Biomedical subjects

B Gopalakrishnan

Publications and source records attributed to B Gopalakrishnan.

At least 19 recordsLinked to original sources

PubMed2005

3D-QSAR studies on antitubercular thymidine monophosphate kinase inhibitors based on different alignment methods.

Three dimensional quantitative structure-activity relationship (3D-QSAR) studies were carried out on deoxythymidine monophosphate (dTMP) derivatives inhibiting thymidine monophosphate kinase (TMPK) in Mycobacterium tuberculosis. Molecular field analysis (MFA) models with three different alignment techniques, namely, least squares, pharmacophore based and receptor based methods were developed. Receptor based MFA model showed better results when compared with least squares and pharmacophore based models. The results help us to understand the nature of substituents required for activity and thereby provide guidelines to design novel and potent inhibitors as antitubercular agents.

Antitubercular Agents↗

PubMed2003

Design of EGFR kinase inhibitors: a ligand-based approach and its confirmation with structure-based studies.

Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were developed for 100 anilinoquinazolines, inhibiting epidermal growth factor receptor (EGFR) kinase. The studies included molecular field analysis (MFA) and receptor surface analysis (RSA). The cross-validated r2 (r2cv) values are 0.81 and 0.79 for MFA and RSA, respectively. The predictive ability of these models was validated by 28 test set molecules. The results of the best QSAR model were further compared with structure-based investigations using docking studies with the crystal structure of EGFR kinase domain. The results helped to understand the nature of substituents at the 6- and 7-positions, thereby providing new guidelines for the design of novel inhibitors.

Aniline Compounds↗

PubMed2003

Synthesis and cyclooxygenase-2 inhibiting property of 1,5-diarylpyrazoles with substituted benzenesulfonamide moiety as pharmacophore: Preparation of sodium salt for injectable formulation.

A series of 1,5-diarylpyrazoles having a substituted benzenesulfonamide moiety as pharmacophore was synthesized and evaluated for cyclooxygenase (COX-1/COX-2) inhibitory activities. Through SAR and molecular modeling, it was found that fluorine substitution on the benzenesulfonamide moiety along with an electron-donating group at the 4-position of the 5-aryl ring yielded selectivity as well as potency for COX-2 inhibition in vitro. Among such compounds 3-fluoro-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1-benzenesulfonamide 3 displayed interesting pharmacokinetic properties along with antiinflammatory activity in vivo. Among the sodium salts tested in vivo, 10, the propionyl analogue of 3, showed excellent antiinflammatory activity and therefore represents a new lead structure for the development of injectable COX-2 specific inhibitors.

Animals↗

PubMed2003

Comparative molecular field analysis (CoMFA) of phthalazine derivatives as phosphodiesterase IV inhibitors.

A comparative molecular field analysis (CoMFA) of phthalazine class of phosphodiesterase IV (PDE IV) inhibitors has been performed to correlate their chemical structures with their observed biological activity. A statistically valid model with good correlative and predictive power is reported. The leave one out cross-validation study gave cross-validation r(2)(cv) of value 0.507 at six optimum components and conventional r(2) of value 0.98. The predictive ability of the model was tested by predicting the seven molecules belonging to the test set giving predictive correlation coefficient of 0.59. This model is potentially helpful in the design of novel and more potent PDE IV inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

3D-QSAR studies on thieno[3,2-d]pyrimidines as phosphodiesterase IV inhibitors.

Cyclic nucleotide phosphodiesterase IV (PDE IV) inhibitors find utility in asthma and Chronic Obstructive Pulmonary Disease (COPD) therapy. A series of 29 thieno[3,2-d]pyrimidines with affinity for PDE IV was subjected to three dimensional quantitative structure activity relationship (3D-QSAR) studies using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA). Both CoMFA and CoMSIA provided statistically valid models with good correlative and predictive power. The incorporation of hydrophobic, hydrogen bond donor and hydrogen bond acceptor fields showed insignificant improvement in CoMSIA model. The 3D-QSAR models provide information for predicting the affinity of related compounds and designing more potent inhibitors.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2002

Computer-aided design of selective COX-2 inhibitors: comparative molecular field analysis, comparative molecular similarity indices analysis, and docking studies of some 1,2-diarylimidazole derivatives.

