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Biomedical subjects

M Hariharan

Publications and source records attributed to M Hariharan.

At least 19 recordsLinked to original sources
PubMed1998

Effects of the T-->R transition on the electrostatic properties of E. coli aspartate transcarbamylase.

Aspartate transcarbamylase is a large (310 kD), multisubunit protein that binds substrates cooperatively and undergoes a large change in quaternary structure when substrates bind. The forces that drive this transition are poorly understood. We evaluated the electrostatic component of these forces by using finite difference and multigrid methods to solve the nonlinear Poisson-Boltzmann equation for complexes of the enzyme with several substrates and substrate analogs. The results have been compared with calculations for the unliganded protein. While pK1/2 values of most ionizable residues fall within 3 pH units of values for model compounds, 31 have pK1/2 values that fall outside the range 0-17. Many of these residues are at the active site, where they interact with the highly charged substrate, in the 80s loop or 240s loop or interact with these loops. The pK1/2 values of eight ionizable residues related by the twofold molecular axes differ by more than 3 pH units, providing additional evidence for asymmetry within the crystal. As in the unliganded structure, a set of residues forms a network in which ionizable groups with Wij values greater than 2 kcal-m(-1) are separated by distances greater than 5 A. Some residues participate in this network in both the unliganded and N-phosphonacetyl-L-aspartate (PALA)-liganded structure, while others are found in only one structure. The network is more extensive in the PALA-liganded structure than in the unliganded structure, but consists of two separate networks in the two halves of the molecule.

Allosteric Regulation↗
PubMed1997

Ionic properties of membrane association by vitamin K-dependent proteins: the case for univalency.

Ionic properties of membrane interaction by prothrombin, protein Z, and other vitamin K-dependent proteins were studied to determine the relevance of a monovalent membrane contact mechanism between one phospholipid headgroup and a calcium-lined pore in the protein [McDonald, J. F., Shah, A. M., Schwalbe, R. A., Kisiel, W., Dahlback, B., and Nelsestuen, G. L. (1997) Biochemistry 36, 5120-5127]. For comparison, multivalent ionic interaction was illustrated by peptides of +3 to +5 net charge and by blood clotting factor V. As expected, the peptides were easily dissociated by salt and gave nominal charge-charge interactions (zazb values) of -13 to -17. Factor V showed much higher binding affinity despite nominal zazb values of about 9. Membrane-bound prothrombin and protein Z showed very low sensitivity to salt as long as calcium was at saturating levels (zazb values of approximately -1.3 to -1.4), appropriate for univalent ionic attraction. Prothrombin contains +3 charge groups (Lys-2, Lys-11, Arg-10) that are absent from the GLA domain (residues 1-35) of protein Z, while protein Z contains -4 charge groups (Gla-11, Asp-34, Asp-35) that are absent in prothrombin. Thus, similar zazb relationships indicated little role for these surface charges in direct membrane contact. Calcium-saturated protein Z bound to phosphatidylcholine (PC) in a manner which indicated the addition of one calcium ion, bringing the total calcium stoichiometry in the protein-membrane complex to at least 8. Protein Z bound to phosphatidic acid (PA) in a manner suggesting the need for a fully ionized phosphate headgroup, a property expected by ion pairing in an isolated environment. Electrostatic calculations showed that the proposed protein site for phosphate interaction was electropositive. The cluster of hydrophobic amino acids (Phe-5, Leu-6, and Val-9) on the surface of prothrombin was electronegative, suggesting a role in the electrostatic architecture of the GLA domain. Overall, membrane binding by vitamin K-dependent proteins appeared consistent with the formation of an ion pair in an isolated environment.

Amino Acid Sequence↗
PubMed1997

Consensus chemistry and beta-turn conformation of the active core of the insect kinin neuropeptide family.

