Biomedical subjects

S Subramaniam

Publications and source records attributed to S Subramaniam.

At least 145 records · Page 8Linked to original sources

PubMed1991

Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization.

We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by approximately 100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experiments, respectively, show the presence of long-lived O-like and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and O intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or O intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis----all-trans reisomerization of retinal is completed during the decay of the N and O intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal.

Alanine↗

PubMed1991

Analysis of the binding site architecture of monoclonal antibodies to morphine by using competitive ligand binding and molecular modeling.

The structural features of mAb directed against the opiate morphine were analyzed by using competitive ligand analog-binding studies, examination of the V region amino acid sequence, and computer-aided molecular modeling of the fragment V region. The antibody response in BALB/c mice to morphine is relatively restricted, in that all of the mAb examined in this study contained the same lambda L chain and very similar H chain V regions. A three-dimensional model of the antimorphine-binding site was constructed by using computational and graphic display techniques. Each of the six complementary-determining regions was constructed by using fragment replacement methods employing canonical loop conformations of known "parent" structures. Experimental competitive ligand-binding data and theoretical modeling suggest that a charged glutamate residue at position H:50 and aromatic side chains of residues H:33W, H:47W, H:58F, H:95W, H:101iY, and L:91W are key features in ionic and hydrophobic interactions with the ligand. This study represents the first use of theoretical and experimental modeling techniques to describe the Ag-binding site of a mouse fragment V region containing a lambda L chain.

Amino Acid Sequence↗

PubMed1991

The reaction of hydroxylamine with bacteriorhodopsin studied with mutants that have altered photocycles: selective reactivity of different photointermediates.

The reaction of the retinylidene Schiff base in bacteriorhodopsin (bR) to the water-soluble reagent hydroxylamine is enhanced by greater than 2 orders of magnitude under illumination. We have used this reaction as a probe for changes in Schiff base reactivity during the photocycle of wild-type bR and mutants defective in proton transport. We report here that under illumination at pH 6, the D85N mutant has a 20-fold lower rate and the D212N mutant has a greater than 4-fold higher rate for the light-dependent reaction with hydroxylamine compared with wild-type bR. In contrast, the reactivities of wild-type bR and the D96N and T46V mutants are similar. It has been previously shown that the D96N and T46V replacements have no significant effect on the kinetics of "M" formation but have dramatic effects on rate of the decay of M. We therefore conclude that the hydroxylamine reaction occurs before formation of the M intermediate. Most likely it occurs at the "L" stage of the cycle and reflects increased water accessibility to the Schiff base due to a light-driven change in protein conformation.

Amino Acid Sequence↗

PubMed1991

Binding of the neuroleptic drug haloperidol to a monoclonal antibody: refinement of the binding site molecular model using canonical structures.

The ligand binding site of a monoclonal antibody (185), which binds the neuroleptic drug haloperidol, has been modelled using canonical structures and energy minimization techniques. This refined modelling protocol has allowed us to predict the variable region loop conformations. Three key residues, H:50(W), H:100a(D) and L:96(Y) appear to create the basis of the electrostatic, pi-pi stacking interactions and hydrogen bonding required for the high affinity binding site characteristics present in this antibody. The use of computer-aided graphics techniques and appropriate three-dimensional modelling permits inspection of the predicted molecular recognition features of the ligand binding site.

Animals↗

PubMed1991

Time-correlation analysis of simulated water motion in flexible and rigid gramicidin channels.

