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Biomedical subjects

C Narasimhan

Publications and source records attributed to C Narasimhan.

At least 19 recordsLinked to original sources
PubMed2004

Clinical efficacy of sildenafil in primary pulmonary hypertension: a randomized, placebo-controlled, double-blind, crossover study.

OBJECTIVES: In a randomized, double-blind, crossover design, we compared the efficacy of sildenafil with placebo in patients with primary pulmonary hypertension (PPH). The primary end point was the change in exercise time on treadmill using the Naughton protocol. Secondary end points were change in cardiac index and pulmonary artery systolic pressure as assessed by Doppler echocardiography and quality of life (QOL) as assessed by a questionnaire. BACKGROUND: Primary pulmonary hypertension is a disorder with limited treatment options. Uncontrolled studies had shown sildenafil to be beneficial in the treatment of PPH. METHODS: After initial clinical evaluation, including Doppler echocardiography and treadmill exercise test, patients were randomized to placebo or sildenafil with dosages ranging from 25 to 100 mg thrice daily on the basis of body weight. The evaluation was repeated after six weeks. Then patients were crossed over to alternate therapy. Final evaluation was performed after another six weeks of treatment. RESULTS: Twenty-two patients completed the study. Exercise time increased by 44% from 475 +/- 168 s at the end of placebo phase to 686 +/- 224 s at the end of sildenafil phase (p < 0.0001). With sildenafil, cardiac index improved from 2.80 +/- 0.9 l/m2 to 3.45 +/- 1.1 l/m(2) (p < 0.0001), whereas pulmonary artery systolic pressure decreased insignificantly from 105.23 +/- 17.82 mm Hg to 98.50 +/- 24.38 mm Hg. There was significant improvement in the dyspnea and fatigue components of the QOL questionnaire. During the placebo phase, one patient died and another had syncope. There were no serious side effects with sildenafil. CONCLUSIONS: Sildenafil significantly improves exercise tolerance, cardiac index, and QOL in patients with PPH.

Adolescent↗
PubMed2004

A high resolution computer model for sound propagation in the human thorax based on the Visible Human data set.

A parallel supercomputer model based on realistic tissue data is developed for sound propagation in the human thorax and the sound propagation behavior is analyzed under various conditions using artificial sound sources. The model uses the Visible Human male data set for a realistic representation of the human thorax. The results were analyzed in time and frequency domains. The analysis suggests that lower frequencies of around 100 Hz are more effectively transmitted through the thorax and that the spatial confinement of sound waves within the thorax results in a resonance effect at around 1500 Hz. The results confirm previous studies that show the size of the thorax plays a significant role in the type of sound generated at the chest wall.

Acoustics↗
PubMed2003

Common supraventricular arrhythmias in Indian population and their management.

Supraventricular tachycardias (SVTs) present as recurrent palpitations. Unlike ventricular tachycardias, these arrhythmias usually carry a benign prognosis. It is important to exclude structural heart disease in these patients. Though vagal manoeuvres and drug therapy are useful for acute termination of SVTs, recurrences are common. Catheter ablation is safe, highly effective and has a curative potential. It obviates the need for long-term medical therapy as it eliminates the arrhythmia in these patients.

Adrenergic beta-Antagonists↗
PubMed2000

Changes in QT dispersion during adenosine infusion.