BACKGROUND: Neuropeptides are examples of small, flexible molecules that bind to receptors and induce signal transduction, thereby eliciting biological activity. The multifunctional insect kinin neuropeptides retain full activity when reduced to only their carboxy-terminal pentapeptide (Phe1-X2-X3-Trp4-Gly5-NH2), thereby allowing extensive structure-function studies and conformational analysis. RESULTS: A combined experimental and theoretical analysis of the insect kinin carboxy-terminal pentapeptide was used to probe the role of each residue, define the bioactive conformation, and design a constrained bioactive analog. Coupling receptor-binding data with two biological activity assays allowed receptor binding and signal transduction to be differentiated. A preferred beta-turn conformation, found for residues 1-4 by molecular dynamics simulations, was tested by designing a conformationally restricted cyclic hexapeptide. This cyclic analog showed a preference for the beta-turn conformation, as shown by a conformational search and nuclear magnetic resonance spectroscopy, and it showed stronger receptor binding but decreased activity relative to highly active linear analogs. CONCLUSIONS: Each residue of the insect kinin carboxy-terminal pentapeptide has a distinct role in conformational preference, specific receptor interactions or signal transduction. The beta-turn preference of residues Phe1-X2-X3-Trp4 implicates this as the bioactive conformation. The amidated carboxyl terminus, required for activity in many neuropeptide families, may be generally important for signal transduction and its inclusion may therefore be essential for agonist design.

Animals↗
PubMed1997

The effect of paroxetine on thiothixene pharmacokinetics.

OBJECTIVE: In this study healthy volunteers received thiothixene with and without a 3-day pretreatment with paroxetine to determine if paroxetine decreased the clearance of thiothixene. METHOD: Ten healthy medication-free volunteers (4 women and 6 men, mean age 38 +/- 12 years) were randomized to receive a single 20 mg oral dose of thiothixene on two separate occasions. On one occasion thiothixene was given concurrently, and following 3 days of pre-treatment with oral paroxetine (20 mg/day). On the other occasion thiothixene was given without paroxetine pre-treatment. The two study days were separated by a minimum period of 2 weeks. On both study days, after the administration of thiothixene, 10 ml blood samples were collected over the next 72 h. RESULTS: None of the pharmacokinetic parameters of thiothixene were significantly altered by a 3-day treatment with paroxetine. DISCUSSION: It is likely that the CYP2D6 isoenzyme is not responsible for a high proportion of thiothixene clearance, but one cannot exclude the possibility that a longer paroxetine pretreatment might have caused some inhibition of thiothixene clearance.

Administration, Oral↗
PubMed1997

Cognitive, behavioral, and motor effects of the NMDA antagonist ketamine in Huntington's disease.

BACKGROUND: Excitotoxicity may contribute to neuronal degeneration in Huntington's disease (HD). N-methyl-D-aspartate (NMDA) receptor antagonists can prevent neuronal degeneration caused by excitotoxicity, but their effects in HD patients are not known. METHODS: We investigated the acute cognitive, behavioral, and motor effects of the NMDA-receptor antagonist ketamine in HD patients. Double-blind infusions of 0.10, 0.40, and 0.60 mg/kg/hr ketamine were given to 10 HD patients on one test day and compared with placebo infusions on a second, identical testing day. Linear mixed-effects models and randomization tests were used to identify whether, and at which dose, a significant change from baseline occurred in outcome variables. RESULTS: We demonstrated that ketamine is well tolerated at low and intermediate subanesthetic doses. Intermediate ketamine doses produced specific decline in memory and verbal fluency. Higher subanesthetic doses caused a significant increase in psychiatric symptoms and impairment of eye movements. CONCLUSIONS: These results describe the spectrum of clinical effects produced by increasing NMDA receptor blockade in HD patients. The clinical effects appearing with higher levels of NMDA receptor blockade can identify the range of doses used in clinical trials of NMDA receptor antagonists.

Adult↗
PubMed1997

Optimization of sustained-release tablet formulations: a four-component mixture experiment.

Sustained-release tablet formulations containing chlorpheniramine maleate were prepared using a mixture of two hydrophilic gums, gamma-carrageenan and cross-linked carboxymethylcellulose sodium, and two filler materials, alpha-lactose monohydrate and dibasic calcium phosphate in different ratios. The objective of the experiment was to find an optimum formulation with the most desirable properties. In order to achieve this, the matrices were evaluated for the time taken to release 80% drug (t80), the release exponent (n), and the crushing strength (h). Several formulations produced near zero-order release of the model drug over a time period of up to 10 hr. Response surface plots for the three dependent variables were generated over the experimental region. the effect of different levels of the components on the response variables was thus visualized. Upon superimposition of the plots it was possible to graphically find a region where the responses were in an optimum range of values. To pinpoint the best formulation, a simultaneous optimum point within this region was located by arbitrarily defining optimum values for each of the responses and using a multi-objective function that combined the three response variables. The observed response variables of the optimum formulation were in good agreement with the predicted values.

