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Biomedical subjects

R Bhat

Publications and source records attributed to R Bhat.

At least 55 records · Page 3Linked to original sources
PubMed1993

Cu(II)-dependent degradation of DNA by riboflavin.

Riboflavin, in the presence of Cu(II), caused breakage of calf thymus DNA and supercoiled pBR322 plasmid DNA. The rate of such DNA degradation was several times greater than that caused by riboflavin alone. The reaction was inhibited under anaerobic conditions, but oxygen could be replaced by the addition of H2O2. Riboflavin reduced Cu(II) to Cu(I), which was shown by using the Cu(I)-sequestering reagent neocuproine, to be an essential intermediate in the DNA degradation reaction. Results obtained with scavengers of active oxygen species and their production by riboflavin and Cu(II) indicated that the species predominantly responsible for DNA breakage is singlet oxygen.

Animals↗
PubMed1993

Value of amnioinfusion in reducing meconium aspiration syndrome.

Infusing normal saline into the uterine cavity, or amnioinfusion, is used to reduce the risk of meconium aspiration syndrome in babies born to women in whom the pregnancy is complicated by thick, meconium-stained amniotic fluid. In this retrospective review of 436 such pregnancies amnioinfusion was performed in 110. In 18.2% of infants in the amnioinfusion group meconium was found in the trachea compared with 29.1% of infants in the untreated group. In the treated group respiratory distress occurred in 2.7%, and meconium aspiration syndrome in 1.8%, whereas in the untreated group these frequencies were 10.1 and 5.5%, respectively. We conclude that saline amnioinfusion in pregnancies complicated by thick, meconium-stained amniotic fluid reduces the risk of respiratory distress in the newborn.

Adult↗
PubMed1993

Identification of residues important for ligand binding to the human 5-hydroxytryptamine1A serotonin receptor.

The functional significance of the conserved amino acids within transmembrane regions II and VII of the human 5-hydroxytryptamine (5-HT)1A receptor was analyzed by oligonucleotide-directed mutagenesis followed by transient expression of the mutated receptor genes in COS-1 cells. The substitution of a conserved asparagine at position 396 (transmembrane region VII) with either alanine, phenylalanine, or valine resulted in a receptor that did not bind the 5-HT1A agonist 8-hydroxy-2-(di-n-[3H]propylamino)tetralin. In contrast, replacement of Asn396 with glutamine did not affect agonist binding. In addition, serine residues at positions 391 and 393 (transmembrane domain VII) were changed to alanine. Changing the less conserved Ser391 to alanine had no effect on ligand binding. However, replacement of the conserved Ser393 with alanine reduced ligand binding by 86%. Replacement of a conserved aspartate at position 82 (transmembrane region II) with alanine also produced a receptor without detectable agonist binding. Protein immunoblotting detected receptor protein of approximately 51 kDa in both wild-type and mutant receptor-expressing cells, indicating that these mutations probably did not affect expression or processing of the protein. Importantly, the sequence of the human 5-HT1A receptor described in this paper differs from the published sequence [Nature (Lond.) 329:75-79 (1987)] in transmembrane region IV. The present sequence encodes a protein of 422 amino acids, instead of the 421-amino acid protein that has been described previously [Nature (Lond.) 329:75-79 (1987)], and has a change in the sequence in transmembrane region IV from ... RPRAL ... to ... RRAAA ..., which corresponds to the published sequence [J. Biol. Chem. 265:5825-5832 (1990)] of the rat 5-HT1A receptor. Moreover, conversion of the transmembrane region IV sequence of the present clone to that of the published sequence by site-directed mutagenesis abolished ligand binding to the receptor.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1992

Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols.

The preferential interactions of bovine serum albumin, lysozyme, chymotrypsinogen, ribonuclease A, and beta-lactoglobulin with polyethylene glycols (PEGs) of molecular weight 200-6,000 have been measured by dialysis equilibrium coupled with high precision densimetry. All the proteins were found to be preferentially hydrated in all the PEGs, and the magnitude of the preferential hydration increased with increasing PEG size for each protein. The change in the chemical potentials of the proteins with the addition of the PEGs had highly positive values, indicating a strong thermodynamic destabilization of the system by the PEGs. A viscosity study of the PEGs showed them to be randomly coiled polymers, as their radii of gyration were related to the molecular weight by Rg = aM0.55. The thickness of the effective shell impenetrable to PEG around protein molecules, calculated from the preferential hydration, was found to vary with PEG molecular weight in similar fashion as the PEG radius of gyration, supporting the proposal (Arakawa, T. & Timasheff, S.N., 1985a, Biochemistry 24, 6756-6762) that the preferential exclusion of PEGs from proteins is due principally to the steric exclusion of PEG from the protein domain, although favorable interactions with protein surface residues, in particular nonpolar ones, may compete with the exclusion. These thermodynamically unfavorable preferential exclusion interactions lead to the action of PEGs as precipitants, although they may destabilize protein structure at higher temperatures.