BACKGROUND: QT dispersion (QTd) measurement during treadmill stress testing has been to shown to improve the accuracy of exercise electrocardiogram (ECG) in the detection of significant coronary artery disease (CAD). HYPOTHESIS: The aim of this study was to determine whether adenosine-induced changes in QTd could predict significant CAD and to assess its efficacy as a diagnostic index in patients undergoing adenosine stress test. METHODS: QT interval measurements were made in 57 consecutive patients undergoing adenosine sestamibi stress test. Patients with an abnormal stress test underwent coronary angiography. Patients with significant disease by coronary angiography (> 70% stenosis) were classified as having CAD (Group 1), and those with normal stress images and/or normal coronaries by angiography were classified as having no CAD (Group 2). RESULTS: QT dispersion increased from 28.2 +/- 4.5 to 43.8 +/- 4.5 ms with a delta QTd of 15.53 +/- 3.68 in Group 1 (p = 0.001) and from 28.4 +/- 2.6 to 34.8 +/- 2.8 ms with a delta QTd of 6.58 +/- 2.21 ms in Group 2 (p = 0.006). Patients in Group 1 had a significantly higher increase in QTd (delta QTd) than the patients in Group 2 (p < 0.03). Addition of delta QTd (> 10 ms) to the ST depression during adenosine infusion would increase the sensitivity of the ECG from 23 to 65% and decrease the specificity from 91 to 70% for diagnosis of significant CAD. CONCLUSIONS: delta QTd is significantly more prolonged in patients with CAD during adenosine infusion. It increases the sensitivity of the stress ECG in diagnosis of CAD during adenosine infusion when used as an adjuvant index.

Adenosine↗
PubMed1999

Reversing the initial phase polarity in biphasic shocks: is the polarity benefit reproducible?

The effect of initial phase polarity (IPP) reversal using biphasic shocks on DFT at the time of implantation of implantable cardioverter defibrillator and the reproducibility of this effect during predischarge testing was evaluated in a randomized fashion. Twenty-two patients with ventricular tachycardia or ventricular fibrillation (VF) who received either the Medtronic 7219D (7 patients), 7219C (12 patients), 7223 (1 patient), or CPI Ventak MINI (2 patients) were studied. The DFT was determined in a randomized fashion at implantation and during predischarge testing using a binary search protocol. Initial shock was delivered at 12 J. If successful, subsequent shock was delivered at 6 J, following which the shock was incremented or decremented by 3 J depending upon the success. The DFT for right ventricular (RV)- and RV + IPP was 10.9 +/- 4.1 J and 11.1 +/- 4.0 J, respectively, at implant (P = ns) and 9.7 +/- 4.3 J and 8.4 +/- 6 J, respectively, (P = ns) at predischarge testing. Of the six patients who had better DFT with RV + at implantation, only one patient maintained the benefit during predischarge testing. The differences observed in IPP in individual patients may not be demonstrable during repeated testing. These findings may have implications on how these devices should be programmed.

Adult↗
PubMed1998

Atrioventricular nodal modification and atrioventricular junctional ablation for control of ventricular rate in atrial fibrillation.

Atrial fibrillation results in several structural and functional changes in the heart that lead to worsening ventricular function. Although restoration of sinus rhythm is the ideal goal, it is not always feasible. Pharmacologic therapy is associated with adverse effects and is not always effective. We have reviewed the current status of nonpharmacologic therapy in the management of rapid ventricular response due to atrial fibrillation. Electrophysiologic studies have confirmed that the posterior inputs to the AV node have a shorter refractory period and are mainly responsible for maintaining rapid ventricular response in atrial fibrillation. AV nodal modification involves ablation of these posterior inputs in a sequential fashion until a significant reduction of ventricular response is achieved. This procedure has been reported to be successful in maintaining the controlled ventricular response in about 70% of the patients over long-term follow-up. Ablation of the AV node with implantation of a permanent pacemaker is a more definitive procedure and simpler to perform. Reduction in ventricular response achieved with this procedure results in improvement of the patient's clinical symptoms as well as the underlying left ventricular function. Nonpharmacologic therapy for control of ventricular rate should be considered for patients with atrial fibrillation, in whom pharmacologic therapy for rate control is ineffective or poorly tolerated.

Atrial Fibrillation↗
PubMed1997

Ventricular tachycardia in valvular heart disease: facilitation of sustained bundle-branch reentry by valve surgery.

