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S Subramaniam

Publications and source records attributed to S Subramaniam.

At least 73 records · Page 4Linked to original sources
PubMed1998

Analytical shape computation of macromolecules: I. Molecular area and volume through alpha shape.

The size and shape of macromolecules such as proteins and nucleic acids play an important role in their functions. Prior efforts to quantify these properties have been based on various discretization or tessellation procedures involving analytical or numerical computations. In this article, we present an analytically exact method for computing the metric properties of macromolecules based on the alpha shape theory. This method uses the duality between alpha complex and the weighted Voronoi decomposition of a molecule. We describe the intuitive ideas and concepts behind the alpha shape theory and the algorithm for computing areas and volumes of macromolecules. We apply our method to compute areas and volumes of a number of protein systems. We also discuss several difficulties commonly encountered in molecular shape computations and outline methods to overcome these problems.

Algorithms↗
PubMed1998

Analytical shape computation of macromolecules: II. Inaccessible cavities in proteins.

The structures of proteins are well-packed, yet they contain numerous cavities which play key roles in accommodating small molecules, or enabling conformational changes. From high-resolution structures it is possible to identify these cavities. We have developed a precise algorithm based on alpha shapes for measuring space-filling-based molecular models (such as van der Waals, solvent accessible, and molecular surface descriptions). We applied this method for accurate computation of the surface area and volume of cavities in several proteins. In addition, all of the atoms/residues lining the cavities are identified. We use this method to study the structure and the stability of proteins, as well as to locate cavities that could contain structural water molecules in the proton transport pathway in the membrane protein bacteriorhodopsin.

Algorithms↗
PubMed1998

Novel euglycemic and hypolipidemic agents. 1.

A series of [[(heterocyclyl)ethoxy]benzyl]-2,4-thiazolidinediones have been synthesized by the condensation of corresponding aldehyde 1 and 2,4-thiazolidinedione followed by hydrogenation. Both unsaturated thiazolidinedione 2 and its saturated counterpart 3 have shown antihyperglycemic activity. Many of these compounds have shown superior euglycemic and hypolipidemic activity compared to troglitazone (CS 045). The indole analogue DRF-2189 (3g) was found to be a very potent insulin sensitizer, comparable to BRL-49653 in genetically obese C57BL/6J-ob/ob and 57BL/KsJ-db/db mice. Pharmacokinetic and tissue distribution studies conducted on BRL-49653 and DRF-2189 (3g) indicate that these drugs are well-distributed in target tissues. On the basis of euglycemic activity as well as enhanced selectivity against reduction of triglycerides in plasma, DRF-2189 (3g) has been selected for further evaluation.

Animals↗
PubMed1998

Brownian dynamics simulations of protein folding: access to milliseconds time scale and beyond.

Protein folding occurs on a time scale ranging from milliseconds to minutes for a majority of proteins. Computer simulation of protein folding, from a random configuration to the native structure, is nontrivial owing to the large disparity between the simulation and folding time scales. As an effort to overcome this limitation, simple models with idealized protein subdomains, e.g., the diffusion-collision model of Karplus and Weaver, have gained some popularity. We present here new results for the folding of a four-helix bundle within the framework of the diffusion-collision model. Even with such simplifying assumptions, a direct application of standard Brownian dynamics methods would consume 10,000 processor-years on current supercomputers. We circumvent this difficulty by invoking a special Brownian dynamics simulation. The method features the calculation of the mean passage time of an event from the flux overpopulation method and the sampling of events that lead to productive collisions even if their probability is extremely small (because of large free-energy barriers that separate them from the higher probability events). Using these developments, we demonstrate that a coarse-grained model of the four-helix bundle can be simulated in several days on current supercomputers. Furthermore, such simulations yield folding times that are in the range of time scales observed in experiments.

Biophysics↗
PubMed1998

High-performance liquid chromatographic determination of the insulin sensitizing agent DRF-2189 in rat plasma.

