Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic anaerobic methanarchaeon from a deep-sea hydrothermal vent, carries an NADH oxidase (Nox) homologue (MJ0649). According to the characteristics described here, MJ0649 represents an unusual member within group 3 of the flavin-dependent disulfide reductase (FDR) family. This FDR group comprises Nox, NADH peroxidases (Npx) and coenzyme A disulfide reductases (CoADRs); each carries a Cys residue that forms Cys-sulfenic acid during catalysis. A sequence analysis identified MJ0649 as a CoADR homologue. However, recombinant MJ0649 (rMJNox), expressed in Escherichia coli and purified to homogeneity an 86 kDa homodimer with 0.27 mol FAD (mol subunit)(-1), showed Nox but not CoADR activity. Incubation with FAD increased FAD content to 1 mol (mol subunit)(-1) and improved NADH oxidase activity 3.4-fold. The FAD-incubated enzyme was characterized further. The optimum pH and temperature were > or =10 and > or =95 degrees C, respectively. At pH 7 and 83 degrees C, apparent Km values for NADH and O2 were 3 microM and 1.9 mM, respectively, and the specific activity at 1.4 mM O2 was 60 micromol min(-1) mg(-1); 62 % of NADH-derived reducing equivalents were recovered as H2O2 and the rest probably generated H2O. rMjNox had poor NADPH oxidase, NADH peroxidase and superoxide formation activities. It reduced ferricyanide, plumbagin and 5,5'-dithiobis(2-nitrobenzoic acid), but not disulfide coenzyme A and disulfide coenzyme M. Due to a high Km, O2 is not a physiologically relevant substrate for MJ0649; its true substrate remains unknown.

Tyr235 of GTP-dependent phosphoenolpyruvate (PEP) carboxykinase is a fully invariant residue. The aromatic ring of this residue establishes an energetically favorable weak anion-quadrupole interaction with PEP carboxylate. The role of Tyr235 in catalysis was investigated via kinetic analysis of site-directed mutagenesis-derived variants. The Y235F change lowered the apparent K-m for PEP by about six-fold, raised the apparent K-m for Mn2+ by about 70-fold, and decreased oxaloacetate (OAA)-forming activity by about 10-fold. These effects were due to an enhanced anion-quadrupole interaction between the aromatic side chain at position 235, which now lacked a hydroxyl group, and PEP carboxylate, which probably increased the distance between PEP and Mn2+ and consequently affected the phosphoryl transfer step and overall catalysis. For the Y235A and Y235S changes, an elimination of the favorable edge-on interaction increased the apparent K-m for PEP by four- and six-fold, respectively, and the apparent K-m for Mn2+ by eight- and six-fold, respectively. The pyruvate kinase-like activity, representing the PEP dephosphorylation step of the OAA-forming reaction, was affected by the substitutions in a similar way to the complete reaction. These observations indicate that the aromatic ring of Tyr235 helps to position PEP in the active site and the hydroxyl group allows an optimal PEP-Mn2+ distance for efficient phosphoryl transfer and overall catalysis. The Y235A and Y235S changes drastically reduced the PEP-forming and OAA decarboxylase activities, probably due to the elimination of the stabilizing interaction between Tyr235 and the respective products, PEP and pyruvate.

We report the first kinetic characterization of human liver cytosolic GTP-dependent phosphoenolpyruvate carboxykinase (GTP-PEPCK), which plays a major role in the development of type 2 diabetes in human. In this work two recombinant forms of the enzyme were studied. One form had a His10-tag and the other was His-tag-free, and with one exception, both exhibited similar kinetic properties. When Mn2+ was used as the sole divalent cation, the His10-tagged enzyme, but not the His-tag-free enzyme, was increasingly inhibited at Mn2+ concentrations greater than 0.7 mM. This inhibition did not pose any problem in kinetic analysis, for within the relevant Mn2+ concentration range the His-tagged human PEPCK behaved almost identically to the tag-free enzyme. This property will bring simplicity and speed to purifying and studying multiple structural variants of this important enzyme. Apparent Km values of tag-free enzyme for phosphoenolpyruvate, GDP and bicarbonate were 450, 79 and 20,600 microM, respectively, while those for oxaloacetate and GTP were 4 and 23 microM, respectively, emphasizing the enzyme's gluconeogenic character. Bicarbonate (>100 mM) inhibited OAA-forming activity, which was a new observation with a GTP-PEPCK. The apparent Km for Mn2+ in the PEP-forming direction was 30-fold lower than that for the OAA-forming direction. Mn2+ and bicarbonate or CO2 might regulate the enzyme in vivo.