BACKGROUND: The clinical characteristics of sustained monomorphic ventricular tachycardia (SMVT), when it develops after valve surgery, have not been described. METHODS AND RESULTS: Between 1985 and 1996, 31 patients (30 men and 1 woman) who had undergone valve surgery were found to have inducible SMVT. Nine patients (29%) had sustained VT due to bundle-branch reentry (BBR) (group 1). Four of these patients had normal left ventricular function, and VT with a right bundle-branch morphology was inducible in 4 patients. Group 2 included 20 patients with inducible myocardial (ie, non-BBR) VT. Coronary artery disease was present in 15 group 2 patients (75%) due to atherosclerotic (n=12) and nonatherosclerotic (n=3) causes. Two patients had both inducible sustained BBR and myocardial VT (group 3). Sustained BBR VT occurred significantly earlier after valve surgery (median, 10 days) than the onset of postoperative myocardial VT (median, 72 months; P<.005). CONCLUSIONS: Myocardial VT was the most common type of inducible SMVT in patients with valvular heart disease. The majority of these patients had underlying coronary artery disease and significant left ventricular dysfunction. However, in almost one third of the patients, sustained BBR VT was the only type of inducible SMVT. This type of VT was facilitated by the valve procedure occurring within 4 weeks after surgery in most patients. In these patients, left ventricular function was relatively well preserved, and the right bundle-branch block type of BBR was frequently induced. Because a curative therapy can be offered to these patients (ie, bundle-branch ablation), BBR should be seriously considered as the mechanism of VT in patients with valvular heart disease, particularly if the arrhythmia occurs soon after valve surgery.

Adult↗
PubMed1997

Comparison of outcome of implantable cardioverter defibrillator implantation in patients with severe versus moderately severe left ventricular dysfunction secondary to atherosclerotic coronary artery disease.

This study was undertaken to assess the feasibility and clinical outcome of implantable cardioverter-defibrillators (ICDs) among patients with coronary artery disease and left ventricular ejection fraction (LVEF) of <20%. The morbidity, mortality, and the long-term survival of 117 patients with LVEF of <20% (group 1) were compared with 321 patients with LVEF of 20% to 40% (group 2). Mortality in the first 30 days after ICD implantation was 0% for group 1 and 0.6% in group 2. Actuarial survival (all cause) at the end of 2, 4, and 5 years were 83%, 70%, and 62%, respectively, in group 1 and 90%, 80%, and 71% in group 2 (p = 0.05). Fifty-five patients (47%) in group 1 and 126 patients (39%) in group 2 received appropriate shocks during follow up. Among the patients in group 1, the overall survival at 2 years after an appropriate shock from an ICD was 92% for patients <60 years of age, 77% for patients ages 60 to 69, and 53% for patients >70 years old. Although the overall survival of patients in group 1 was slightly lower compared with those in group 2, in a multivariate analysis, the EF was not an independent predictor of poor survival. The ICD can be implanted with acceptable operative morbidity and mortality in selected patients with LVEF of <20%.

Aged↗
PubMed1997

Identification and functional characterization of an active-site lysine in mevalonate kinase.

We report the construction of an expression plasmid for rat mevalonate kinase and the overexpression of recombinant enzyme in Escherichia coli. The homogeneous enzyme had a specific activity of 30 units/mg and an observed subunit molecular mass of 42 kDa. The Michaelis constants (Km) for DL-potassium mevalonate (288 microM) and for ATP (1.24 mM) were in agreement with values reported for enzymes isolated from rat liver (Tanaka, R. D., Schafer, B. L., Lee, L. Y., Freudenberger, J. S., and Mosley, S. T. (1990) J. Biol. Chem. 265, 2391-2398). Recombinant rat mevalonate kinase was inactivated by the lysine-specific reagent, pyridoxal phosphate (PLP). ATP (5 mM) afforded protection against inactivation, suggesting reaction of PLP with an active-site lysine. Mapping, isolation, and Edman degradation of the ATP-protectable peptide from [3H]PLP-inactivated borohydride-reduced mevalonate kinase allow assignment of lysine 13, a residue invariant in known mevalonate kinase sequences, as the modification site. These results represent the first identification of an active-site residue in mevalonate kinase. The function of lysine 13 was evaluated by replacing this residue with methionine. Vm of the mutant protein is diminished by 56-fold, suggesting that lysine 13 facilitates catalysis. Kd values of wild-type and mutant proteins for ATP were determined in electron spin resonance competition experiments. The observed 56-fold diminution in affinity for the mutant enzyme supports an additional role for lysine 13 in stabilization of ATP binding.