A high-performance liquid chromatographic method for the determination of DRF-2189, using troglitazone as internal standard, is described. A dichloromethane-ethyl acetate solvent mixture (6:4, v/v) was used as the extraction solvent. A Kromasil C18 column with a mobile phase consisting of 0.05 M phosphate buffer-acetonitrile-methanol (22.5:37.5:40) (pH 5.0) was used at a flow-rate of 1.0 ml/min. The eluate was monitored by using fluorescence detection with excitation and emission wavelengths at 292 nm and 325 nm, respectively. Ratio of peak area of analyte to internal standard was used for quantification of plasma samples. Using this method, the absolute recovery of DRF-2189 from rat plasma was >95% and the limit of quantitation was 50 ng/ml. The intra-day relative standard deviation (R.S.D.) ranged from 1.74 to 7.24% at 1 microg/ml and 1.86 to 3.83% at 10 microg/ml. The inter-day R.S.D.s were 8.34 and 4.91% at 1 and 10 microg/ml, respectively. The method was applied to measure plasma concentrations of DRF-2189 in pharmacokinetic studies in Wistar rats.

Animals↗
PubMed1998

Species identification of Mycoplasma bovis and Mycoplasma agalactiae based on the uvrC genes by PCR.

The DNA repair genes uvrC from Mycoplasma bovis and Mycoplasma agalactiae type strains were cloned and their nucleotide sequences were established. These sequences were used to design polymerase chain reaction (PCR) primer pairs for M. bovis and M. agalactiae. Each primer pair amplified a 1-6 kb fragment of the uvrC gene in the respective species. The specificity of the primer pairs for the two species was demonstrated through the lack of cross-amplifications in heterologous PCR reactions and in reactions using DNA from other mycoplasma species. Subsequent restriction enzyme analysis of the amplified uvrC gene segments from type and field strains of M. bovis and M. agalactiae showed that the uvrC genes are well conserved in both species but differ significantly between the two species. The diagnostic PCR assay enabled unambiguous identification of M. bovis and M. agalactiae strains isolated from geographically diverse places, even in cases where 16S rRNA gene sequence analysis was unable to discriminate between the two species.

Animals↗
PubMed1998

N-methyl-D-aspartate receptor channel block by the enantiomeric 6,7-benzomorphans BIII 277 CL and BIII 281 CL.

BIII 277 CL ((-)-2R-[2 alpha, 3(R*),6 alpha]-3-(2-methoxypropyl)-6,11, 11-trimethyl-2,6-methano-1,2,3,4,5,6-hexahydro-3-benzazocin-9-ol hydrochloride) is a novel benzomorphan with neuroprotective and anticonvulsant properties that exhibits high affinity binding to the N-methyl-D-aspartate (NMDA) receptor but, in contrast to other structurally related benzomorphans, low affinity for mu opiate and sigma sites. Whole-cell voltage-clamp and single-channel recording were used to study the interaction of BIII 277 CL and its enantiomer BIII 281 CL with native NMDA receptors in cultured hippocampal neurons. BIII 277 CL and BIII 281 CL produced a slow use-dependent block of whole-cell NMDA receptor currents. Once block was established, recovery was slow (< 50% in > or = 40 min). The steady-state IC50 (nH) values derived from logistic fits to concentration-block isotherms obtained at -60 mV were 5.3 nM (0.67) and 58 nM (1.2), respectively. The benzomorphans had no effect on currents evoked by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and gamma-aminobutyric acid but minimally inhibited kainate-evoked currents at high (> or = 30 microM) concentrations. BIII 277 CL and BIII 281 CL failed to bind and block closed NMDA receptor channels, and the block was occluded by Mg++, consistent with an open channel-blocking mechanism. Steady-state block was diminished by depolarization; analysis of the voltage-dependence of block indicated that BIII 281 CL binds within the channel at a site that senses 46% of the transmembrane electric field. Recordings of single NMDA receptor channels in outside-out membrane patches confirmed the slow, persistent blocking action obtained in whole-cell recordings. In addition, at high concentrations, flickering of the unitary currents was observed consistent with a low-affinity channel-blocking action. Taking the present data in conjunction with previously obtained structure-activity information for N-substituted benzomorphans, a three-mode-blocking model was developed in which there are three interaction sites for binding of the high-affinity ligand BIII 277 CL. In this model, the drug can bind in one of three modes by docking at one, two or all three interaction points but cannot transition between modes. The model further proposes that the lower-affinity enantiomer BIII 281 CL binds in modes with one and two but not all three interaction points docked. We conclude that BIII 277 CL and BIII 281 CL are potent and selective, use-dependent (uncompetitive) channel-blocking NMDA receptor antagonists. The substantially higher affinity that BIII 277 CL exhibits for the NMDA receptor in comparison with its enantiomer and other benzomorphans appears to be due to stabilization of binding at three sites within the channel.