Comparative molecular field analysis and comparative molecular similarity indices analysis were performed on 114 analogues of 1,2-diarylimidazole to optimize their cyclooxygenase-2 (COX-2) selective antiinflammatory activities. These studies produced models with high correlation coefficients and good predictive abilities. Docking studies were also carried out wherein these analogues were docked into the active sites of both COX-1 and COX-2 to analyze the receptor ligand interactions that confer selectivity for COX-2. The most active molecule in the series (53) adopts an orientation similar to that of SC-558 (4-[5-(4-bromophenyl)-3-trifluoromethyl-1H-1-pyrozolyl]-1-benzenesulfonamide) inside the COX-2 active site while the least active molecule (101) optimizes in a different orientation. In the active site, there are some strong hydrogen-bonding interactions observed between residues His90, Arg513, and Phe518 and the ligands. Additionally, a correlation of the quantitative structure-activity relationship data and the docking results is found to validate each other and suggests the importance of the binding step in overall drug action.

Binding Sites↗

PubMed2000

The zinc finger protein schnurri acts as a Smad partner in mediating the transcriptional response to decapentaplegic.

In Drosophila, a BMP-related ligand Decapentaplegic (Dpp) is essential for cell fate specification during embryogenesis and in imaginal disc development. Dpp signaling culminates in the phosphorylation and nuclear translocation of Mothers against dpp (Mad), a receptor-specific Smad that can bind DNA and regulate the transcription of Dpp-responsive genes. Genetic analysis has implicated Schnurri (Shn), a zinc finger protein that shares homology with mammalian transcription factors, in the Dpp signal transduction pathway. However, a direct role for Shn in regulating the transcriptional response to Dpp has not been demonstrated. In this study we show that Shn acts as a DNA-binding Mad cofactor in the nuclear response to Dpp. Shn can bind DNA in a sequence-specific manner and recognizes sites within a well-characterized Dpp-responsive promoter element, the B enhancer of the Ultrabithorax (Ubx) gene. The Shn-binding sites are relevant for in vivo expression, since mutations in these sites affect the ability of the enhancer to respond to Dpp. Furthermore we find that Shn and Mad can interact directly through discrete domains. To examine the relative contribution of the two proteins in the regulation of endogenous Dpp target genes we developed a cell culture assay and show that Shn and Mad act synergistically to induce transcription. Our results suggest that cooperative interactions between these two transcription factors could play an important role in the regulation of Dpp target genes. This is the first evidence that Dpp/BMP signaling in flies requires the direct interaction of Mad with a partner transcription factor.

Animals↗

PubMed1999

Secretion of glutathione S-transferase isoforms in the seminiferous tubular fluid, tissue distribution and sex steroid binding by rat GSTM1.

The seminiferous tubular fluid (STF) provides the microenvironment necessary for spermatogenesis in the adluminal compartment of the seminiferous tubule (ST), primarily through secretions of the Sertoli cell. Earlier studies from this laboratory demonstrated the presence of glutathione S-transferase (GST) in STF collected from adult rat testis and in the spent media of ST cultures. This study describes the cellular source, isoform composition and possible function of GSTs in the STF. The major GST isoforms present in STF in vivo share extensive N-terminal similarity with rat GSTM1 (rGSTM1), rGSTM2, rGSTM3 and rGST-Alpha. Molecular masses of rGSTM2, rGSTM3 and rGST-Alpha from liver and testis sources were similar, unlike STF-GSTM1, which was larger by 325 Da than its liver counterpart. Peptide digest analysis profiles on reverse-phase HPLC between liver and STF isoforms were identical, and N-terminal sequences of selected peptides obtained by digestion of the various isoforms were closely similar. The above results confirmed close structural similarity between liver and STF-GST isoforms. Active synthesis and secretion of GSTs by the STs were evident from recovery of radiolabelled GST from the spent media of ST cultures. Analysis of secreted GST isoforms showed that GST-Alpha was not secreted by the STs in vitro, whereas there was an induction of GST-Pi secretion. Detection of immunostainable GST-Mu in Sertoli cells in vitro and during different stages of the seminiferous epithelium in vivo, coupled with the recovery of radiolabelled GST from Sertoli cell-culture media, provided evidence for Sertoli cells as secretors of GST. In addition, STF of 'Sertoli cell only' animals showed no change in the profile of GST isoform secretion, thereby confirming Sertoli cells as prime GST secretors. Non-recovery of [35S]methionine-labelled GSTs from germ cell culture supernatants, but their presence in germ cell lysates, confirm the ability of the germ cells to synthesize, but not to release, GSTs. Functionally, STF-GSTM1 appeared to serve as a steroid-binding protein by its ability to bind to testosterone and oestradiol, two important hormones in the ST that are essential for spermatogenesis, with binding constants of <9.8x10(-7) M for testosterone and 9x10(-6) M for oestradiol respectively.