Adenosine Triphosphate↗
PubMed1997

Inactivation of 3-hydroxy-3-methylglutaryl-CoA synthase and other Acyl-CoA-utilizing enzymes by 3-Oxobutylsulfoxyl-CoA.

3-Oxobutylsulfoxyl-CoA has been produced by oxidation of S-3-oxobutyl-CoA, the thioether analog of acetoacetyl-CoA. Avian hydroxymethylglutaryl-CoA (HMG-CoA) synthase is inactivated by oxobutylsulfoxyl-CoA in a time-dependent fashion. Protection against inactivation is afforded by the substrate, acetyl-CoA, suggesting that inactivation involves modification of the enzyme's active site. Pretreatment of HMG-CoA synthase with the inactivator blocks the enzyme's ability to form Michaelis and acetyl-S-enzyme intermediates, supporting the hypothesis that modification is active-site directed. Incubation of enzyme with oxobutylsulfoxyl-[32P]CoA followed by precipitation with trichloroacetic acid indicates that inactivation correlates with stoichiometric formation of a covalent adduct between enzyme and a portion of the inactivator that includes the CoA nucleotide. The observation of reagent partitioning suggests that HMG-CoA synthase catalyzes conversion of oxobutylsulfoxyl-CoA into a reactive species that modifies the protein. Treatment of inactivated enzyme with DTT or other mercaptans restores enzyme activity and reverses the covalent modification with release of CoASH. Oxobutylsulfoxyl-CoA inactivates beta-ketothiolase and HMG-CoA lyase in a process that is also reversed by DTT. These three enzymes all contain active site cysteines, suggesting that inactivation results from disulfide formation between a cysteine and the CoA moiety of the inhibitor. The data are consistent with the hypothesis that enzymatic cleavage of oxobutylsulfoxyl-CoA results in the transient formation of a sulfenic acid derivative of CoA which subsequently reacts to form a stable disulfide linkage to protein.

Acyl Coenzyme A↗
PubMed1996

Rhodobacter sphaeroides phosphoribulokinase: binary and ternary complexes with nucleotide substrate analogs and effectors.

Rhodobacter sphaeroides phosphoribulokinase (PRK) binds ATP substrate, as well as spectroscopically active ATP analogs (trinitrophenyl-ATP and ATP gamma S-acetamidoproxyl), to form stable binary complexes. Stoichiometric binding of these nucleotide triphosphates in PRK's substrate site is observed not only with wild-type enzyme but also with D42A and D169A mutants. The demonstration that these mutants contain a full complement of functional substrate binding sites indicates their substantial structural integrity and underscores the significance of their markedly diminished catalytic activity [Charlier et al. (1994) Biochemistry 33, 9343-9350]. Similarly, PRK forms a stable binary complex with the allosteric activator NADH. The negative allosteric effector AMP displaces activator NADH but not substrate from their respective binary complexes with enzyme. When trinitrophenyl-ATP, a fluorescent nucleotide triphosphate that functions as an alternative PRK substrate, forms a binary complex with enzyme, its fluorescence emission is enhanced and lambda max shifted from approximately 557 to 545 nm. Upon formation of a binary PRK-NADH complex, the fluorescence emission of the dinucleotide effector is also enhanced and the lambda max shifted from approximately 460 to 440 nm. PRK forms stable ternary complexes containing NADH and either ATP or trinitrophenyl-ATP. Due to energy transfer, NADH fluorescence in the ternary complex with trinitrophenyl-ATP is markedly quenched, allowing an estimation of the spatial separation between this novel donor/acceptor pair.

Adenosine Triphosphate↗
PubMed1995

Utility of a novel spin-labeled nucleotide in investigation of the substrate and effector sites of phosphoribulokinase.