Carrageenan↗
PubMed1997

Controlled-release tablet matrices from carrageenans: compression and dissolution studies.

This study investigates the potential of two commercial carrageenans, Gelcarin GP-379 (iota-carrageenan) and Viscarin GP-209 (lambda-carrageenan) to be used for the preparation of controlled-release tablet matrices. Tablets were compressed on an instrumented Stokes single punch machine and compression characteristics of the carrageenans were analyzed. Heckel plots using out-of-die tablet densities were linear with calculated yield pressures of 81.3 MPa and 105.2 MPa for iota- and lambda-carrageenan, respectively. Drug release from tablet formulations that contained equal amounts of the two carrageenans had near zero-order release profiles. There was little or no effect of tablet compression pressure on the drug release profiles from 70 to 175 MPa. As drug loading was increased from 5 to 20%, the diffusional exponent decreased from 1.056 to 0.678. Thirty percent drug loading resulted in breakup of tablets during dissolution and departure from zero-order release. Multiple regression analysis was used to predict the time for 50% release as a function of the concentration of the two carrageenans and a third filler material, microcrystalline cellulose. Predicted values were in good agreement with observed values and R2 for the final cubic model was 0.9984.

Carrageenan↗
PubMed1996

Molecular structure, conformational analysis, and structure-activity studies of Dendrotoxin and its homologues using molecular mechanics and molecular dynamics techniques.

Three-dimensional structures of Dendrotoxin (DtX), Toxin-I (DpI), and Toxin-K (DpK) were determined using molecular mechanics and molecular dynamics techniques. The overall molecular conformation and protein folding of the three dendrotoxins are very similar to the published crystal structures of bovine pancreatic trypsin inhibitor (BPTI) and alpha-DtX. Major secondary structural regions of the dendrotoxins are stable without much fluctuation during the dynamics simulation; the regions corresponding to the turns and bends (rich in lysines and arginines) exhibit more fluctuations. The conformational angles and the C alpha...C alpha' distances of the three disulfides (in each of the dendrotoxins) are different from each other. Comparative model building studies, involving the dendrotoxins and the proteinases, reveal that the key interactions (observed in BPTI-trypsin complex) needed for anti-protease activity are absent due to structural differences between the dendrotoxins and BPTI at the anti-protease loop; this explains the inability of the dendrotoxins to inhibit proteinases. The model also suggests that the solvent-exposed beta-turn region, rich in lysines (residues 26-28), might bind directly to the extracellular anionic sites of the receptors (K+ channels) by ionic interactions. The strikingly homologous cysteine distribution (Cys-x-x-x-Cys) in DtX, DpI, and DpK, at the C-terminus, induces the occurrence of a characteristic conformational motif, consisting of an alpha-helix (in an amphiphilic environment) stabilized by two disulfides, one involving a cysteine at the beta-strand, and the other at the N-terminus. This amphiphilic secondary structural element seems to provide the rigid frame work needed for exposing the proposed active site region of the dendrotoxins to the anionic sites of the K+ channel receptors.

Amino Acid Sequence↗
PubMed1996

The role of melatonin in the pathogenesis of adolescent idiopathic scoliosis.

STUDY DESIGN: A matched, case-control study comparing melatonin production in female patients with and without adolescent idiopathic scoliosis. OBJECTIVES: To determine whether melatonin production is decreased in adolescent idiopathic scoliosis. SUMMARY OF BACKGROUND DATA: A central etiology for idiopathic scoliosis has never been established. Previous authors have produced experimental scoliosis in chickens after pinealectomy, preventable by administration of melatonin. They suggested that a defect in melatonin synthesis might be involved in the pathogenesis of human idiopathic scoliosis. METHODS: Nine female adolescents with no medical problems, normal neurologic examinations, radiographic idiopathic scoliosis of 15-40 degrees, and Risser Stage I-III were in the patient group. Eighteen healthy adolescent girls with no medical problems, a negative school screening, and no family history of scoliosis were control subjects. Patients and control subjects were matched for age, weight, Tanner stage, sleep duration, and light exposure by multiple linear regression. Nighttime and daytime urine samples were analyzed for melatonin by high-performance liquid chromatography. RESULTS: Although nighttime melatonin levels were significantly higher than daytime levels in all volunteers (P < 0.00002), there were no significant differences in nighttime (P > 0.63) or daytime (P > 0.78) melatonin levels between patients and control subjects, even after matching by multiple linear regression analysis. A statistical analysis demonstrated that if a melatonin deficiency of 25% or more did exist in patients with scoliosis compared with control subjects, the likelihood that it would have been detected in this study was more than 98%. CONCLUSION: Although melatonin deficiency may cause scoliosis in the chicken, this study suggests that it is not a mechanism in the pathogenesis of adolescent idiopathic scoliosis in humans.

