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Biomedical subjects

S Subramaniam

Publications and source records attributed to S Subramaniam.

At least 55 records · Page 3Linked to original sources
PubMed2000

Protein A-activated rat splenic lymphocyte proliferation involves tyrosine kinase-phospholipase C-protein kinase C pathway.

Protein A (PA) of Staphylococcus aureus was long been known for its affinity towards the Fc domain of immunoglobulin G. It is now well established that PA is a potent biological response modifier showing simultaneously antitumor, antitoxic, and anticarcinogenic properties. This bacterial protein was also observed to stimulate production of cytokines. But the molecular mechanism(s) of immunocyte activation by PA still remained essentially unknown. In this report, we demonstrate a hitherto undescribed role of PA as a signal inducer in rat splenic lymphocytes. Our studies describe that PA induces transition of G0/G1 to S and G2/M phases of cell cycle, thus ultimately stimulating splenic lymphocyte proliferation. It has also been revealed that PA binds to rat splenic lymphocytes in a dose dependent manner and stimulates proliferation via tyrosine kinase-phospholipase C (PLC)-Ca2+-dependent protein kinase C (PKC) pathway. These observations will be of valid help in correlating the immunostimulatory activities of PA with the molecular mechanism(s) of its action.

Animals↗
PubMed1999

Electron crystallography of bacteriorhodopsin with millisecond time resolution.

The goal of time-resolved crystallographic experiments is to capture dynamic "snapshots" of molecules at different stages of a reaction pathway. In recent work, we have developed approaches to determine determined light-induced conformational changes in the proton pump bacteriorhodopsin by electron crystallographic analysis of two-dimensional protein crystals. For this purpose, crystals of bacteriorhodopsin were deposited on an electron microscopic grid and were plunge-frozen in liquid ethane at a variety of times after illumination. Electron diffraction patterns were recorded either from unilluminated crystals or from crystals frozen as early as 1 ms after illumination and used to construct projection difference Fourier maps at 3.5-A resolution to define light-driven changes in protein conformation. As demonstrated here, the data are of a sufficiently high quality that structure factors obtained from a single electron diffraction pattern of a plunge-frozen bacteriorhodopsin crystal are adequate to obtain an interpretable difference Fourier map. These difference maps report on the nature and extent of light-induced conformational changes in the photocycle and have provided incisive tools for understanding the molecular mechanism of proton transport by bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1999

Knowledge-based interaction potentials for proteins.

We discuss the derivation of atomic-level potentials of mean force from the known protein structures and their applicability for structural evaluation applications. In the derivation process, rigorous density estimation methodology is used to estimate the probability density functions (PDFs) for the distributions of interatomic distances in the protein structures. Potentials of mean force are then derived from these density functions using simple Boltzmann's relation. We also test the potentials against pairs of current and superseded protein structures in the Protein Data Bank. Using PDF potentials to evaluate each structure pair, we are able to identify, with high accuracy, which of the two structures is of higher resolution or better quality. This result shows that the PDF potentials are sensitive to details in protein structures as the current and superseded atomic coordinates generally do not differ by more than 1 A in root-mean-square deviation, and that the PDF potentials could potentially be used for X-ray structure refinement and protein structure prediction.

Databases, Factual↗
PubMed1999

Molecular fingerprinting of Riemerella anatipestifer by repetitive sequence PCR.

Riemerella anatipestifer is a gram-negative rod-shaped bacterium associated with epizootic infections in poultry. A total of 35 R. anatipestifer isolates including the type strain ATCC11845T, reference and field strains for 18 different serotypes were characterized by repetitive sequence based-PCR (rep-PCR) with outwardly-directed primers based on the repetitive extragenic palindromic (REP) consensus sequence. This technique was applied by using either extracted genomic DNA or preparation of whole bacterial cells harvested directly from plate cultures. Rep-PCR discriminated the R. anatipestifer isolates into 19 electrophoretic types. DNA fingerprints obtained from rep-PCR of extracted genomic DNA or from preparations of whole cells yielded comparable patterns. Substantial variation was seen among the rep-PCR fingerprints of different serotypes. Moreover, different polymorphisms of the rep-PCR fingerprints were evident among epidemiologically unrelated isolates of the same serotype. These results suggest the presence of repetitive extragenic palindromic-like elements within the genome of R. anatipestifer that can be used in some isolates to discriminate between different strains belonging to the same serotype. Rep-PCR may serve as a useful molecular tool for subtyping R. anatipestifer isolates for epidemiologic investigations. The whole cell procedure offers the advantage of ease of performance requiring only small quantities of cells.

Animals↗
PubMed1999

Protein conformational changes in the bacteriorhodopsin photocycle.

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation. Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.

Bacteriorhodopsins↗
PubMed1999

Subordinate-level object classification reexamined.