The activated spin-label 3-(2-bromoacetamido)proxyl modifies the sulfur atom of phosphorothioate-containing AMP, ADP, and ATP analogs in a facile reaction that produces a new series of spin-labeled nucleotides. One of these products, adenosine 5'-O-(S-acetamidoproxyl 3-thiotriphosphate) (ATP gamma SAP), has been evaluated as a structural probe for Rhodobacter sphaeroides phosphoribulokinase (PRK). When incubated with affinity-purified enzyme that contains tightly bound substrate ATP, ATP gamma SAP binds noncooperatively to the allosteric site (n = 1; KD = 8 microM). Probe bound in this site is displaced (K1/2 = 100 microM) by the allosteric effector, NADH, at concentrations comparable to those required for enzyme activation (Ka = 133 microM). In the presence of NADH, when PRK's substrate site is vacant, ATP gamma SAP binds in a cooperative mode (Hill coefficient approximately 2.9; KD = 20 microM). In the absence of NADH, ATP gamma SAP mimics ATP by exhibiting nonequilibrium binding to PRK. The observations with phosphoribulokinase, together with the straightforward nature of the methodology documented for synthesis and isolation of this class of spin-labeled nucleotides, suggest that these analogs have potentially wide application as structural probes.

Adenosine Triphosphate↗
PubMed1995

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: testing the function of the active site cysteine by site-directed mutagenesis.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase is affinity labeled by 2-butynoyl-CoA; peptide sequence analysis demonstrates C237 to be the site of modification [Hruz et al. (1992) Biochemistry 31, 6842-6847]. In order to evaluate whether C237 functions in the chemistry of hydroxymethylglutaryl-CoA cleavage, cassette mutagenesis has been employed to alter wild-type DNA to encode serine or alanine at residue 237. ESR measurements indicate that the purified mutant enzymes bind stoichiometric amounts of the spin-labeled substrate analog, R.CoA, which has been established as a competitive inhibitor. Binding affinities measured with C237S (Kd = 92 microM) and C237A (Kd = 97 microM) lyases are comparable to that observed with wild-type lyase. The rotational dynamics of R.CoA bound to mutant enzymes are also very similar to those for R.CoA bound to wild-type lyase. These observations suggest that the mutant enzymes are structurally intact. In view of this demonstrated structural integrity, it is significant that the VmaxS of C237A and C237S are approximately 4 x 10(4)- and approximately 725-fold lower, respectively, than the value measured for wild-type hydroxymethylglutaryl-CoA lyase. The C237S enzyme exhibits a Km = 53 microM for substrate; this value is only 2-fold higher than the Km of the wild-type enzyme. Additionally, we report that the residual activity in C237S hydroxymethylglutaryl-CoA lyase is unaffected by 2-butynoyl-CoA under conditions which support inactivation of wild-type enzyme. These results are consistent with an active site assignment to C237, confirming the prediction based on the affinity labeling/peptide mapping data.(ABSTRACT TRUNCATED AT 250 WORDS)

Acyl Coenzyme A↗
PubMed1995

Evaluation of cysteine 266 of human 3-hydroxy-3-methylglutaryl-CoA lyase as a catalytic residue.

The role of cysteine 266 in human 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase, a residue that is homologous to a cysteine mapped to the active site of prokaryotic HMG-CoA lyase by protein chemistry approaches, has been investigated by site-directed mutagenesis. Both the wild-type human enzyme and a C323S variant, in which a regulatory sulfhydryl has been eliminated without any negative effect on catalytic activity (Roberts, J. R., Narasimhan, C., Hruz, P. W., Mitchell, G. A., and Miziorko, H. M. (1994) J. Biol. Chem. 269, 17841-17846), were used as models. Mutant enzymes C266A, C266A/C323S, C266S, and C266S/C323S were overexpressed in Escherichia coli and purified to homogeneity. In all cases, kinetic characterization indicated that the Km value for HMG-CoA was not substantially different from the value measured using wild-type human lyase, suggesting that no serious structural perturbation occurs upon replacing Cys-266. A dissociable divalent cation (Mn2+ or Mg2+), which is required for activity in both native and C323S enzymes, is also an essential component for activity in each of the Cys-266 mutants. The structural integrity of the human mutants was further indicated by Mn2+ binding studies, which demonstrate similarities not only in the activator cation binding stoichiometries, but also in the KD values for Mn2+ as determined for wild-type and mutant C266A or C266S proteins. Purified C266A and C266A/C323S mutants both displayed approximately 1.3 x 10(4)-fold diminution in specific activity, while the kcat value was diminished in both C266S and C266S/C323S by approximately 9.9 x 10(2)-fold. This large diminution in catalytic efficiency in enzyme variants that display no substantial structural perturbations is in accord with an active-site assignment to Cys-266 and qualifies its sulfhydryl group for consideration as a component of the catalytic apparatus.

