Biomedical subjects

V Jayaraman

Publications and source records attributed to V Jayaraman.

At least 19 recordsLinked to original sources

PubMed2001

Stereochemistry of quinoxaline antagonist binding to a glutamate receptor investigated by Fourier transform infrared spectroscopy.

The stereochemistry of the interactions between quinoxaline antagonists and the ligand-binding domain of the glutamate receptor 4 (GluR4) have been investigated by probing their vibrational modes using Fourier transform infrared spectroscopy. In solution, the electron-withdrawing nitro groups of both compounds establish a resonance equilibrium that appears to stabilize the keto form of one of the cyclic amide carbonyl bonds. Changes in the 6,7-dinitro-2,3-dihydroxyquinoxaline vibrational spectra on binding to the glutamate receptor, interpreted within the framework of a published crystal structure, illuminate the stereochemistry of the interaction and suggest that the binding site imposes a more polarized electronic bonding configuration on this antagonist. Similar spectral changes are observed for 6-cyano-7-dinitro-2,3-dihydroxyquinoxaline, confirming that its interactions with the binding site are highly similar to those of 6,7-dinitro-2,3-dihydroxyquinoxaline and leading to a model of the 6-cyano-7-dinitro-2,3-dihydroxyquinoxaline-S1S2 complex, for which no crystal structure is available. Conformational changes within the GluR ligand binding domain were also monitored. Compared with the previously reported spectral changes seen on binding of the agonist glutamate, only a relatively small change is detected on antagonist binding. This correlation between the functional effects of different classes of ligand and the magnitude of the spectroscopic changes they induce suggests that the spectral data reflect physiologically relevant conformational processes.

Excitatory Amino Acid Antagonists↗

PubMed2001

Effect of trypsin-chymotrypsin (Chymoral Forte D.S.) preparation on the modulation of cytokine levels in burn patients.

The present study was carried out in burn patients administered with Trypsin-Chymotrypsin (Chymoral Forte D.S.) preparation to observe if the acute-phase protein levels in the serum are modulated through the synthesis of IL-1beta and IL-6 and if the severity of the inflammatory phase could be regulated. The effects of Trypsin-Chymotrypsin preparation on the modulation of cytokine levels particularly, IL-6 and IL-1beta were analyzed in serum samples of 15 burn patients and compared with untreated controls. Significant differences in cytokine levels (P<0.05) were observed in untreated burn patients and Trypsin-Chymotrypsin preparation treated patients. There were significant variations in serum IL-6 and IL-1beta on the day of admission and post burn day 10 in treated as well as untreated burn patients. Patients with a higher percentage of total body surface area and sepsis showed a significant increase in IL-1beta and IL-6 in the serum. An increase in serum levels of both cytokines was observed on post burn day 1 and a significant decrease was observed in Trypsin-Chymotrypsin preparation treated patients on days 7 and 10. The possible role of the inflammatory cytokines in the pathophysiology of burns is discussed.

Adolescent↗

PubMed2000

Mechanism-based discovery of ligands that counteract inhibition of the nicotinic acetylcholine receptor by cocaine and MK-801.

Nicotinic acetylcholine receptors (AChR) belong to a family of proteins that form ligand-gated transmembrane ion channels. They are involved in the fast transmission of signals between cells and the control of intercellular communication in the nervous system. A variety of therapeutic agents and abused drugs, including cocaine, inhibit the AChR and monoamine transporters and interfere with nervous system function. Here we describe a mechanism-based approach to prevent this inhibition. We had previously developed presteady-state kinetic (transient kinetic) techniques, with microsecond-to-millisecond time resolutions, for investigations of reactions on cell surfaces that allow one to determine the effects of inhibitors not only on the channel-opening probability but also on the opening and closing rates of the AChR channel. The transient kinetic measurements led to two predictions. (i) Ligands that bind to a regulatory site on the closed-channel conformation of the AChR with higher affinity than to the site on the open-channel form shift the equilibrium toward the closed-channel form, thereby inhibiting the receptor. (ii) Ligands that bind to a regulatory site with an affinity for the open conformation equal to or higher than their affinity for the closed conformations are expected not to inhibit the receptor and to displace inhibitors. The identification of such ligands in a combinatorial library of RNA ligands is reported. The implication of this approach to other protein-mediated reactions in which an inhibitor changes the equilibrium between active and inactive conformations is discussed.