The roles of Asp75, Asp78, and Glu83 of the 75DPSDVARVE83 element of Mycobacterium smegmatis GTP-dependent phosphoenolpyruvate (PEP) carboxykinase (GTP-PEPCK) were investigated. Asp78 and Glu83 are fully conserved in GTP-PEP-CKs. The human PEPCK crystal structure suggests that Asp78 influences Tyr220; Tyr220 helps to position bound PEP, and Glu83 interacts with Arg81. Experimental data on other PEPCKs indicate that Arg81 binds PEP, and the phosphate of PEP interacts with Mn2+ of metal site 1 for catalysis. We found that D78A and E83A replacements severely reduced activity. E83A substitution raised the apparent Km value for Mn2+ 170-fold. In contrast, Asp75 is highly but not fully conserved; natural substitutions are Ala, Asn, Gln, or Ser. Such substitutions, when engineered, in M. smegmatis enzyme caused the following. 1) For oxaloacetate synthesis, Vmax decreased 1.4-4-fold. Km values for PEP and Mn2+ increased 3-9- and 1.2-10-fold, respectively. Km values for GDP and bicarbonate changed little. 2) For PEP formation, Vmax increased 1.5-2.7-fold. Km values for oxaloacetate increased 2-2.8-fold. The substitutions did not change the secondary structure of protein significantly. The kinetic effects are rationalized as follows. In E83A the loss of Glu83-Arg81 interaction affected Arg81-PEP association. D78A change altered the Tyr220-PEP interaction. These events perturbed PEP-Mn2+ interaction and consequently affected catalysis severely. In contrast, substitutions at Asp75, a site far from bound PEP, brought subtle effects, lowering oxaloacetate formation rate but enhancing PEP formation rate. It is likely that Asp75 substitutions affected PEP-Mn2+ interaction by changing the positions of Asp78, Arg81, and Glu83, which translated to differential effects on two directions.

Pre-event identification of a specific cardiac substrate abnormality in a patient is an important step in preventing sudden cardiac death (SCD) in the pediatric and adult population. Certain cardiac substrate abnormalities can render the patient "at risk" for SCD and strategies for prevention of SCD in children must involve the identification and subsequent modification of these cardiac substrates.

Pediatric cardiovascular centers should aim to provide high-quality therapeutic outcomes for infants and children with congenital and acquired heart diseases. This policy statement describes critical elements and organizational features of centers in which high-quality outcomes have the greatest likelihood of occurring. Center elements include noninvasive diagnostic modalities, cardiac catheterization, cardiovascular surgery, and cardiovascular intensive care. These elements should be organizationally united in centers in which pediatric cardiac physician specialists and specialized pediatric staff work together to achieve and surpass existing quality-of-care benchmarks.

We report a case of 2:1 atrioventricular block associated with acquired long QT syndrome. A newborn presented to our neonatal intensive care unit with intermittent bradycardia due to 2: 1 atrioventricular block. Initial evaluation showed QT prolongation and significant electrolytic abnormalities. After correction of the electrolytic imbalance, the QT interval normalized and atrioventricular block resolved. Compared to congenital long QT syndrome with 2: 1 atrioventricular block, acquired long QT syndrome with comparable atrioventricular block has a benign prognosis, provided treatment is initiated quickly.

Ventricular tachycardia is a rare arrhythmia in the pediatric population that occurs in structurally normal and abnormal hearts. The treatment of ventricular tachycardia is aimed at reducing symptoms and the risk of sudden death. The intracardiac electrophysiology study and radiofrequency ablation have come to the forefront of diagnostic and therapeutic options for patients with ventricular arrhythmias. The indications for radiofrequency ablation are debated, but it has been shown to be an effective and safe means of eliminating ventricular tachycardia. The purpose of this review is to outline the mechanisms and electrophysiologic characteristics of ventricular tachycardia, discuss the forms common to the pediatric population and summarize the indications, success and complications of radiofrequency ablation for ventricular tachycardia.

The long QT syndrome should be considered when evaluating patients with syncope. Prolongation of the QT interval and abnormalities of T wave morphology due to abnormal ventricular repolarization characterize the syndrome. In the past decade, molecular genetics has revealed that abnormal repolarization is the result of gene mutations encoding integral ion channels that generate the cardiac action potential. Eight subgroups of long QT syndrome associated with five genes have been described to date. The explosion in research in this area has led to a greater understanding of the clinical expression of autosomal dominant (Romano-Ward syndrome), autosomal recessive (Jervell and Lange-Nielsen syndrome) and acquired forms of the disease. This has also led to investigation in the area of genotype-specific therapy. The purpose of this review is to outline the strides made in the field of molecular genetics and update the reader on the recent advances in diagnosis and treatment of the long QT syndrome.