Molecular dynamics simulations have been done on a system consisting of the polypeptide membrane channel former gramicidin, plus water molecules in the channel and caps of waters at the two ends of the channel. In the absence of explicit simulation of the surrounding membrane, the helical form of the channel was maintained by artificial restraints on the peptide motion. The characteristic time constant of the artificial restraint was varied to assess the effect of the restraints on the channel structure and water motions. Time-correlation analysis was done on the motions of individual channel waters and on the motions of the center of mass of the channel waters. It is found that individual water molecules confined in the channel execute higher frequency motions than bulk water, for all degrees of channel peptide restraint. The center-of-mass motion of the chain of channel waters (which is the motion that is critical for transmembrane transport, due to the mandatory single filing of water in the channel) does not exhibit these higher frequency motions. The mobility of the water chain is dramatically reduced by holding the channel rigid. Thus permeation through the channel is not like flow through a rigid pipe; rather permeation is facilitated by peptide motion. For the looser restraints we used, the mobility of the water chain was not very much affected by the degree of restraint. Depending on which set of experiments is considered, the computed mobility of our water chain in the flexible channel is four to twenty times too high to account for the experimentally measured resistance of the gramicidin channel to water flow. From this result it appears likely that the peptide motions of an actual gramicidin channel embedded in a lipid membrane may be more restrained than in our flexible channel model, and that these restraints may be a significant modulator of channel permeability. For the completely rigid channel model the "trapping" of the water molecules in preferred positions throughout the molecular dynamics run precludes a reasonable assessment of mobility, but it seems to be quite low.

Gramicidin↗

PubMed1991

Molecular dynamics computations and solid state nuclear magnetic resonance of the gramicidin cation channel.

This paper reports on a coupled approach to determining the structure of the gramicidin A ion channel, utilizing solid state nuclear magnetic resonance (NMR) of isotopically labeled gramicidin channels aligned parallel to the magnetic field direction, and molecular dynamics (MD). MD computations using an idealized right-handed beta-helix as a starting point produce a refined molecular structure that is in excellent agreement with atomic resolution solid state NMR data. The data provided by NMR and MD are complementary to each other. When applied in a coordinated manner they provide a powerful approach to structure determination in molecular systems not readily amenable to x-ray diffraction.

Amino Acid Sequence↗

PubMed1991

Quantitative autoradiographic characterization of the binding of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ([3H]MK-801) in rat brain: regional effects of polyamines.

The distribution and properties of N-methyl-D-aspartate (NMDA) receptors, labeled with [3H](+)-5-methyl-10, 11-dihydro-5H-dibenzo[a,d]cyclohepten-5 5,10-imine (MK-801), were examined in rat brain. In sections of brain mash, the kinetics of association and dissociation of [3H]MK-801 were monophasic in the presence of 100 microM glutamate and glycine, and Scatchard transformations of saturation isotherms resulted in linear plots. Inhibition of the binding of [3H]MK-801 by other noncompetitive antagonists produced competition curves with Hill coefficients close to 1.0, consistent with a simple bimolecular interaction between the radioligand and the receptor. Scatchard plots based upon densitometric measurements of [3H]MK-801 binding in serial sections of rat brain were also linear, with dissociation constant values ranging from 5.0 to 8.4 nM in different regions at the level of the hippocampus. The distribution of [3H]MK-801 binding sites paralleled the distribution of NMDA displaceable L-[3H]glutamate binding sites. One exception was the cerebellar granule cell layer, where the density of binding sites for [3H]MK-801 was extremely low. The relative density of [3H]MK-801 to NMDA displaceable L-[3H]glutamate binding sites was approximately 1 to 2, consistent with the existence of two transmitter recognition sites per NMDA receptor. The modulatory effects of polyamines on [3H]MK-801 binding were studied in washed brain sections. The polyamine agonists spermine and spermidine enhanced [3H]MK-801 binding in all regions studied, with increases ranging from 18% in the thalamus to 106% in the ventromedial striatum. The effects of spermine and spermidine in these regions were highly correlated. Diethylenetriamine, which blocks the effects of spermidine, by itself produced decreases in the binding of [3H]MK-801 in most regions ranging from 5 to 21% but increased binding in parts of the striatum by 3 to 22%. The decrease in binding produced by diethylenetriamine in different brain regions was negatively correlated with the increase in binding produced by the agonists, suggesting that variability in the residual concentration of endogenous polyamines contributes to the regional variability of agonist effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Studies on the changes in plasma lipids and lipoproteins in CMF treated breast cancer patients.