Animals↗

PubMed2000

Ligand--protein interactions in the glutamate receptor.

Fourier transform infrared spectroscopy was used to investigate ligand-protein interactions in the ligand-binding domain of the GluR4 glutamate receptor subunit. Glutamate binding induces more extensive secondary structural changes in the ligand-binding domain than does kainate binding. Glutamate also alters the hydrogen bonding strength of the single free cysteine side chain in the domain, while kainate does not. On the other hand, the interaction of a binding site arginine residue with kainate appears to be stronger than that with glutamate. These results identify chemical and structural differences that may explain the different functional characteristics of the two agonists acting on ionotropic glutamate receptors. In doing so, they complement and extend recent crystallographic structures of the ligand-binding domain.

Binding Sites↗

PubMed2000

Fourier transform infrared spectroscopic characterization of a photolabile precursor of glutamate.

Recently, it has been demonstrated that Fourier transform infrared spectroscopy (FTIR) detects conformational changes in the glutamate receptor ligand-binding domain that are associated with agonist binding. Combined with flash photolysis, this observation offers the prospect of following conformational changes at individual protein and agonist moieties in parallel and with high temporal resolution. Here, we demonstrate that gamma(alpha-carboxy-2-nitrobenzyl) glutamate (caged glutamate) does not interact with the protein, and that following photolysis with UV light the FTIR difference spectrum indicated changes in the protein tertiary and secondary interactions. These changes were similar to those observed for the protein upon addition of free glutamate. Thus, caged glutamate and its photolysis by-products are inert in this system, whereas the released glutamate exhibits full activity. Difference spectra of caged glutamate and of reaction analogs permitted identification of and correction for FTIR signals arising from the photolytic reaction and confirmed that its products are indeed glutamate and 2-nitrosophenyl glyoxalic acid.

Animals↗

PubMed1999

How fast does the gamma-aminobutyric acid receptor channel open? Kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.

The gamma-aminobuytric acid(A) (GABA(A)) receptor is a membrane-bound protein that mediates signal transmission between neurons through formation of chloride ion channels. GABA is the activating ligand, which upon binding to the receptor triggers channel opening in the microsecond time domain and reversible desensitization of the receptor in the millisecond time region. We have investigated the channel-opening mechanism for this receptor in rat hippocampal neurons before the protein desensitizes by using a rapid flow method (cell-flow) with a 10 ms time resolution and a laser-pulse photolysis technique with a approximately 30 micros time resolution to determine the rate and equilibrium constants for channel opening and closing. Two different forms of the receptor, namely, a rapidly and a slowly desensitizing form, exist in the rat hippocampal cells and are characterized by their different rates for desensitization. At 250 microM GABA the rate constant for desensitization was 2.3 +/- 0.4 s(-)(1) for the rapidly desensitizing form and 0.4 +/- 0.1 s(-)(1) for the slowly desensitizing form. The dissociation constant of GABA from the site controlling channel opening was 100 +/- 40 microM for the rapidly desensitizing form and 120 +/- 60 microM for the slowly desensitizing form. The rate constants for channel closing did not differ significantly for the two forms, 85 +/- 20 s(-)(1) for the rapidly desensitizing and 100 +/- 60 s(-)(1) for the slowly desensitizing form. However, the channel-opening rate constant differed by a factor of 3, 1840 +/- 160 s(-)(1) for the rapidly desensitizing and 6700 +/- 330 s(-)(1) for the slowly desensitizing form. This difference in the rate constant for channel opening for the two forms, determined by the laser-pulse photolysis technique, is reflected as a shift in the channel-opening equilibrium constant, which is 7 +/- 5 and 20 +/- 15 for the rapidly and slowly desensitizing forms respectively, determined by the cell-flow method. These constants, together with the concentration of GABA and the concentration of receptor sites in the membrane, determine the number of channels that open as a function of GABA concentration, and the rate at which they open and close. These constants play an important role in determining the rate of the transmembrane ion flux and, therefore, the receptor-controlled changes in transmembrane voltage that trigger signal transmission.