Amino Acid Sequence↗
PubMed1994

P. mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: electron paramagnetic resonance investigation of the copper binding site.

The copper binding site in Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase has been investigated by multifrequency electron spin resonance spectroscopy. Methodology has been developed to introduce copper in vitro into the isolated apoenzyme. The X-band EPR of Cu2+ (mixed isotopes or 63Cu2+) introduced in this way is very similar to the EPR spectra of samples in which copper is introduced during protein expression. g parallel and A parallel values in the X-band spectra support the prediction of nitrogen ligands to the tightly bound copper. In the g parallel region of S-band EPR spectra (mI = -1/2) of lyase-bound 63Cu2+, superhyperfine interactions due to nitrogen ligands are observed. Computer simulation with appropriate g values and A values and strain parameters was used to satisfactorily model both the X-band and the S-band spectra. The g parallel value of 2.282 and the A parallel value of 470 MHz are consistent with two nitrogen and two oxygen donor atoms for a square planar type 2 copper center. By simulating the mI = -1/2 line of the S-band spectrum, the superhyperfine features could be well modeled. This simulation approach was also used to distinguish between two and three nitrogen donor atoms. Based on the intensity patterns of the superhyperfine lines and the estimated coupling constants, it is concluded that at least two (and probably only two) nitrogen donor atoms are liganded to the tightly bound copper in HMG-CoA lyase. Additionally, kinetic experiments demonstrate that a spin-labeled substrate analog (R.CoA) is a competitive inhibitor of HMG-CoA lyase (KI = 98 microM). ESR titration experiments indicate that R.CoA binds to lyase with an equilibrium dissociation constant of 103 microM. Bound spin label exhibits a rotational correlation time, tau c, of 20 ns, in agreement with the value predicted for immobilization on a protein composed of two 32-kDa subunits.

Acyl Coenzyme A↗
PubMed1994

3-Hydroxy-3-methylglutaryl-CoA lyase: expression and isolation of the recombinant human enzyme and investigation of a mechanism for regulation of enzyme activity.

cDNA encoding the mature form of human hydroxy-methylglutaryl-CoA (HMG-CoA) lyase, a mitochondrial matrix protein, has been used to prepare expression plasmids appropriate for production of this protein in Escherichia coli. Using a T7 RNA polymerase-based pET system, HMG-CoA lyase was overexpressed but largely recovered in an insoluble, catalytically inactive form. In contrast, an expression plasmid (pTrcHL-1), derived from pTrc99a, supported production of soluble, active enzyme. A synthetic oligonucleotide cassette was employed to produce an enzyme variant in which cysteine was replaced by serine at position 323. Both wild-type and C323S HMG-CoA lyases were isolated in homogeneous form and characterized. The function of Cys-323 in influencing catalytic activity in vitro has been investigated by comparing the response of wild-type and C323S lyases to oxidation and reduction. Additionally, the consequences of treatment of these enzymes with the sulfhydryl-directed bifunctional reagent, o-phenylenedimaleimide have been determined. The results support the hypothesis that a thiol/disulfide exchange mechanism affects enzyme activity in vitro and indicate that Cys-323 residues on adjacent subunits of the homodimeric native enzyme are suitably positioned to form an intersubunit cross-link upon oxidative inactivation and disulfide formation.

Amino Acid Sequence↗