Adolescent↗
PubMed1996

Urinary catecholamine excretion and behavioral differences in ADHD and normal boys.

Urinary catecholamine excretion was assessed in 15 boys with attention-deficit/hyperactivity disorder (ADHD) and 16 normal controls during a defined physical and mental task. Dihydroxyphenylalanine, dopamine, norepinephrine (NE), epinephrine (EPI), 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxyphenylglycol (DOPEG) concentrations were assayed by high-pressure liquid chromatography with electrochemical detection. The urinary concentration of DOPEG, an NE metabolite that has not been previously measured in ADHD, was significantly lower in the ADHD subjects than in the normal controls. There was also a trend for lower urinary EPI levels in the hyperactive boys. Stepwise multiple regression analyses demonstrated that DOPEG and EPI each contributed significantly to the variance in the behavioral symptoms within the full sample. The results are consistent with previous reports of abnormal metabolism of norepinephrine and epinephrine in ADHD. These neurochemical findings may be due to differences between ADHD and normal boys in neuronal (central or peripheral) or nonneuronal (e.g., adrenal, renal) activity. The results are also consistent with prior findings in normal children of an inverse relationship between EPI excretion and inattentive, restless behaviors. Together, these findings suggest caution in ascribing metabolite changes to ADHD or to ADHD-like behaviors that may be seen in normal children.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1994

Identification of a novel integrin binding site in fibronectin. Differential utilization by beta 3 integrins.

Fibronectin (Fn) binding to the integrins alpha IIb beta 3 and alpha v beta 3 involves the Arg-Gly-Asp sequence. The identification of other regions of Fn that interact with alpha IIb beta 3 suggests a potential mechanism for differential ligand recognition by integrins. We report here the identification of an 11-residue peptide sequence from the 9th type III repeat of Fn (3Fn9), which inhibits ligand binding to alpha IIb beta 3 by interacting directly with this receptor. Mutational analysis demonstrated that this same region was involved in the formation of epitopes for two anti-Fn mAbs that inhibit Fn binding to alpha IIb beta 3, thus emphasizing the role of this site in the macromolecule. Molecular modeling of the 3Fn9-10 modules suggested that Fn residues Asp1373-Thr1383 are at least 25 A distant from the Arg-Gly-Asp site and therefore does not directly interact with it. The 3Fn9 site was differentially recognized by the beta 3 integrin family. The Asp1373-Thr1383 peptide failed to inhibit ligand binding to alpha v beta 3, a recombinant Fn Ala1235-Ser1436 fragment was not recognized by alpha v beta 3, and addition of the 3Fn9 module to the amino terminus of the 3Fn10 did not affect the potency of inhibition of Fn binding to alpha v beta 3. Thus, a novel integrin recognition site in the 3Fn9 module of Fn that is differentially recognized by the beta 3 integrins has been localized within the residues Asp1373-Thr1383.

Amino Acid Sequence↗
PubMed1993

Systematic approach to the development of plasma amino acid analysis by high-performance liquid chromatography with ultraviolet detection with precolumn derivatization using phenyl isothiocyanate.

A reversed-phase liquid chromatographic method for the separation of 26 phenylthiocarbamyl derivatives of amino acids in human plasma in ca. 35 min. is described. The method used a C18 column (150 x 4.6 mm I.D., 3 micron) thermostatted at 41 degrees C, and a simple multistep linear gradient of two solvents. Solvent A was 0.05 M sodium acetate (pH 5.1)-acetonitrile (98:2, v/v), and solvent B was water-acetonitrile (40:60, v/v). A simple and successful approach to the optimization of the conditions for the separation of the 26 amino acid derivatives was realized. In the initial phase of development, the composition of the gradient, its timings, the column temperature, the flow-rate and the mobile phase compositions were optimized. At the end the influence of pH was studied, and this approach led to a clear resolution of the 26 amino acids. The method was validated by accuracy, precision, and recovery studies, by analyzing patient samples, and by comparing the quality control sample results with the classical ion-exchange method.