Animals↗
PubMed1998

The role of copper, molybdenum, selenium, and zinc in nutrition and health.

Copper, zinc, selenium, and molybdenum are involved in many biochemical processes supporting life. The most important of these processes are cellular respiration, cellular utilization of oxygen, DNA and RNA reproduction, maintenance of cell membrane integrity, and sequestration of free radicals. Copper, zinc, and selenium are involved in destruction of free radicals through cascading enzyme systems. Superoxide radicals are reduced to hydrogen peroxide by superoxide dismutases in the presence of copper and zinc cofactors. Hydrogen peroxide is then reduced to water by the selenium-glutathione peroxidase couple. Efficient removal of these superoxide free radicals maintains the integrity of membranes, reduces the risk of cancer, and slows the aging process. On the other hand, excess intake of these trace elements leads to disease and toxicity; therefore, a fine balance is essential for health. Trace element--deficient patients usually present with common symptoms such as malaise, loss of appetite, anemia, infection, skin lesions, and low-grade neuropathy, thus complicating the diagnosis. Symptoms for intoxication by trace elements are general, for example, flu-like and CNS symptoms, fever, coughing, nausea, vomiting, diarrhea, anemia, and neuropathy. A combination of observation, medical and dietary history, and analyses for multiple trace elements is needed to pinpoint the trace element(s) involved. Serum, plasma, and erythrocytes may be used for the evaluation of copper and zinc status, whereas only serum or plasma is recommended for selenium. Whole blood is preferred for molybdenum. When trace element levels are inconsistent with medical evaluations, a test for activity of the suspected enzyme(s) would support the differential diagnosis. Furthermore, it is important to differentiate whether trace element deficiency or toxicity is the primary cause of the disorder, or is secondary to other underlying diseases. Only successful treatment of the primary disorder will lead to complete recovery. In the event of sample contamination during collection or analysis, the physician may be misled by falsely elevated results. Royal blue top evacuated tubes containing negligibly low concentrations of the trace element or acid-washed plastic sterilized syringes should be used for blood, serum, or plasma collection. Powdered gloves must be avoided. When possible, mineral supplements are not to be administered to the patient for a minimum of 3 days prior to sample collection. Serum and plasma specimens are to be transported in acid-washed polypropylene and polyethylene tubes. Analysis is performed in a controlled environment to minimize or eliminate contamination. During analysis, all laboratory wares should be acid-washed for decontamination. A detailed description of these precautions may be found in reviews by Aitio and Jarvisalo and by Chan and Gerson. Copper and zinc analysis on serum and plasma are commonly performed by flame atomic absorption spectrometry, inductively coupled plasma-atomic emission spectrometry, and inductively coupled plasma-mass spectrometry. Serum and plasma selenium levels are determined by graphite furnace atomic absorption with Zeeman background correction and neutron activation analysis. Molybdenum levels are best determined by neutron activation and highly sensitive inductively coupled plasma-mass spectrometry. The reader is referred to reviews by Tsalev and Jarvis.

Adolescent↗
PubMed1998

Drug testing as part of the war on drugs.

Drug testing programs in the United States may broadly be classified as mandatory (such as under DOT or Nuclear Regulatory Commission regulation) or nonmandatory. In the first group, a regulated employer is required by federal regulations to test. In the second, the employer chooses to test for reasons other than the federal requirements. It always was intended that the federal program would be the model for testing by private employers, and that mandatory testing would be extended to private employers who are regulated by various agencies of the federal government. This has happened. In addition, private employers who are not required to test under federal authority have instituted employee drug testing programs closely modeled on the federal program. Whether mandated or not, a well-designed and implemented drug testing program is a valuable tool in the effort to fight drugs in the workplace.

Government↗
PubMed1997

pH dependence of antibody/lysozyme complexation.