The classification of a table as round rather than square, a car as a Mazda rather than a Ford, a drill bit as 3/8-inch rather than 1/4-inch, and a face as Tom have all been regarded as a single process termed "subordinate classification." Despite the common label, the considerable heterogeneity of the perceptual processing required to achieve such classifications requires, minimally, a more detailed taxonomy. Perceptual information relevant to subordinate-level shape classifications can be presumed to vary on continua of (a) the type of distinctive information that is present, nonaccidental or metric, (b) the size of the relevant contours or surfaces, and (c) the similarity of the to-be-discriminated features, such as whether a straight contour has to be distinguished from a contour of low curvature versus high curvature. We consider three, relatively pure cases. Case 1 subordinates may be distinguished by a representation, a geon structural description (GSD), specifying a nonaccidental characterization of an object's large parts and the relations among these parts, such as a round table versus a square table. Case 2 subordinates are also distinguished by GSDs, except that the distinctive GSDs are present at a small scale in a complex object so the location and mapping of the GSDs are contingent on an initial basic-level classification, such as when we use a logo to distinguish various makes of cars. Expertise for Cases 1 and 2 can be easily achieved through specification, often verbal, of the GSDs. Case 3 subordinates, which have furnished much of the grist for theorizing with "view-based" template models, require fine metric discriminations. Cases 1 and 2 account for the overwhelming majority of shape-based basic- and subordinate-level object classifications that people can and do make in their everyday lives. These classifications are typically made quickly, accurately, and with only modest costs of viewpoint changes. Whereas the activation of an array of multiscale, multiorientation filters, presumed to be at the initial stage of all shape processing, may suffice for determining the similarity of the representations mediating recognition among Case 3 subordinate stimuli (and faces), Cases 1 and 2 require that the output of these filters be mapped to classifiers that make explicit the nonaccidental properties, parts, and relations specified by the GSDs.

Classification↗
PubMed1999

Combined Monte Carlo and molecular dynamics simulation of fully hydrated dioleyl and palmitoyl-oleyl phosphatidylcholine lipid bilayers.

We have applied a new equilibration procedure for the atomic level simulation of a hydrated lipid bilayer to hydrated bilayers of dioleyl-phosphatidylcholine (DOPC) and palmitoyl-oleyl phosphatidylcholine (POPC). The procedure consists of alternating molecular dynamics trajectory calculations in a constant surface tension and temperature ensemble with configurational bias Monte Carlo moves to different regions of the configuration space of the bilayer in a constant volume and temperature ensemble. The procedure is applied to bilayers of 128 molecules of POPC with 4628 water molecules, and 128 molecules of DOPC with 4825 water molecules. Progress toward equilibration is almost three times as fast in central processing unit (CPU) time compared with a purely molecular dynamics (MD) simulation. Equilibration is complete, as judged by the lack of energy drift in 200-ps runs of continuous MD. After the equilibrium state was reached, as determined by agreement between the simulation volume per lipid molecule with experiment, continuous MD was run in an ensemble in which the lateral area was restrained to fluctuate about a mean value and a pressure of 1 atm applied normal to the bilayer surface. Three separate continuous MD runs, 200 ps in duration each, separated by 10,000 CBMC steps, were carried out for each system. Properties of the systems were calculated and averaged over the three separate runs. Results of the simulations are presented and compared with experimental data and with other recent simulations of POPC and DOPC. Analysis of the hydration environment in the headgroups supports a mechanism by which unsaturation contributes to reduced transition temperatures. In this view, the relatively horizontal orientation of the unsaturated bond increases the area per lipid, resulting in increased water penetration between the headgroups. As a result the headgroup-headgroup interactions are attenuated and shielded, and this contributes to the lowered transition temperature.

Lipid Bilayers↗
PubMed1999

Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. I. Structure of the molecular complex.

This paper reports on a simulation of a gramicidin channel inserted into a fluid phase DMPC bilayer with 100 lipid molecules. Two lipid molecules per leaflet were removed to insert the gramicidin, so the resulting preparation had 96 lipid molecules and 3209 water molecules. Constant surface tension boundary conditions were employed. Like previous simulations with a lower lipid/gramicidin ratio (Woolf, T. B., and B. Roux. 1996. Proteins: Struct., Funct., Genet. 24:92-114), it is found that tryptophan-water hydrogen bonds are more common than tryptophan-phospholipid hydrogen bonds. However, one of the tryptophan NH groups entered into an unusually long-lived hydrogen bonding pattern with two glycerol oxygens of one of the phospholipid molecules. Comparisons were made between the behavior of the lipids adjacent to the channel with those farther away. It was found that hydrocarbon chains of lipids adjacent to the channel had higher-order parameters than those farther away. The thickness of the lipid bilayer immediately adjacent to the channel was greater than it was farther away. In general, the lipids adjacent to the membrane had similar orientations to those seen by Woolf and Roux, while those farther away had similar orientations to those pertaining before the insertion of the gramicidin. A corollary to this observation is that the thickness of the hydrocarbon region adjacent to the gramicidin was much thicker than what other studies have identified as the "hydrophobic length" of the gramicidin channel.