In a comparative study on the effect of CMF (Combination of Cyclophosphamid, Methotroxate and 5-Fluorouracil) on the pre and postmenopausal women with breast cancer, a number of differences were observed in circulating plasma lipid concentration. Plasma lipids, phospholipids, triglycerides, cholesterol and free fatty acids levels were all lower in blood obtained from CMF treated breast cancer patients. HDL-cholesterol level were significantly increased in these patients. These differences remained when the patient groups were subdivided according to their menopausal status. HDL cholesterol had been significantly increased in pre and post menopausal CMF treated breast cancer patients when compared with untreated breast cancer patients.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed1991

Autopsy examination of firearm fatalities.

As general pathologists in Malaysia are required to perform medico-legal autopsies, this paper is written with the hope that it may serve as a guide to the less initiated. An account of elementary ballistics is included because it is essential for understanding the features of wounds produced by firearms. A complete autopsy examination including a systematic approach to the injuries is advocated. The recognition and interpretation of firearm injuries can often be difficult and it is essential that all features of injuries and other relevant findings are carefully observed and recorded. Relevant observations, documentation and opinions necessary for accurate determination of the true circumstances of death are discussed.

Autopsy↗

PubMed1990

Impaired cyclic AMP generation in outer medullary tubules of gentamicin-treated rats.

We have examined the effects of chronic gentamicin treatment on arginine8-vasopressin (AVP)-dependent cyclic AMP (cAMP) metabolism in rat medullary collecting tubules (oMCT) and medullary thick ascending limbs of Henle's loop (mTALH). Gentamicin attenuated AVP-stimulated cAMP accumulation to a greater extent in the mTALH (delta -51%) than in the oMCT (delta -25%). The mechanism of attenuation differed between segments, and could not be attributed to either direct inhibition of adenylate cyclase activity nor direct potentiation of cAMP-phosphodiesterase activity. These data suggest that the gentamicin-induced decrease in renal concentrating ability may be due at least in part to reduced AVP-dependent cAMP accumulation in the oMCT and mTALH.

1-Methyl-3-isobutylxanthine↗

PubMed1990

Ultraviolet-visible transient spectroscopy of bacteriorhodopsin mutants. Evidence for two forms of tyrosine-185----phenylalanine.

The photocycle kinetics of the bacteriorhodopsin mutant Tyr-185----Phe has been investigated by UV-visible transient spectroscopy. Flash-induced spectral changes were measured from 100 ns to 500 ms using a gated optical multichannel analyzer on protein samples that were reconstituted in vesicles with Halobacterium halobium lipids. Tyr-185----Phe exhibits a pH-dependent absorbance spectrum reflecting contributions from two different species. At pH 6, the dominant photocycling species has a lambda max near 610 nm although the absorption maximum of light-adapted Tyr-185----Phe is at 581 nm. This red-shifted species does not form any M-like intermediate and undergoes a photocycle similar to that observed for deionized blue membrane. At pH 8, the dominant photoactive form exhibits a lambda max near 550 nm. This purple species, which is blue shifted 20 nm relative to wild-type bacteriorhodopsin, exhibits a photocycle similar to the wild type. However, M formation occurs in 8 microseconds, approximately three times faster than wild-type bacteriorhodopsin at pH 8. In addition, an unusually long lived intermediate absorbing at 610 nm is observed at high pH. In the UV region, a broad band near 300-310 nm is absent in the mutant relative to wild type, consistent with earlier measurements made at low temperature which suggest that Tyr-185 undergoes a change in protonation. Steady-state proton pumping action spectra indicate that the 550 nm species does transport protons but that the blue species is inactive. These results are discussed in terms of a model that hypothesizes that Tyr-185 is located close to the bacteriorhodopsin chromophore and stabilizes the interaction of helices F and G through formation of a polarizable bond with Asp-212.