The issue of the proper use of radiofrequency catheter ablation in infants and small children is controversial. Rarely, catheter ablation techniques are indicated to cure malignant arrhythmias in the pediatric population. Indications, techniques, complications and success rates are reviewed.

Pediatric primary care practitioners are truly on the front line in the diagnosis and treatment of many childhood arrhythmias. The increased vigilance of these primary care providers, combined with advances in the recording technologies of arrhythmias, has resulted:in an increased number of infants, children, and adolescents who are being correctly diagnosed with rhythm disturbances. This article deals with the general principles of pediatric arrhythmia diagnosis and treatment, and individually discusses some of the more frequently encountered arrhythmias in children.

There is much interest in QT dispersion for noninvasive risk stratification of patients at risk of arrhythmias. However, little is known about the genesis of abnormal QT dispersion. In particular, whether eccentric ventricular depolarization, as seen in preexcitation, can lead to abnormal dispersion of repolarization is unknown. We studied 24 children aged 1–19 years (mean ± SD, 11 ± 5 years) with manifest preexcitation due to Wolff‐Parkinson‐White syndrome who had successful catheter ablation. Standard ECGs done preablation, early postablation (< 1 week), mid postablation (> 1 week, < 2 months), and late postablation (> 2 months) were reviewed. The QRS duration prior to ablation ranged from 90–160 ms (mean ± SD, 123 ±21 ms). On the preablation ECG, the JT and JTc dispersions showed no relationship to the QRS duration (r = 0.04 and 0.07, respectively). There was no change in JT dispersion when the preablation (42 ±15 ms) ECG was compared to early (43 ±15 ms), mid (44 ±13 ms), and late postablation (48 ± 19 ms) ECGs. There was no significant change in JTc dispersion as well. Thus, JT dispersion is unrelated to QRS duration and unaffected by catheter ablation in patients with Wolff‐Parkinson‐White syndrome. Eccentric ventricular depolarization does not lead to abnormal dispersion of repolarization.

The differential developmental effects of hypoxia on antegrade fast and slow and retrograde conduction through the atrioventricular junction are unknown. This study describes the effects of hypoxia on fast and slow antegrade atrioventricular node, infra-Hisian and retrograde conduction in immature and mature hearts during premature pacing protocols in excise, perfused adult and neonatal rabbits. The results are: (1) antegrade conduction delay through the atrioventricular node is the same developmentally, but delay through the His-Purkinje system is greater in adults; (2) hypoxia reduces the extra delay in the His-Purkinje system in adults; (3) fast atrioventricular node conduction is more sensitive to hypoxia in neonates than in adults, and slow atrioventricular node conduction is more sensitive to hypoxia in adults than in neonates, and (4) retrograde atrioventricular node conduction is more resistant to hypoxia in neonates than in adults.

. Catheter ablation in children requires placement of multiple large femoral venous sheaths and catheters. Magnetic resonance angiography (MRA) was used to evaluate the effect of indwelling lines on femoral venous blood flow. Between October 1993 and February 1994 a total of 17 patients scheduled for catheter ablation underwent venous MRA. Two-dimensional time-of-flight MRA was performed 12–70 hours after catheterization on all patients. All patients received intravenous heparin during the procedure and had aspirin therapy instituted after ablation. Eighteen catheter ablations and MRA studies were performed on the 17 patients (one patient underwent repeat ablation). There were 7 females and 10 males, with a mean age of 14.8 ± 4.2 years (range 8–21 years). Patients had three venous sheaths inserted in the left femoral vein (5F, 6F, and 7F with external diameters measuring 1.7, 2.0, and 2.3 mm, respectively) and one sheath in the right femoral vein (7F). Four patients (22%) had altered venous flow (two complete obstructions and two partial obstructions) following catheterization. None of these patients experienced symptoms or complications. It was concluded that there is an increased incidence (22%) of venous obstruction following catheter ablation, but there are no related complications. Venous MRA provides a rapid, noninvasive method for evaluating venous flow abnormalities and possibly detects patients at risk for complications.