Biophysical Phenomena↗
PubMed1999

Simulation study of a gramicidin/lipid bilayer system in excess water and lipid. II. Rates and mechanisms of water transport.

A gramicidin channel in a fluid phase DMPC bilayer with excess lipid and water has been simulated. By use of the formal correspondence between diffusion and random walk, a permeability for water through the channel was calculated, and was found to agree closely with the experimental results of Rosenberg and Finkelstein (Rosenberg, P.A., and A. Finkelstein. 1978. J. Gen. Physiol. 72:327-340; 341-350) for permeation of water through gramicidin in a phospholipid membrane. By using fluctuation analysis, components of resistance to permeation were computed for movement through the channel interior, for the transition step at the channel mouth where the water molecule solvation environment changes, and for the process of diffusion up to the channel mouth. The majority of the resistance to permeation appears to occur in the transition step at the channel mouth. A significant amount is also due to structurally based free energy barriers within the channel. Only small amounts are due to local friction within the channel or to diffusive resistance for approaching the channel mouth.

Biological Transport, Active↗
PubMed1999

pH dependence of antibody: hapten association.

Monoclonal antibody NC6.8 is specific for the superpotent sweetener, N-(p-cyanophenyl)-N'-(diphenylmethyl)-guanidiniumacetic++ + acid. The three-dimensional structure of the complex shows the close proximity of complementary charged residues on the antibody and groups of the hapten. As a result, association is dependent on the pH, dielectric, and ionic strength of the medium. Continuum electrostatics methods are used to calculate the pH-dependent association energetics of NC6.8 with the superpotent sweetener. In addition to providing a titration profile, the calculations quantitatively assess the relative influence of charged groups on the energetics of association. Models of site directed mutants are constructed to probe the influence of each charged interface residue on the pH-dependent energetics of association. Examination of electrostatic contribution to free energy of association in mutant complexes, where the key acidic residues on the antibody are neutralized, shows that charge complementarity at the combining site is an important requirement for hapten binding. Also, based on the pKa values of several combining site tyrosine residues, aromatic pi-stacking and van der Waal's contacts between the antibody and hapten contribute to the specificity of the complex.

Acetates↗
PubMed1999

The structure of bacteriorhodopsin: an emerging consensus.

Six different sets of coordinates have been recently published for bacteriorhodopsin, with reported resolutions ranging from 3.5 A to 2. 3 A. Three of these are the result of electron crystallographic investigations of two-dimensional crystals of bacteriorhodopsin, whereas the others are from X-ray crystallographic studies of three-dimensional crystals of bacteriorhodopsin. How similar are these models? Are the structure determinations using X-ray diffraction data more accurate than those determined by electron crystallography? Is any one of these coordinate sets closer to the 'real' structure of bacteriorhodopsin than the others? Does the availability of newer models bring us closer to understanding how bacteriorhodopsin really works? These questions, as well as some related issues, are currently being explored.

Bacteriorhodopsins↗
PubMed1999

Polymorphism of dextromethorphan oxidation in South Indian subjects.

One hundred fifty-six unrelated healthy South Indian subjects were phenotyped according to their ability to metabolize dextromethorphan to its O-demethylated metabolite dextrorphan. Each volunteer was administered 25 mg oral dextromethorphan hydrobromide (19.3 mg dextromethorphan). Urine was collected during an 8-hour period after drug administration and was analyzed for dextromethorphan and dextrorphan by HPLC with fluorescence detection. This analysis was performed with and without previous deconjugation. The log10 (metabolic ratio), calculated as the ratio of dextromethorphan to dextrorphan, was bimodally distributed, and it was inferred that the frequency of occurrence of poor metabolizers of dextromethorphan in South Indian subjects is 3.2%. Phenotype assignment remained the same with both methods of analysis. Furthermore, a fairly good correlation (Spearman rank order correlation coefficient [r(s)] = 0.61; P < .0001) was observed between the log-transformed metabolic ratio derived from both methods.

Adolescent↗
PubMed1999

Oral bioavailability and pharmacokinetics of the new insulin sensitizer DRF-2189 in Wistar rats.