Amino Acid Sequence↗

PubMed1990

Protonation state of Asp (Glu)-85 regulates the purple-to-blue transition in bacteriorhodopsin mutants Arg-82----Ala and Asp-85----Glu: the blue form is inactive in proton translocation.

Previous studies with site-specific mutants of bacteriorhodopsin have demonstrated that replacement of Asp-85 or Arg-82 affects the absorption spectrum. Between pH 5.5 and 7, the Asp-85----Glu and Arg-82----Ala mutants exist in a pH-dependent equilibrium between purple (lambda max approximately 550/540 nm) and blue (lambda max approximately 600/590 nm) forms of the pigment. Measurement of proton transport as a function of wavelength in reconstituted vesicles shows that proton-pumping activities for the above mutants reside exclusively in their respective purple species. For both mutants, formation of the blue form with decreasing pH is accompanied by loss of proton transport activity. The Asp-85----Asn mutant displays a blue chromophore (lambda max approximately 588 nm), is inactive in proton translocation from pH 5 to 7.5, and shows no transition to the purple form. In contrast, the Asp-212----Asn mutant is purple (lambda max approximately 555 nm) and shows no transition to a blue chromophore with decreasing pH. The experiments suggest that (i) the pKa of the purple-to-blue transition is directly influenced by the pKa of the carboxylate at residue 85 and (ii) the relative strengths of interaction between the protonated Schiff base, Asp-85, Asp-212, and Arg-82 make a major contribution to the regulation of color and function of bacteriorhodopsin.

Alanine↗

PubMed1990

Cyclic AMP inhibits inositol polyphosphate production and calcium mobilization in neuroblastoma X glioma NG108-15 cells.

In the neuroblastoma X glioma hybrid cell line NG108-15, bradykinin (BK) receptor stimulation induced a rapid and concentration-dependent rise in cytosolic free Ca2+ levels, as measured with the Ca2(+)-sensitive fluorescent dye fura-2. The Ca2+ transient was present in the absence of extracellular Ca2+ and was associated with a concentration-dependent production of inositol phosphates, particularly inositol trisphosphate (InsP3). Pretreatment of intact NG108-15 cells with forskolin or dibutyryl-cAMP plus isobutylmethylxanthine reduced BK-stimulated InsP3 production and the increase in cytosolic free Ca2+. Membranes prepared from forskolin- and [3H]inositol-pretreated NG108-15 cells also showed a diminished production of InsP3 elicited by guanosine 5'-[gamma-thio]triphosphate, NaF, or BK plus GTP. On the other hand, the Ca2+ sensitivity of membrane-associated phosphoinositide-specific phospholipase C (PI-PLC) was unaffected by forskolin pretreatment of intact NG108-15 cells. Collectively, these results suggest that A-kinase may inhibit receptor-mediated and postreceptor stimulation of PI-PLC in neuron-like cells, perhaps by impairing the coupling between a guanine nucleotide-binding protein and PI-PLC.

Adenylyl Cyclases↗

PubMed1989

Superoxide dismutase: fluctuations in the structure and solvation of the active site channel studied by molecular dynamics simulation.

The molecular dynamics (MD) simulation of superoxide dismutase (SOD) in water is carried out for a total of 23 ps. The simulation system is a 26 A sphere centered at the active site of SOD, including 1602 atoms from SOD and 1761 water molecules. There is no gross deviation from the x-ray structure for the average MD structure. The structure and potential fluctuations around the active site are examined. The results provide new insight to the interactions between SOD and its substrate superoxide.

Binding Sites↗

PubMed1989

Water and polypeptide conformations in the gramicidin channel. A molecular dynamics study.

