calculated by optimizing the filter coefficients which maximized dominant frequency power, (2) autocorrelation (AC), with the rate estimated as the inverse of the signal phase shift generating the largest autocorrelation coefficient, and (3) ensemble average (EA), with the rate estimated by summing successive signal segments and selecting segment length yielding maximum power. Rate measurements were compared between groups, at baseline and with additive interference, having similar frequency content to the electrograms, to test the robustness of the different methods. Results: From pooled data (N = 168 recording sites), a significantly higher LA dominant frequency was found in persistent versus paroxysmal patients using each method (P < 0.001), with a mean value for all methods of 6.23 ± 0.08 Hz versus 5.32 ± 0.10 Hz, respectively. At the highest additive interference level, the rate measurement error was significantly greater in SDF as compared with EA (P = 0.010) and ODF (P = 0.035), and at all interference levels SDF had the largest error of any method. Conclusions: SDF appears less robust to additive interference, compared to the ODF and EA methods of estimating the activation rate at CFAE sites in this small group of patients. Use of optimized filter coefficients for DF measurement, or use of correlative methods such as EA, that reinforce the signal rather than filtering the noise, may improve calculation of activation rates. (J Cardiovasc Electrophysiol, Vol. 21, pp. 133‐143, February 2010)     

Clinical Evaluation of Morphology Discrimination: An Algorithm for Rhythm Discrimination in Cardioverter Defibrillators

The aim of this study was to test the new morphology discrimination diagnostic algorithm for ICDs that differentiates supraventricular tachycardias (SVTs) from VTs by analysis of ventricular depolarization complexes morphology. Twenty-five patients implanted with a St. Jude Ventritex single chamber ICD were studied during electrophysiological evaluation at predischarge and were followed for 7 +/- 4 months. Sensitivity and specificity for VT detection and overall diagnostic accuracy of the morphology discrimination algorithm were calculated on 326 detected events. At electrophysiological evaluation, the algorithm was tested during 67 episodes of right atrial pacing, during 119 episodes of RV pacing (at basal interventricular septum and RV apex) and during 27 episodes of sustained AF: specificity was 98%, sensitivity was 66%, and diagnostic accuracy was 80%. All episodes of AF were correctly diagnosed as SVT. Exclusion of detections related to pacing at the basal interventricular septum, resulted in a specificity of 98%, a sensitivity of 85%, and a diagnostic accuracy of 93%. During follow-up, evaluation of the morphology discrimination algorithm on 113 spontaneous episodes (31 VTs, 31 AF, 7 SVTs, and 44 sinus tachycardias) exhibited a specificity of 89%, a sensitivity of 100%, and a diagnostic accuracy of 92%. In conclusion, the morphology discrimination algorithm exhibits a high specificity in discriminating VTs from SVTs, although with a corresponding reduction in sensitivity. The preliminary experience on spontaneous episodes is promising. To correct for the reduction in sensitivity, it is advisable to use this algorithm in parallel with other algorithms for rhythm discrimination (sudden onset, stability) coupled with extended high rate.     

High pressure liquid chromatography and electrospray ionization mass spectrometry are advantageously integrated into a two‐levels approach to detection and identification of haemoglobin variants

Detecting and correctly identifying haemoglobin (Hb) variants is typically achieved by a two‐levels laboratory approach. We report our experience in dealing with 91 Hb variants, including a number of frequent and a few rare variants. Screening included akaline agarose gel electrophoresis (AGE), ion‐exchange automated high‐performance liquid chromatography (HPLC) and a test for deoxyhaemoglobin solubility. Identification was based on electrospray ionization–mass spectrometry (ESI–MS). Our results confirmed the advantages of HPLC over AGE for screening, because of the occurrence of some electrophoretically ‘silent’ variants. ESI–MS permitted the definitive identification of 90 of the 91 variants included in the study, in some cases (e.g. HbS) through the application of a simple protocol (direct injection of the sample), in other cases requiring the application of more demanding procedures (purification of the variant chain and peptide analysis after enzymatic or chemical cleavage). In an additional case (Hb J‐Oxford), ESI–MS assay did not lead to definitive identification, but gave indications for designing the appropriate primers to focus DNA sequence analysis on the specific region of the gene. Deoxyhaemoglobin solubility test was positive only in the presence of HbS. We conclude that HPLC and ESI–MS are advantageously integrated into a two‐level analytical system for the detection and confirmation of variant Hbs.