Association between proteins often depends on the pH and ionic strength conditions of the medium in which it takes place. This is especially true in complexation involving titratable residues at the complex interface. Continuum electrostatics methods were used to calculate the pH-dependent energetics of association of hen egg lysozyme with two closely related monoclonal antibodies raised against it and the association of these antibodies against an avian species variant. A detailed analysis of the energetic contributions reveals that even though the hallmark of association in the two complexes is the presence of conserved charged-residue interactions, the environment of these interactions significantly influences the titration behavior and concomitantly the energetics. The contributing factors include minor structural rearrangements, buried interfacial area, dielectric environment of the key titratable residues, and geometry of the residue dispositions. Modeled structures of several mutant complexes were also studied so as to further delineate the contribution of individual factors to the titration behavior.

Amino Acid Sequence↗
PubMed1997

Expression, stability, and membrane integration of truncation mutants of bovine rhodopsin.

Premature termination of protein synthesis by nonsense mutations is at the molecular origin of a number of inherited disorders in the family of G protein-coupled seven-helix receptor proteins. To understand how such truncated polypeptides are processed by the cell, we have carried out COS-1 cell expression studies of mutants of bovine rhodopsin truncated at the first 1, 1.5, 2, 3, or 5 transmembrane segments (TMS) of the seven present in wild-type opsin. Our experiments show that successful completion of different stages in the cellular processing of the protein [membrane insertion, N-linked glycosylation, stability to proteolytic degradation, and transport from the endoplasmic reticulum (ER) membrane] requires progressively longer lengths of the polypeptide chain. Thus, none of the truncations affected the ability of the polypeptides to be integral membrane proteins. C-terminal truncations that generated polypeptides with fewer than two TMS resulted in misorientation and prevented glycosylation at the N terminus, whereas truncations that generated polypeptides with fewer than five TMS greatly destabilized the protein. However, all of the truncations prevented exit of the polypeptide from the ER. We conclude that during the biogenesis of rhodopsin, proper integration into the ER membrane occurs only after the synthesis of at least two TMS is completed. Synthesis of the next three TMS confers a gradual increase in stability, whereas the presence of more than five TMS is necessary for exit from the ER.

Amino Acid Sequence↗
PubMed1997

Reducing the flexibility of retinal restores a wild-type-like photocycle in bacteriorhodopsin mutants defective in protein-retinal coupling.

The thermal re-isomerization of retinal from the 13-cis to the all-trans state is a key step in the final stages of the photocycle of the light-driven proton pump, bacteriorhodopsin. This step is greatly slowed upon replacement of Leu-93, a residue in van der Waals contact with retinal. The most likely role of this key interaction is that it restricts the flexibility of retinal. To test this hypothesis, we have exchanged native retinal in Leu-93 mutants with bridged retinal analogs that render retinal less flexible by restricting free rotation around either the C10-C11 (9,11-bridged retinal) or C12-C13 (11,13-bridged retinal) single bonds. The effect of the analogs on the photocycle was then determined spectroscopically by taking advantage of the previous finding that the decay of the O intermediate in the Leu-93 mutants provides a convenient marker for retinal re-isomerization. Time-resolved spectroscopic studies showed that both retinal analogs resulted in a dramatic acceleration of the photocycling time by increasing the rate of decay of the O intermediate. In particular, exchange of native retinal in the Leu-93 --> Ala mutant with the 9,11-bridged retinal resulted in an acceleration of the decay of the O intermediate to a rate similar to that seen in wild-type bacteriorhodopsin. We conclude that the protein-induced restriction of conformational flexibility in retinal is a key structural requirement for efficient protein-retinal coupling in the bacteriorhodopsin photocycle.

Alanine↗
PubMed1997

Electron diffraction studies of light-induced conformational changes in the Leu-93 --> Ala bacteriorhodopsin mutant.

We previously have presented evidence for prominent structural changes in helices F and G of bacteriorhodopsin during the photocycle. These changes were determined by carrying out electron diffraction analysis of illuminated two-dimensional crystals of wild-type bacteriorhodopsin or the Asp-96 --> Gly mutant that were trapped at a stage in the photocycle after light-driven proton release, but preceding proton uptake from the aqueous medium. Here, we report structural analysis of the long-lived O intermediate observed in the photocycle of the Leu-93 --> Ala mutant, which accumulates after the release and uptake of protons, but before the reisomerization of retinal to its initial all-trans state. Projection Fourier difference maps show that upon illumination of the Leu-93 --> Ala mutant, significant structural changes occur in the vicinity of helices C, B, and G, and to a lesser extent near helix F. Our results suggest that (i) all four helices that line the proton channel (B, C, F, and G) participate in structural changes during the late stages of the photocycle, and (ii) completion of the photocycle involves significant conformational changes in addition to those that are associated with steps in proton transport.