Animals↗

PubMed1999

Inhibition of nicotinic acetylcholine receptor by philanthotoxin-343: kinetic investigations in the microsecond time region using a laser-pulse photolysis technique.

The mechanism of inhibition of a nicotinic acetylcholine receptor (nAChR) in BC(3)H1 muscle cells by philanthotoxin-343 (PhTX-343), a synthetic analogue of philanthotoxin-433, a wasp toxin, was investigated using a laser-pulse photolysis technique with microsecond time resolution and in a carbamoylcholine concentration range of 20-100 microM and PhTX-343 concentration range of 0-200 microM. The rate constant for nAChR channel opening determined by the chemical kinetic techniques decreased with increasing PhTX-343 concentration, whereas there was no significant effect on the rate constant for channel closing. The resulting decrease in the channel-opening equilibrium constant accounted quantitatively for the inhibition of the receptor by the toxin. Single-channel current measurements suggest an additional step in which the open channel:inhibitor complex isomerizes to a nonconducting receptor form. Cell-flow experiments with a time resolution of 10 ms indicate that this isomerization step is only important at very high inhibitor concentrations. The inhibitor binds to the open-channel receptor form, with an affinity that is at least 5 times smaller than that for the closed-channel form. This indicates that receptor inhibition mainly involves the binding of PhTX-343 to the closed-channel form of the receptor. PhTX-343, and an analogue of this polyamine, had no effect when applied to the inside of the cell membrane. However, there was significant inhibition of the nAChR when these compounds were applied to the outside of the cell membrane, indicating an extracellular site for inhibition. Furthermore, increasing the transmembrane potential results in a decrease in the ability of PhTX-343 to inhibit the receptor. This observation is related to the voltage dependence of the effect of PhTX-343 on the rate constant for nAChR channel opening with increasing transmembrane voltage (-60 to 50 mV). This suggests that the voltage dependence of inhibition mainly reflects the effect of transmembrane voltage on the rate constant of channel opening and, therefore, the channel-opening equilibrium constant. PhTX-343 competes with cocaine and procaine for its binding site. The finding that this toxin, which binds to a common inhibitory site with compounds such as cocaine, exerts its effect by decreasing the channel-opening equilibrium constant suggests an approach for the development of therapeutic agents. A compound that binds to this regulatory site but does not affect the channel-opening equilibrium constant may be developed. Such a compound can displace an abused drug such as cocaine and thereby alleviate the toxic effect of this compound on the organism.

Animals↗

PubMed1999

Synthesis and photochemistry of a photolabile precursor of N-methyl-D-aspartate (NMDA) that is photolyzed in the microsecond time region and is suitable for chemical kinetic investigations of the NMDA receptor.