Amino Acids↗
PubMed1993

Noradrenergic response to intravenous yohimbine in patients with depression and comorbidity of depression and panic.

Adrenergic response following infusions of yohimbine or normal saline was evaluated in 9 control subjects, 8 patients suffering from a major depressive episode (MDE), and 12 patients suffering from concurrent MDE and panic disorder (MDE + P). Blood was drawn at -20, 0, 5, 10, 20, 45, and 90 min following the infusions, and assayed for norepinephrine (NE) and 3-methoxy-4-hydroxy-phenyl glycol (MHPG). Although the patient groups exhibited higher baseline NE concentrations, and a greater NE area under the plasma concentration versus time curve (AUC0-90) during the yohimbine infusion, the differences were not statistically significant. Baseline NE was significantly correlated with the NE AUC0-90 in all three groups, suggesting that, although the NE system may be dysregulated in the MDE and MDE + P patients, the NE system still appears to respond somewhat predictably following a challenge, even though the actual magnitude of response may vary.

Adult↗
PubMed1993

Assay of human plasma cortisone by liquid chromatography: normal plasma concentrations (between 8 and 10 a.m.) of cortisone and corticosterone.

A high-performance liquid chromatographic method using ultraviolet detection to quantitate human plasma concentrations of cortisone simultaneously with cortisol and corticosterone is described. The method is based on the use of an octadecyl silica column (100 mm x 2 mm I.D., 3 microns), an ultraviolet absorbance detector (242 nm) with a 10 mm path length flow-cell, and a mobile phase composed of water-tetrahydrofuran-acetonitrile (82:10:8, v/v) containing 5 ml/l triethylamine and citric acid to adjust the pH of the buffer to 6.5. Flumethasone is used as the internal standard. The detection limit of the method for the three steroids is 300 ng/l using a 1-ml sample. The average inter-assay coefficient of variation for cortisone is 3.3% and the average recovery is 100.8%. Possible interferences from common drugs and endogenous and exogenous steroids in the method have been studied. Plasma concentrations (drawn from 8 to 10 a.m.) of cortisone and corticosterone for 43 normal volunteers have been determined.

Chromatography, High Pressure Liquid↗
PubMed1993

Phenelzine produces subsensitivity to nicotine.

1. The authors attempted to detect a possible effect of treatment with phenelzine on a physiological response to nicotine in the rat. 2. Positive findings in an animal model suggest the feasibility of more complicated experiments in animals and the possibility of studies involving human subjects. 3. Treatment of Sprague Dawley rats (n = 10) with phenelzine sulfate (15.0 mg/kg ip) every 48 hours for 14 days was associated with a 73.3% decrease in the hypothermic response to nicotine. 4. Treatment with phenelzine did not enhance the rate of elimination of nicotine. 5. The authors discuss a possible relationship between changes in nicotinic mechanisms and the therapeutic actions of drugs used to treat affective illness.

Animals↗
PubMed1993

Differences between smokers and never-smokers in sensitivity to nicotine: a preliminary report.

Sensitivity to nicotine was explored using test doses administered via intra-nasal aerosol in 10 smokers and 10 never-smokers. Smokers received 1.50 mg nicotine (in 2 sprays, < 5 seconds apart, one spray per nostril); never-smokers received either 0.50 mg (n = 3) or 0.25 mg (n = 7) nicotine. Accumulation of nicotine in plasma, per unit dose administered, was nearly four times greater in never-smokers than in smokers, indicating differences in pharmacokinetic tolerance. To examine sensitivity to nicotine without this confound, peak physiological reactivity (heart rate and blood pressure changes) was divided by peak plasma nicotine increment and the ratio was expressed as a function of cotinine level prior to dosing, thereby relating sensitivity to nicotine to history of exposure. In smokers, functional sensitivity to nicotine was inversely related to customary nicotine intake, replicating previous findings for light and heavy smokers. The observation that never-smokers were not much more sensitive to nicotine than light smokers is notable given the disparity in previous history of exposure.

Administration, Intranasal↗