The influence of flecainide (0.1, 0.5, 1.0, and 2.0 µg/mL)on atrioventricular (AV) conduction was studied in neonatal and adultperfused rabbit hearts using extracellular bipolar surface electrograms andpremature atrial and ventricular pacing. Flecainide produced a concentrationand rate-related increase in the steady-state nodal conduction(AHmin and an increase in slow AH conduction(AHmax) in both age groups. The drug produced significantincreases in the refractory periods of the atrium, AV node, His-Purkinjesystem, and ventricular myocardium. The neonatal refractory periods weresignificantly greater at lower or the same drug concentrations than those ofthe adult. The neonatal Wenckebach cycle length was significantly greaterwith a lower concentration of drug (0.5 µg/mL) than was the adultWenckebach cycle length. The His-Purkinje system steady-state conductiontime (HVmin was increased by a lower concentration of drug inthe neonate (0.5 µg/mL) as compared with 2.0 µg/mL inthe adult. These data show that across a wide range of AV conductionparameters, the neonatal preparations responded to a lower concentration offlecainide than did the adult preparations. These findings may, in part, bethe basis for the reported greater efficacy of the drug in children than inadults.

Background: The use of inhaled beta(2)-agonists for bronchodilation in the treatment of lower airway obstruction is accepted worldwide. These agents are used for symptomatic relief of lower airway obstruction and, as well, can be employed prophylactically in exercise-induced bronchospasm. Cardiac dysrhythmias, specifically the long QT syndrome, have been associated with cardiac events precipitated by sympathomimetics. There are reports of documented long QT syndrome in association with syncope in children; however, there are no reports of beta(2)-agonist-induced syncope in the absence of long QT syndrome.Objective: To determine predisposing cardiac factors resulting in syncope associated with inhaled beta(2)-agonist use.Method: Case report. The index case was evaluated for cardiac pathology through non-invasive techniques, cardiac catheterization, and electrophysiologic studies. Electrophysiologic studies included provocative challenge with parenteral adrenergic agents.Results: Non-invasive studies were unremarkable. There was no evidence of prolonged QT syndrome or support for vasopressor syncope. Electrophysiologic studies revealed reproducible polymorphic ventricular tachycardia. This predisposition required a ventricular stimulation program of higher intensity while on mexilitine.Conclusions: This case of syncope associated with inhaled, short-acting beta(2)-agonist resulted from a hyperexcitable conduction system rather than the presence of a long QT syndrome.

Prolonged QRS duration on the electrocardiogram has been found to predict adverse arrhythmic events in patients late after repair of tetralogy of Fallot. Whether QRS duration can also predict inducible ventricular tachycardia (VT) at electrophysiologic study is unknown. Between 1984 and 1995 we studied 135 survivors of tetralogy of Fallot surgery whose age at surgery was 34 days to 37 years (3.7 +/- 3.9, median 2.5) and age at electrophysiologic study was 1.4 to 43 years (9.7 +/- 8.2, median 6.7). QRS duration was 80 to 240 ms (137 +/- 29) and > or = 180 ms in 9 patients. Sustained VT was induced in 22 patients (monomorphic in 17). Induced sustained monomorphic VT was related to QRS duration, right ventricular dimension, H-V interval, and presence of symptoms. QRS duration was also related to induced sustained monomorphic VT by multivariate analysis. QRS duration > or = 180 ms was 35% sensitive and 97% specific for induced sustained monomorphic VT. QRS duration was related to induced sustained monomorphic VT even when only asymptomatic patients were analyzed. A QRS duration > or = 180 ms was 100% sensitive and 96% specific for detecting clinical VT. Prolonged QRS duration on the electrocardiogram is associated with induced sustained monomorphic VT on electrophysiologic study. The finding of prolonged QRS duration should suggest the need for further testing to determine the risk of adverse arrhythmic events in patients after repair of tetralogy of Fallot, even if they are asymptomatic.

OBJECTIVE To investigate the efficacy of radiofrequency catheter ablation (RFCA) as an alternative nonpharmacological therapy for tachycardia-induced cardiomyopathy. DESIGN A retrospective study of 8 pediatric patients (age range, 10 months to 21 years) who underwent RFCA for an incessant supraventricular tachycardia-induced cardiomyopathy. A patient's tachycardia was considered incessant if the tachycardia was present more than 75% of the time. The left ventricular shortening fraction, as measured by echocardiography, before and after ablation, was used as the index of cardiac function. Cardiomyopathy was defined as a left ventricular shortening fraction of 28% or less. RESULTS Following RFCA, 7 patients had total resolution of their tachycardia and were discharged from the hospital with no antiarrhythmic medications. The remaining patient's tachycardia was modified by the catheter ablation and was subsequently controlled with flecainide acetate. With follow-up ranging from 9 months to 3 years, all patients have normal cardiac function as documented by echocardiography. No significant morbidity resulted from the catheter ablations. CONCLUSIONS Tachycardia-induced cardiomyopathy is amenable to "curative" therapy with RFCA. Ventricular function returns to normal after the successful catheter ablation procedure.