Animals↗

PubMed1998

Inhibition of sperm glutathione S-transferase leads to functional impairment due to membrane damage.

The role of glutathione S-transferase (GST) in the defense mechanisms of sperm is not known. We report here interference with normal motility, acrosome reaction and fertilizing ability of the goat sperm as a consequence of inhibition of GST activity. That these functional impairments were due to membrane changes was evident from the alteration in the lipid peroxidation status of these cells after GST inhibitor treatment. Increased reactive oxygen species production by the cell which occurred when GST activity was suppressed may be the mediator for membrane damage. The data argue for a role of GST in maintaining sperm membrane status.

Acrosome↗

PubMed1998

Studies on glutathione S-transferases important for sperm function: evidence of catalytic activity-independent functions.

Our earlier studies reported the identification of a rat testicular protein of 24 kDa with significant similarity at the N-terminus with Mu class glutathione S-transferases (GSTs). Treatment of goat sperm with antisera against this protein identified immunoreactive sites on the spermatozoa and inhibited in vitro fertilization of goat oocytes by the antibody-treated sperm. The above observations indicated the presence of GST-like molecule(s) important for fertility related events on goat spermatozoa. In this study, we report the purification of goat sperm GSTs (GSP1) which were purified by glutathione affinity chromatography and were enzymically active towards 1-chloro-2,4,-dinitrobenzene, a general GST substrate, and ethacrynic acid, a substrate for Pi class GSTs. GSP1 resolved into three major components on reverse-phase HPLC: peaks 1 and 2 with molecular masses of 26.5 kDa and peak 3 with a molecular mass of 25.5 kDa, as determined by SDS/PAGE. Multiple attempts to obtain N-terminal sequences of the first two peaks failed, indicating N-terminal block; however, they reacted to specific anti-Mu-GST antisera on Western blots and ELISA, and not to anti-Pi-GST antisera, which provides evidence for the presence of Mu-GST-reactive sites on peaks 1 and 2. The third component showed 80% N-terminal similarity with human and rat GSTP1-1 over an overlap of 15 amino acids, and reacted to anti-Pi-specific antisera in ELISA. Sperm labelled with antibodies against a 10-mer and an 11-mer peptide, designed from the N-terminal sequences of Mu and Pi class GSTs respectively, showed the presence of both Mu- and Pi-GST on goat sperm surface at distinct cellular domains. Selective inhibition of Pi class GST by the Pi-specific antisera, either at 0 h or at 3 h after initiation of sperm capacitation, leads to a reduction in fertilization rates. In contrast, the inhibition of Mu class GST by specific antisera at 0 h does not inhibit fertilization, although such treatment at 3 h after the initiation of capacitation reduces fertilization rates. The results indicate that both Pi- and Mu-GSTs are involved in fertilization, but the Mu-GST sites essential for fertilization are exposed only after 3 h of capacitation. The enzymic activity of GSP1 or live spermatozoa is not inhibited by the two antisera. The inability of the antibodies to cause such inhibition indicates that the reduction in fertilization rates and acrosome reaction caused by the antibodies is through a mechanism which does not interfere with the catalytic activity of the molecule. Therefore we established the presence of Pi and Mu class GST on goat sperm, their localization and their possible function in fertility-related events.

Animals↗

PubMed1998

Insect resistance of transgenic tobacco expressing an insect chitinase gene.

Chitinase expression in the insect gut normally occurs only during moulting, where the chitin of the peritrophic membrane is presumably degraded. Thus, insects feeding on plants that constitutively express an insect chitinase gene might be adversely affected, owing to an inappropriately timed exposure to chitinase. This hypothesis was tested by introducing a cDNA encoding a tobacco hornworm (Manduca sexta) chitinase (EC 3.2.1.14) into tobacco via Agrobacterium tumefaciens-mediated transformation. A truncated but enzymatically active chitinase was present in plants expressing the gene. Segregating progeny of high-expressing plants were compared for their ability to support growth of tobacco budworm (Heliothis virescens) larvae and for feeding damage. Both parameters were significantly reduced when budworms fed on transgenic tobacco plants expressing high levels of the chitinase gene. In contrast, hornworm larvae showed no significant growth reduction when fed on the chitinase-expressing transgenics. However, both budworm and hornworm larvae, when fed on chitinase-expressing transgenic plants coated with sublethal concentrations of a Bacillus thuringiensis toxin, were significantly stunted relative to larvae fed on toxin-treated non-transgenic controls. Foliar damage was also reduced. Plants expressing an insect chitinase gene may have agronomic potential for insect control.