The amino acid L-glutamate is a major neurotransmitter at excitatory synapses within the central nervous system. Neuronal responses to glutamate are mediated by at least three receptor types, one of which is the NMDA subtype, named for its specific ligand N-methyl-D-aspartic acid. Neurotransmitter receptors are transmembrane proteins that can form ion channels upon binding a specific ligand and are involved in many physiological activities of the brain and in some neurological disorders. Elucidating the mechanisms of the formation of transmembrane receptor-channels and of receptor regulation and inhibition is necessary for understanding nervous system function and for designing potential therapeutic agents. This has been hampered by the lack of rapid reaction techniques suitable for investigating protein-mediated reactions on cell surfaces. Recently a laser-pulse photolysis technique was developed to study the chemical reactions of channel-forming receptor proteins in the microsecond-to-millisecond time region. To apply the technique to NMDA1 receptors a photolabile NMDA precursor (beta-DNB NMDA) was synthesized. In this precursor the side chain carboxylate was protected as a photosensitive 2,2'-dinitrobenzhydryl ester. Photolysis with 308 nm laser light generated free NMDA with a time constant of 4.2 +/- 0.1 microseconds at pH 7 and a photolysis quantum yield of 0.18 +/- 0.05. In rat hippocampal neurons the beta-DNB NMDA (250 microM) neither activated endogenously expressed receptors nor potentiated or inhibited the NMDA response. Equilibration of hippocampal neurons in the whole-cell current recording mode with 250 microM caged precursor followed by a pulse of 333 nm laser light resulted in a rapid current rise with a rate constant of 100 s-1 due to opening of NMDA-activated receptor-channels. The caged NMDA precursor described here now makes it possible to investigate the mechanism of NMDA receptors in the micro- to millisecond time region.

Animals↗

PubMed1999

Physicochemical properties of extracellular matrix proteins in post-burn human granulation tissue.

Wound healing is a finely controlled biological process involving a series of complex cellular interactions. Following inflammation, the wound bed matrix is gradually replaced by granulation tissue followed by the long slow process where collagen accumulates and restores tensile strength. The studies revealed that human granulation tissue varied in many aspects in comparison with normal skin. In granulation tissue the molecular organization of collagen showed an increased amount of type III collagen resembling embryonic tissue. The presence of type V collagen with three distinct chains was the characteristic feature of granulation tissue. The physicochemical properties of collagen extracted from granulation tissue showed the influence of proteoglycans during collagen aggregation and these proteoglycans from the major non-collagenous proteins during the proliferative phase of healing.

Burns↗

PubMed1999

Carbohydrate analysis of bradyrhizobial (NC92) lipopolysaccharides by high performance-anion exchange chromatography with pulsed amperometric detection.

Composition analysis of monosaccharides of Sepharose 4B purified NC 92 LPS and the polysaccharides fractions from Sephadex G-50 chromatography was performed by high performance anion exchange chromatography using pulsed amperometric detection. Rhamnose, mannose, galactose and glucose are present in a substantial amount in the purified LPS (Pk I). High molecular weight purified polysaccharides (PS I) obtained after sephadex gel filtration of the purified LPS (Pk I) acid hydrolysate showed an increase in glucose:galactose ratio. This indicates the presence of the peanut root lectin (PRA II) specific sugar in higher proportion on the O-antigen part of the LPS molecule, which may aid in the critical recognition reaction.

Bradyrhizobium↗

PubMed1998

Channel-opening mechanism of a kainate-activated glutamate receptor: kinetic investigations using a laser-pulse photolysis technique.

Kainate is an excitatory neurotransmitter that binds to the kainate and AMPA receptor subtypes of the glutamate receptor and triggers the formation of cation permeable transmembrane channels in these receptors. In the present report the channel-opening mechanism of the AMPA receptors by kainate has been determined in rat hippocampal neurons using two different kinetic methods, namely, the rapid-flow method (cell-flow) with a 10 ms time resolution and a laser-pulse photolysis technique with a approximately 65 microseconds time resolution. The whole-cell currents induced by kainate, using the cell-flow method, are nondesensitizing and inhibited significantly by CNQX and hence pertain to activation of the AMPA receptors and not the kainate receptors. The cell-flow measurements were used to evaluate the constants pertaining to the minimum mechanism that could account for the concentration of the receptor in the open-channel form over a 500-fold range of kainate concentration. These constants, namely, the intrinsic dissociation constant of kainate from the AMPA receptor and the channel-opening equilibrium constant, were determined to be 140 +/- 30 microM and 8 +/- 2, respectively. On the other hand, the kinetics of the steps leading to channel opening was evaluated using the laser-pulse photolysis techniques. In this technique whole-cell currents were obtained by releasing kainate in the submillisecond time scale near the cell by photolysis of N-(alpha-carboxy-2-nitrobenzyl) kainate. The concentration of the released kainate was calculated by comparing the whole-cell currents obtained from the laser-pulse photolysis experiments with the whole currents obtained with 100 microM kainate on the same cell using cell-flow measurements. The rate constants for channel opening and closing were then determined from the observed rate constants for the current rise obtained as a function of kainate concentration. These rates were 5000 +/- 2000 and 640 +/- 30 s-1, respectively. The rate and equilibrium constants obtained in the present report allow an evaluation of the fraction of the receptors in the open-channel form as a function of time and kainate concentration, hence providing insight into the role of kainate in neuronal signal transmission.