Theoretical studies of ion channels address several important questions. The mechanism of ion transport, the role of water structure, the fluctuations of the protein channel itself, and the influence of structural changes are accessible from these studies. In this paper, we have carried out a 70-ps molecular dynamics simulation on a model structure of gramicidin A with channel waters. The backbone of the protein has been analyzed with respect to the orientation of the carbonyl and the amide groups. The results are in conformity with the experimental NMR data. The structure of water and the hydrogen bonding network are also investigated. It is found that the water molecules inside the channel act as a collective chain; whereas the conformation in which all the waters are oriented with the dipoles pointing along the axis of the channel is a preferred one, others are also accessed during the dynamics simulation. A collective coordinate involving the channel waters and some of the hydrogen bonding peptide partners is required to describe the transition of waters from one configuration to the other.

Gramicidin↗

PubMed1989

Replacement of aspartic residues 85, 96, 115, or 212 affects the quantum yield and kinetics of proton release and uptake by bacteriorhodopsin.

Recently, a number of aspartic acid mutants of bacteriorhodopsin have been shown to be defective in steady-state proton transport. Here we report time-resolved measurements of light-induced proton release and uptake for these mutants. Proton transfers between the protein and the aqueous phase were directly monitored by measuring changes in the bulk conductivity of a micellar solution of bacteriorhodopsin. For the Asp-96----Asn mutant, proton uptake was slowed by greater than 1 order of magnitude with no observable effect on the release step. For Asp-85----Asn, H+ uptake occurred with normal kinetics, but the yield was significantly lower compared with either the Asp-96----Asn mutant or wild type, especially at pH 6. Substitution of glutamate for Asp-85 or Asp-96 had smaller but detectable effects on the kinetics and quantum yield of proton movements. Both asparagine and glutamate substitutions of aspartates at positions 115 and 212 lowered the proton quantum yields. Of these, only the Asp-115----Asn mutant showed an effect on the proton release step, and only the Asp-212----Glu mutation decreased the proton uptake rate. These experiments imply an obligatory role for Asp-96 in H+ uptake in the normal operation of the bacteriorhodopsin proton pump. The results also indicate that the amino acid substitutions affect the kinetics of either H+ release or H+ uptake, but not both. This implies that the two steps occur independently of each other after initiation of the photocycle.

Asparagine↗

PubMed1989

A comparison of the digestion and absorption of cocoa butter and palm kernel oil and their effects on cholesterol absorption in rats.

Digestion and absorption of cocoa butter and palm kernel oil and their effect on cholesterol absorption were studied in adult male rats. Duodenal and thoracic duct catheters were inserted surgically into the anesthetized rats. After an overnight fast, animals were given a single duodenal dose of an aqueous emulsion containing [1,2-3H]cholesterol and one of the following: corn oil, cocoa butter or palm kernel oil. Digestion and absorption were estimated by recovering the total fatty acids in the thoracic duct lymph over a 24-h collection period (after subtraction of the baseline "endogenous fatty acids" in the lymph). Intestinal absorption of cholesterol into the thoracic duct lymph was reduced significantly (P less than 0.05) in the presence of cocoa butter, compared to absorption when palm kernel oil or corn oil was administered. Compared to the absorption of corn oil (arbitrarily defined as 100%), the absorption of palm kernel oil and cocoa butter was 82 and 63%, respectively. The present study suggests that palm kernel oil absorption was not significantly different from that of corn oil. The lower absorbability of cocoa butter and its inhibitory effect on cholesterol absorption may explain in part why cocoa butter is less hypercholesterolemic and atherogenic than other equally saturated fats.

Analysis of Variance↗

PubMed1988

Engineering, mosquitoes and filariasis: a case report.

The results of larval surveys were used to assess the relative numbers of mosquitoes breeding in different types of habitat and in different parts of the town of Pondicherry, India. The results illustrate an effective method to set priorities for mosquito control by identifying the most significant breeding sites in a town, and show that they are not necessarily the most obvious, the most extensive or those intuitively most likely.

Animals↗