Chymotrypsinogen↗
PubMed1992

Ontogeny of endothelin and its receptors in rat brain.

The ontogeny of endothelin (ET) system in rats was studied in preterm (18 days of gestation), term (21 days of gestation) and 1 week post term rats. Brains were dissected out and (1) processed for the estimation of endogenous ET-1 by RIA and (2) membranes were prepared for radioreceptor binding. Receptor characteristics, affinity (Kd) and density (Bmax) were determined using [125I] ET-1 and [125I] SRT 6b (which is structurally similar to ET) and cold ET-1 or SRT 6b as displacer. ET levels were found to be 25.66 +/- 3.18 pg/g protein in preterm, 47.37 +/- 5.31 pg/g protein in term and 48.30 +/- 1.90 pg/g protein in post term rats. ET levels were significantly lower in preterm as compared to term and post term rats. Preterm, term and post term rats showed single high affinity binding site for both [125I] ET-1 and [125I] SRT 6b. The Kd values for [125I] ET-1 and [125I] SRT 6b binding were similar in preterm, term and post term rats. The Bmax values of both [125I] ET-1 and [125I] SRT 6b binding were found to be similar in preterm and term rats while they were significantly higher in post term rats. In adult (4 month old) rats the Kd values were similar to neonatal rats while the Bmax values were significantly lower than the post term neonatal rats. It is concluded that ET and its receptors are developmentally regulated and there is a possibility that endogenous ET is involved in the regulation of ET receptor density.

Animals↗
PubMed1992

Morphine metabolism in acutely ill preterm newborn infants.

To examine the manner in which morphine is metabolized in acutely ill premature infants, we measured the levels of morphine, morphine-3- and -6-glucuronides, and codeine in timed urine specimens and paired plasma specimens at 4 hours and 24 hours after a single dose of morphine in 16 preterm infants (less than 32 weeks of gestational age). A large amount of unmetabolized morphine was found in the urine in 13 (81.2%) of the 16 infants at 4 hours; in 12 of them, morphine was excreted even at 24 hours. Urinary morphine levels varied greatly; the coefficient of variation was 130% at 4 hours and 118% at 24 hours. Codeine was not found in any of the infants. In 10 (62.5%) of the 16 infants, at least one metabolite was found in either plasma or urine. Plasma and urinary levels of morphine conjugates also varied greatly among these 10 infants (coefficient of variation: 109% to 317%). All six infants (37.5%) who had no metabolites excreted large amounts of unmetabolized morphine in the urine for up to 24 hours. Birth weight, gestational age, postnatal age, systemic blood pressure, and other clinical or physiologic variables did not differ significantly between the 10 infants who had morphine conjugates and the six who did not. We conclude that (1) nearly two thirds of acutely ill preterm infants born at less than 32 weeks of gestational age conjugate morphine; (2) irrespective of their ability to produce morphine conjugates, preterm infants excrete large amounts of morphine unmetabolized, as late as 24 hours after a single dose; (3) morphine handling patterns are highly variable among premature infants, and no obvious factors account for the variability; and (4) such variability in morphine handling in general, and the production of the highly potent morphine-6-glucuronide in particular, could explain the variance in morphine pharmacokinetic measures and in the clinical responses to morphine during the newborn period.

Chromatography, High Pressure Liquid↗
PubMed1992

Photoinduction of strand scissions in DNA by kojic acid: role of transition metal ions and oxygen free radical intermediates in the reaction.

Kojic acid (5-hydroxy-2-hydroxymethyl-gamma-pyrone) is a bacterial metabolic product used intensively in the food industry. In the presence of visible light and molecular oxygen it was found to cause breakage of calf thymus DNA. Such degradation was considerably enhanced in the presence of the transition metal ions Fe(III), Fe(II) and Cu(II). The cleavage of DNA in the presence of Fe(III) did not appear to have any preferred site(s) or sequence(s) for strand scission. Kojic acid catalysed the reduction of transition metal which in the case of Cu(II) was found to play an essential role in the degradation of DNA. Kojic acid also reduced oxygen to superoxide and hydroxyl radicals were formed in the presence of metal ions. The involvement of these active oxygen species in the reaction was established by the inhibition of DNA breakage by superoxide dismutase, catalase, iodide, mannitol, formate and sodium azide.

Animals↗
PubMed1991

High-performance liquid chromatographic determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in biological samples using multi-wavelength forward optical detection.

An isocratic high-performance liquid chromatographic method has been developed for the determination of morphine, morphine-3-glucuronide, morphine-6-glucuronide and codeine in plasma, urine and cerebrospinal fluid. The use of an efficient solid-phase extraction procedure together with a forward optical scanning detector allows a detection limit of 500 pg/ml. The method was evaluated by examination of biological samples taken from newborn infants following the intravenous administration of morphine sulfate.

Chromatography, High Pressure Liquid↗
PubMed1990

Preferential interactions determine protein solubility in three-component solutions: the MgCl2 system.