Animals↗

PubMed1998

Interaction of peanut root lectin (PRA II) with rhizobial lipopolysaccharides.

Sugar specific binding of peanut root lectin (PRA II) to peanut specific bradyrhizobial lipopolysaccharides (LPS) was demonstrated by gel retardation assay and lectin based ELISA. Sephadex G-50 gel purified high molecular weight polysaccharides from NC 92 LPS bind PRA II most efficiently. Binding of NC 92 LPS only to PRA II and not to PNA, SBA and PSL by Western blot analysis suggests that this lectin-LPS interaction is tissue as well as species specific.

Enzyme-Linked Immunosorbent Assay↗

PubMed1998

The efficacy of trypsin: chymotrypsin preparation in the reduction of oxidative damage during burn injury.

This study was mainly aimed to investigate the efficacy of trypsin:chymotrypsin to elicit anti-oxidant properties. In our earlier studies it was observed that the enzyme preparation exhibited an anti-inflammatory action as there was a remarkable reduction in oedema formation and tissue destruction. This led to further study on the amount of lipid peroxidation products formed and the levels of enzymatic and non-enzymatic anti-oxidants and relative trace element contents of copper, selenium, iron and zinc during administration of the enzyme preparation. Decreased formation of lipid peroxidation products was observed in treated group in comparison with the untreated group. Higher levels of enzymatic anti-oxidants mainly super oxide dismutase, catalase, glutathione peroxidase and glutathione-s-transferase and non-enzymatic antioxidant namely ceruloplasmin persisted for a longer period of time in the treated group than in the untreated group. No statistical significance was observed in non-enzymatic antioxidants viz. ascorbic acid and tocopherol levels in both the groups. Increased serum copper and selenium levels in the treated group could be related to higher levels of the ceruloplasmin and glutathione peroxidase observed in the treated group. The above studies support the finding that treatment with the enzyme preparation reduced tissue destruction leading to decreased formation of free radicals and subsequent effective scavenging of free radicals by the higher levels of enzymatic and non-enzymatic anti-oxidants.

Adult↗

PubMed1998

Partial characterization of the lipopolysaccharides of peanut specific Bradyrhizobium strains.

Hexoses, heptose, hexuronic acids, 2-keto-3-deoxyoctonic acid (Kdo) contents of the lipopolysaccharides (LPS) isolated from peanut (A. hypogaea) specific Bradyrhizobium strains NC 92, IGR 92, IGR 40 and IGR 6 were found to vary considerably. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed a heterogeneous LPS population and distinct heterogeneity was observed in NC and IGR strains. Sepharose gel filtration chromatography removed the hexose contaminants from NC 92 LPS. Polysaccharides obtained by mild acid hydrolysis of Sepharose purified NC 92 LPS were further purified on Sephadex G-50 gel filtration. Immunoblot analysis indicated cross reactivity amongst the peanut specific Bradyrhizohium strains but not with B. japonicum(SB 16). Antibodies raised against native NC 92 LPS showed higher binding towards the Sephadex purified polysaccharides suggesting the antigenic property of polysaccharide moiety in LPS molecule.

Arachis↗

PubMed1997

Action of trypsin:chymotrypsin (Chymoral forte DS) preparation on acute-phase proteins following burn injury in humans.