The pharmacokinetics of the new insulin sensitizing agent, DRF-2189 ([5-[4-[2-(1-indolyl) ethoxy]phenyl]methyl]thiazolidine-2,4-dione, CAS 172647-53-9) were studied in male Wistar rats following oral doses of 1, 3 and 10 mg/kg as suspension in 0.25% carboxymethylcellulose. Drug was extracted from plasma samples using a solvent mixture containing ethylacetate and dichloromethane (3:2) and analyzed by high-performance liquid chromatography with fluorescence detection. DRF-2189 was absorbed slowly, attaining maximum levels at 2-3 h, and was eliminated with a half-life (t1/2) of about 3 h. The Cmax and AUC(0-infinity) increased linearly (r2 = 0.99) with the dose, while the elimination half-life (t1/2) was independent of the dose. An intravenous pharmacokinetic study of DRF-2189 was carried out in Wistar rats at a dose of 3.0 mg/kg. The pharmacokinetic parameters AUC(0-infinity), t1/2, plasma clearance (Cl) and volume of distribution (Vd) were found to be 49.52 micrograms x h/ml, 2.99 h, 16.31 ml/h and 45.11 ml. respectively. Oral bioavailability (f) of DRF-2189 in Wistar rats was 44%. Based on pharmacokinetic studies, DRF-2189 is a good choice for further development.

Animals↗
PubMed1999

Protein structure determination using a database of interatomic distance probabilities.

The accelerated pace of genomic sequencing has increased the demand for structural models of gene products. Improved quantitative methods are needed to study the many systems (e.g., macromolecular assemblies) for which data are scarce. Here, we describe a new molecular dynamics method for protein structure determination and molecular modeling. An energy function, or database potential, is derived from distributions of interatomic distances obtained from a database of known structures. X-ray crystal structures are refined by molecular dynamics with the new energy function replacing the Van der Waals potential. Compared to standard methods, this method improved the atomic positions, interatomic distances, and side-chain dihedral angles of structures randomized to mimic the early stages of refinement. The greatest enhancement in side-chain placement was observed for groups that are characteristically buried. More accurate calculated model phases will follow from improved interatomic distances. Details usually seen only in high-resolution refinements were improved, as is shown by an R-factor analysis. The improvements were greatest when refinements were carried out using X-ray data truncated at 3.5 A. The database potential should therefore be a valuable tool for determining X-ray structures, especially when only low-resolution data are available.

Computer Simulation↗
PubMed1999

Perinatal sidestream cigarette smoke exposure and the developing pulmonary surfactant system in rats.

1. Epidemiological studies have strongly implicated passive smoking with increased incidence of various respiratory diseases in children. Our earlier studies have shown that chronic exposure to tobacco smoke significantly changes the composition and the surface activity of the pulmonary surfactant in adult rats. The aim of the present study was to determine if perinatal exposure to sidestream cigarette smoke influences the composition and function of pulmonary surfactant system in developing rat pups. 2. Pregnant Sprague Dawley rats were exposed to sidestream cigarette smoke in a whole body exposure chamber for a total of 6 h each day and the pups born to these dams were further exposed to sidestream smoke for 2 h/day during postnatal period. Exposure of animals to smoke was ascertained by measuring their plasma cotinine. Surfactant analysis was performed on bronchoalveolar lavage fluids (BALF) collected from pups on postnatal day 1, 3, 7, 14, 21 and 35. The phospholipid (PL) content, percentage disaturated phosphatidylcholine (DSPC) and surfactant proteins (SP-A and SP-B) in BALF surfactant preparations from sham and smoke-exposed pups were compared. 3. Significant differences between the two groups were observed at two exposure points: a reduced level of SP-A on day 1, and, a higher level of SP-A and PLs on day 21, in smoke-exposed pups. Surface activity analysis of the surfactant films by pulsating bubble surfactometer did not show any significant differences between the sham and smoke-exposed groups at any exposure point. 4. The results indicate that perinatal exposure to sidestream smoke is capable of producing biochemical changes in the composition of pulmonary surfactant at different stages of development but these changes do not influence surface tension reducing properties of the surfactant.

Animals↗
PubMed1998

Direct molecular level measurements of the electrostatic properties of a protein surface.

In this work, we used direct measurements with the surface force apparatus to determine the pH-dependent electrostatic charge density of a single binding face of streptavidin. Mean field calculations have been used with considerable success to model electrostatic potential fields near protein surfaces, but these models and their inherent assumptions have not been tested directly at the molecular level. Using the force apparatus and immobilized, oriented monolayers of streptavidin, we measured a pI of 5-5.5 for the biotin-binding face of the protein. This differs from the pI of 6.3 for the soluble protein and confirms that we probed the local electrostatic features of the macromolecule. With finite difference solutions of the linearized Poisson-Boltzmann equation, we then calculated the pH-dependent charge densities adjacent to the same face of the protein. These calculated values agreed quantitatively with those obtained by direct force measurements. Although our study focuses on the pH-dependence of surface electrostatics, this direct approach to probing the electrostatic features of proteins is applicable to investigations of any perturbations that alter the charge distribution of the surfaces of immobilized molecules.

Binding Sites↗