Animals↗

PubMed1997

Mechanism(S) of interferon inhibitory activity in blood from patients with AIDS and patients with lupus erythematosus with vasculitis.

We have earlier reported that patients suffering from acquired immuno-deficiency syndrome (AIDS), systemic lupus erythematosus (SLE) with vasculitis, Wegner granulomatosis and certain types of late stage cancer have interferon inhibitory activity in their serum. The purpose of this study was to identify the factor(s) involved in this interferon inhibitory activity. Twenty patients with advanced AIDS, twenty patients with SLE and vasculitis and twenty normal healthy controls between ages 25-40 years were studied. In contrast to normal, healthy controls, significant interferon inhibitory activity was found in AIDS and SLE patients. This appears to be largely due to: (a) increased soluble circulating interferon alpha/beta receptors, (b) increased prostaglandin E2 levels which inhibits interferon and (c) a interferon inhibitory protein. Further understaging of the nature of interferon inhibitory activity in the patient's sera and development of anti-interferon inhibitory agents would greatly enhance interferons potential as a treatment modality.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1995

A testicular protein important for fertility has glutathione S-transferase activity and is localized extracellularly in the seminiferous tubules.

A 24-kDa protein isolated by preparative gel electrophoresis from rat testes was reported by us as an active immunogen in rats. Anti-24-kDa antibodies inhibited murine sperm-oocyte binding in vitro. Here, we show similarity at the NH2 terminus shared by this protein purified on Sephadex G-75 followed by anion exchange high performance liquid chromatography with glutathione S-transferase (GST)-mu subunits. This protein purified by glutathione affinity chromatography also demonstrated similarity to GST-mu NH2 terminus in a 30-amino-acid overlap. Both proteins showed activity toward the GST substrate 1-chloro-2,4-dinitrobenzene (Km of 33 microM and 50 microM) which was inhibited by 17 beta-estradiol 3-sulfate. Antisera against both proteins recognized liver GST-mu on Western blots and sperm acrosome of multiple species immunocytochemically. Both antisera significantly inhibited in vitro fertilization of goat oocytes by sperm preincubated with them while anti-liver GST sera did not. GST activity was localized on rat sperm, seminiferous tubular fluid, and Sertoli cells. Seminiferous tubular fluid 24-kDa protein shared similarity to the NH2 terminus of GST-mu subunits in a 20-amino-acid overlap. Time-dependent accumulation of GST was detected in the spent culture medium of seminiferous tubules from rats of different ages suggesting secretion.

Amino Acid Sequence↗

PubMed1992

Carbohydrate binding specificity of the Tn-antigen binding lectin from Vicia villosa seeds (VVLB4).

2-Dansylamino-2-deoxy-D-galactose (GalNDns) is a useful fluorescent probe to study the interaction of non-fluorescent sugars with the B4 lectin from Vicia villosa seeds (VVLB4). Binding of the lectin to GalNDns leads to a 5.2-fold increase in Dansyl fluorescence with a concomitant 10 nm blue shift in its emission maximum. The strong binding of GalNDns (Ka = 7.33 x 10(4) M-1 at 20 degrees C) is due to a favourable entropic contribution to the association process. Among the other sugars studied, GalNAc alpha 1-O-Ser followed by Me alpha GalNAc are the best ligands. 2-Deoxygalactose, galactosamine and galactose are 2013, 469 and 130 times weaker ligands, respectively, as compared to GalNAc, whereas GalNDns is about 2.44 times more potent than GalNAc, indicating that substitutions at the C-2 position of GalNAc have a considerable influence on the binding affinities. Equatorial orientation of the hydroxyl group at C-3 and axial orientation at C-4 as in galactose are important for the interaction with VVLB4. The C-6 hydroxyl group is not indispensable. The binding site of the lectin is directed exclusively towards monosaccharides alone. Interestingly enough, despite its preference for Me alpha GalNAc over Me beta GalNAc, in oligosaccharides, the lectin prefers terminal beta-linked GalNAc as compared to the alpha-linked one.

Antigens↗

PubMed1992

Do antigenic peptides have a unique sense of direction inside the MHC binding groove? A molecular modelling study.