Bacteriorhodopsins↗
PubMed1997

Studies of Rh1 metarhodopsin stabilization in wild-type Drosophila and in mutants lacking one or both arrestins.

We have used Drosophila mutants which are deficient in one or both of the arrestins present in photoreceptor cells to critically test the requirements for arrestin in the stabilization of Rh1 metarhodopsin under in vitro and in vivo conditions. Heads from flies illuminated with blue light were homogenized to obtain membranes or micellar extracts, and the amount of metarhodopsin present was quantitated by spectroscopic methods. Compared to wild-type, approximately 64% Rh1 metarhodopsin was recovered in flies deficient in arrestin-1 (arr1(1) mutant), approximately 38% in flies deficient in arrestin-2 (arr2(3) mutant), and approximately 6% in flies deficient in both arrestin-1 and arrestin-2 (arr1(1), arr2(3) double mutant). In contrast, no decrease was observed in the amounts of Rh1 metarhodopsin recovered from illuminated flies which were deficient either in the eye-specific phosphatase (rdgC mutant) or in the eye-specific phospholipase C (norpA(H24) and norpA(H52) mutants). Further, reconstitution experiments in total head homogenates showed that metarhodopsin produced in the arr1(1), arr2(3) double mutant could be stabilized upon the addition of exogenous arrestin-2. These studies provide definitive evidence that arrestin binding stabilizes Rh1 metarhodopsin under in vitro conditions. To test whether arrestin was also required to stabilize metarhodopsin in intact photoreceptor cells, metarhodopsin was generated in arr1(1), arr2(3) double mutant flies by in vivo illumination, and after a wait period of 20 min, converted back into rhodopsin by further illumination with red light. Quantitation of the regenerated rhodopsin in extracts from Drosophila heads showed no significant change in the level of rhodopsin recovered by this illumination protocol. Together, these experiments demonstrate that in disrupted photoreceptor cells, metarhodopsin is not stabilized unless arrestin is present, but in intact photoreceptor cells, significant metarhodopsin stabilization occurs even in the absence of bound arrestin.

Animals↗
PubMed1997

Computation of molecular electrostatics with boundary element methods.

In continuum approaches to molecular electrostatics, the boundary element method (BEM) can provide accurate solutions to the Poisson-Boltzmann equation. However, the numerical aspects of this method pose significant problems. We describe our approach, applying an alpha shape-based method to generate a high-quality mesh, which represents the shape and topology of the molecule precisely. We also describe an analytical method for mapping points from the planar mesh to their exact locations on the surface of the molecule. We demonstrate that derivative boundary integral formulation has numerical advantages over the nonderivative formulation: the well-conditioned influence matrix can be maintained without deterioration of the condition number when the number of the mesh elements scales up. Singular integrand kernels are characteristics of the BEM. Their accurate integration is an important issue. We describe variable transformations that allow accurate numerical integration. The latter is the only plausible integral evaluation method when using curve-shaped boundary elements.

Biophysical Phenomena↗
PubMed1997

Expression of bacteriorhodopsin in Sf9 and COS-1 cells.

We report studies on the expression of the archaebacterial membrane protein bacteriorhodopsin in Sf9 insect cells and in COS-1 mammalian cells. In both cell systems, the apoprotein bacterio-opsin was expressed at levels of approximately 1 microgram/10(6) cells. Immunofluorescence studies showed that the expressed protein was accumulated in the endoplasmic reticulum. However, upon addition of all-trans retinal to membranes isolated from either Sf9 or COS-1 cells expressing bacterio-opsin, the characteristic bacteriorhodopsin chromophore (lambda max at approximately 560 nm) was rapidly generated. This is in contrast to bacterio-opsin expressed in E. coli, which cannot be functionally reconstituted with retinal unless it is first denatured, and then renatured in vitro. These studies demonstrate that the bacterio-opsin expressed is correctly folded and show that localization of a heterologously expressed membrane protein in the endoplasmic reticulum does not necessarily imply that it is misfolded.

Animals↗