A study was carried out to investigate the efficacy of trypsin: chymotrypsin (Chymoral forte DS) preparation on burn patients by analysing the changes taking place in serum acute-phase proteins. Serum proteins were analysed qualitatively and quantitatively for both control and enzyme-treated groups by the methods of Western Blot, ELISA and Turbidimetric assays. Furthermore, the trypsin inhibitory capacity (TIC) of the sera was also determined. Significant differences were observed between a control group of patients and a parallel group treated with trypsin: chymotrypsin preparation. During the first phase of burn wounds an initial rise was seen in C-reactive protein, alpha 1-antitrypsin, alpha 2-macroglobulin and TIC in both the groups. In the following days, enzyme preparation inhibited the rise in C-reactive protein titres and enhanced the rise in alpha 1-antitrypsin, alpha 2-macroglobulin and TIC. The above studies clearly indicate that the changes in serum acute-phase proteins between the control and treated groups reflect the anti-inflammatory activity and hence the therapeutic efficacy of Chymoral forte DS.

Acute-Phase Proteins↗

PubMed1997

Management of partial-thickness burn wounds by amniotic membrane: a cost-effective treatment in developing countries.

Burns incidence in our country has been on the increase over the past two decades. Cost-effective management of burn wounds has become the prerogative as our annual budget for burn care is limited. We have used human amniotic membrane procured from HbSAg, HIV-seronegative mothers undergoing caesarean section as a temporary biological dressing on superficial and deep partial-thickness burns. The advantages of amniotic membrane cover are reduction in pain, early drying of the wound and epithelialization. This type of wound management has been used in 350 cases. It has reduced the number of days stay in hospital and the bulky dressings that are conventional. Considering the patient acceptability, reduced hospital stay and reduction in cost, we find that treatment of superficial and deep partial-thickness burns with amniotic membrane is ideal for a developing country.

Adult↗

PubMed1995

Hemoglobin allostery: resonance Raman spectroscopy of kinetic intermediates.

The end states, R and T, of the allosteric transition in hemoglobin (Hb) are structurally well characterized, but there is little information on intermediate structures along the allosteric pathway. These intermediates were examined by means of time-resolved resonance Raman spectroscopy in the nanosecond-to-microsecond interval after HbCO photolysis. Complementary spectra of the heme group and of the tyrosine and tryptophan residues were recorded during laser excitation at 436 and 230 nanometers. These spectra reveal a sequence of interleaved tertiary and quaternary motions during the photocycle, motions involving the proximal and distal helices, and the alpha 1 beta 2 subunit interface. This sequence leads to a modified form of the T state, in which the alpha 1 beta 2 interface is deformed as a result of two carbon monoxide molecules binding to the same dimer within the tetramer.

Allosteric Regulation↗

PubMed1995

FT-IR difference spectroscopy of hemoglobins A and Kempsey: evidence that a key quaternary interaction induces protonation of Asp beta 99.

Fourier transform infrared difference spectra are reported for the CO adduct of human hemoglobin versus deoxyHb, in H2O and D2O. In addition to the well-known CO stretching and S-H(D) stretching bands, the difference spectra reveal numerous bands in the 1200-1700 cm-1 region, a number of which are assigned. Several amide modes are identified via their frequencies and D2O sensitivities. Bands arising from histidine protonation have also been found via comparison of the difference spectra at different pH(D) values, with the aid of aqueous histidine spectra. Of particular interest is the observation of a negative band at 1697 cm-1, which is assigned to the C = O stretch of carboxylic acid. This carboxylic acid is tentatively identified as the side chain of Asp beta 99, because it is missing in the difference spectrum of Hb Kempsey, a mutant in which Asp beta 99 is replaced by Asn. Asp beta 99 forms a critical contact with Tyr alpha 42 across the alpha 1 beta 2 interface in deoxyHb, which is broken upon ligation. Protonation of Asp beta 99 in deoxyHb is consistent with UV resonance Raman evidence that Tyr alpha 42 is the acceptor rather than the donor of the quaternary H-bond.

Adult↗