In the models suggested recently for antigenic peptides binding in the alpha 1, alpha 2 groove of MHC class I molecules, the orientation of the peptide has been shown uniquely as: the N----C vector of the peptide being parallel to the N----C vector of the alpha 1 helix of MHC. Here, we demonstrate that the reverse orientation of the peptide is equally probable. This hypothesis is supported by molecular modelling calculations and computer graphic analyses on a murine class I MHC molecule H-2Kd and its complexes with a restricted peptide RYLENGKETLQ. Analysis of the complementary interactions between the peptide residues and the amino acid side chains lining the MHC groove shows that the binding orientation of the peptide may be allele-specific and could depend on the sequence and structure of the antigenic epitope.

Amino Acid Sequence↗

PubMed1991

Barley aleurone layer cell protoplasts as a transient expression system.

Protoplasts were prepared from barley aleurone layers using 'Onozuka' cellulase digestion and purification through a Percoll gradient. Protoplasts prepared by this procedure had a viability ranging from 60% to 80% during the first two days of culture. They were responsive to gibberellic acid (GA) as measured by the stimulation of alpha-amylase synthesis. The GA stimulation was counteracted by abscisic acid (ABA). In the presence of polyethylene glycol (PEG), the protoplasts took up exogenously added plasmid DNA containing the reporter gene coding for chloramphenicol acetyl transferase (CAT) linked to a 35S promoter from cauliflower mosaic virus (CaMV) or to barley alpha-amylase gene promoters and expressed CAT activity. Therefore, barley aleurone layer protoplasts are suitable for analysis of hormone-responsive elements in hydrolase genes.

Abscisic Acid↗

PubMed1990

Thermodynamic analysis of ligand binding to winged bean (Psophocarpus tetragonolobus) acidic agglutinin reveals its specificity for terminally monofucosylated H-reactive sugars.

The sugar-specific binding of N-dansylgalactosamine to WBA II (n = 2; Ka = 5.6 x 10(3) M-1; delta H = -21 kJ.mol-1; delta S = -21.3 J.mol-1.K-1) was utilized in substitution titrations for evaluating the association constants for the interaction of sugars with the lectin. An axial hydroxyl at C-4 and equatorial hydroxyls at C-3 and C-6 as in D-galacto configuration are crucial for binding. Both axial and equatorial hydroxyls are tolerated at C-2. Conformationally akin disaccharides such as lactose, N-acetyllactosamine, Gal beta 1-3GlcNAc, and Gal beta 1-3GalNAc show similar affinities. 2'-Fucosyllactose and H-disaccharide display 146 and 13 times stronger affinity over lactose and galactose, yet fucose by itself is devoid of activity. An interesting feature, noted for the first time, in protein-sugar interactions is the positive entropy change for the binding of 2'-fucosyllactose, suggesting that nonpolar interactions play an important role in stabilization of the lectin-sugar complex. 3-Fucosyllactose, lactodifucotetraose, lacto-N-fucopentaose II and III are inactive, whereas lacto-N-fucopentaose I has 14-fold lower affinity as compared with 2'-fucosyllactose. Conformational analysis indicates that the substitution at subterminal glucose or GlcNAc by L-fucose in either alpha 1-3 or alpha 1-4 linkage leads to its projection so as to sterically hinder the access of 3'-fucosyllactose, lactodifucotetraose, and lacto-N-fucopentaose II and III to the binding site of winged bean agglutinin II. Similarly the projection of alpha 1-3 linked Gal/GalNAc also leads to steric hindrance and hence prevents the binding of blood group A and B reactive sugars. Considering its unique specificity winged bean agglutinin II should be useful in the isolation and characterization of terminally monofucosylated H-reactive oligosaccharides from those that are difucosylated or internally fucosylated.

Calorimetry↗

PubMed1988

Theoretical studies on alpha-helix--DNA interactions.

The interaction energies between (Ala)10 and alpha-helix fragment and different nucleotide sequences in right-handed B-form have been optimized using semi-empirical potential energy functions. The energies are calculated for two different orientations of the alpha-helix, viz., when the alpha-helix axis taken in the N----C direction is (i) parallel and (ii) antiparallel to the 5'-3' ascending strand of DNA, proximal to it. When both the DNA molecule as well as the alpha-helix are treated as rigid molecules it is found that a polyalanine alpha-helix has slightly more favourable contacts when it is in the proximity of a four nucleotide sequence of 5'-(N-A-T-N)-3' type, where N is either a purine or a pyrimidine. However, when the two interacting molecules are allowed to undergo local structural variations then the interaction energy appears to be independent of the base sequence confirming the non-specific nature of these interactions.

Base Composition↗