The correlation between protein solubility and the preferential interactions of proteins with solvent components was critically examined with aqueous MgCl2 as the solvent system. Preferential interaction and solubility measurements with three proteins, beta-lactoglobulin, bovine serum albumin, and lysozyme, resulted in similar patterns of interaction. At acid pH (pH 2-3) and lower salt concentrations (less than 2 M), the proteins were preferentially hydrated, while at higher salt concentrations, the interaction was either that of preferential salt binding or low salt exclusion. At pH 4.5-5, all three proteins exhibited either very low preferential hydration or preferential binding of MgCl2. These results were analyzed in terms of the balance between salt binding and salt exclusion attributed to the increase in the surface tension of water by salts, which is invariant with conditions. It was shown that the increase in salt binding at high salt concentration is a reflection of mass action, while its decrease at acid pH is due to the electrostatic repulsion between Mg2+ ions and the high net positive charge on the protein. The preferential interaction pattern was paralleled by the variation of protein solubility with solvent conditions. Calculation of the transfer free energies from water to the salt solutions for proteins in solution and in the precipitate showed dependencies on salt concentration. This indicates that the nature of interactions between proteins and solvent components is the same in solution and in the solid state, which implies no change in protein structure during precipitation. Analysis of the transfer free energies and preferential interaction parameter in terms of the salting-in, salting-out, and weak ion binding contributions has led to the conclusions that, when the weak ion binding contribution is small, the predominant protein-salt interaction must be that of preferential salt exclusion most probably caused by the increase of the surface tension of water by addition of the salt. A necessary consequence of this is salting-out of the protein, if the protein structure is to remain unaltered.

Calorimetry↗
PubMed1990

Why preferential hydration does not always stabilize the native structure of globular proteins.

The observed preferential hydration of proteins in aqueous MgCl2 solutions at low pH and low salt concentration (Arakawa et al., 1990) prompted a scrutiny of possible protein stabilization by MgCl2 under the same conditions, in view of earlier observations in aqueous solutions of sugars, amino acids, and a number of salts that preferential hydration is usually accompanied by the stabilization of the native structure of globular proteins. The results of thermal transition experiments on five proteins (ribonuclease A, lysozyme, beta-lactoglobulin, chymotrypsinogen, and bovine serum albumin) revealed neither significant stabilization nor destabilization of the protein structures by MgCl2 both at acid conditions (except for ribonuclease A, which was stabilized, but to a much smaller extent than by MgSO4) and at higher pH at which MgCl2 displayed little preferential hydration. This was in contrast to the great stabilizing action of MgSO4 at the same conditions. 2-Methyl-2,4-pentanediol (MPD), which gives a very large preferential hydration of native ribonuclease A at pH 5.8 [Pittz & Timasheff (1978) Biochemistry 17, 615-623], was found to be a strong destabilizer of that protein at the same conditions. Analysis of the preferentially hydrating solvent systems led to their classification into two categories: those in which the preferential hydration is independent of solution conditions and those in which it varies with conditions. The first always stabilize protein structure, while the second do not. In the first category the predominant interaction is that of cosolvent exclusion, determined by solvent properties, with the protein being essentially inert. In the second category interactions are determined to a major extent by the chemical nature of the protein surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Chymotrypsinogen↗
PubMed1990

Pharmacokinetics of a single dose of morphine in preterm infants during the first week of life.

We studied morphine pharmacokinetics after a single intravenous dose of 0.1 mg/kg in 20 newborn infants, who were born at 26 to 40 weeks of gestation and were less than 5 days of age. In the 10 infants whose gestational age was less than or equal to 30 weeks, the mean (+/- SD) distribution half-life was 50 +/- 35 minutes, elimination half-life was 10 +/- 3.7 hours, and clearance was 3.39 +/- 3.28 ml/kg/min; the corresponding values for the three term infants were 19 +/- 8 minutes, 6.7 +/- 4.6 hours, and 15.5 +/- 10 ml/kg/min, respectively. The data suggested a trend of decreasing values for distribution and elimination half-lives with increasing gestation, but a considerable degree of variation was seen. The morphine clearance rate increased as a function of gestational age at a rate of 0.9 ml/kg/min per week of gestation. Between 18% and 22% of the drug was found to be protein bound. Four hours after the dose, the drug level remained greater than or equal to 12 ng/ml in 8 of 10 infants born at greater than or equal to 31 weeks of gestation. In 8 of 10 infants born at less than or equal to 30 weeks of gestation, similar levels were maintained at 8 hours after the initial dose. We conclude that (1) there is a marked degree of variation in morphine pharmacokinetics during the neonatal period, (2) nearly 80% of the intravenously infused drug remains free, which might explain the high sensitivity to morphine in this age group, and (3) during the first week of age, adequate blood levels can be maintained by administration of morphine at 4- to 6-hour intervals in term infants and at less frequent intervals in very premature infants (less than or equal to 30 weeks of gestation).

Blood Proteins↗