Biomedical subjects

C Mandal

Publications and source records attributed to C Mandal.

At least 37 records · Page 2Linked to original sources

PubMed1998

Rapid diagnosis of Indian visceral leishmaniasis using achatininH, a 9-O-acetylated sialic acid binding lectin.

We have developed a rapid, accurate, and inexpensive hemagglutination assay (HA) for diagnosis of Indian visceral leishmaniasis (VL) using achatininH, a 9-O-acetylated sialic acid binding lectin. The assay is based on the selective binding of achatininH to 9-O-acetylated derivatives of sialic acid (9-O-AcSA), which appears exclusively on the erythrocytes of patients with VL. Blood samples from 35 patients with demonstrable splenic or bone marrow amastigotes were assayed by HA and compared with an ELISA. All cases were correctly diagnosed by HA, and were in concordance with the ELISA. The increase in 9-O-AcSA was corroborated by direct fluorimetric estimation of sialic acid and its 9-O-acetylated derivative. Therefore, 9-O-acetylated derivatives of sialic acid, absent in normal erythrocytes, serve as an important biomarker, and indirectly reflect the presence of the parasite. Accordingly, this assay can be used for detection of active kala-azar cases and merits clinical consideration.

Biomarkers↗

PubMed1997

Role of zinc in tRNA-acceptor stem binding by glutamyl-tRNA synthetase from E.coli: a molecular modeling study.

A model of the N-terminal half of glutamyl-tRNA synthetase from E. coli was constructed on the basis of similarity in sequence and function of Glutaminyl- and Glutamyl-tRNA synthetases. The glutaminyl-tRNA synthetase does not contain any zinc atom, but glutamyl-tRNA synthetase from E. coli contains one atom of zinc. The specific role of zinc is not yet known. In this article, molecular modeling is employed to show that the zinc atom is well outside the contact region of the acceptor stem of tRNA. The placement of a zinc atom at a significant distance from the tRNA acceptor stem indicates that the role of zinc is likely to be indirect and structural.

Amino Acid Sequence↗

PubMed1996

Microheterogeneity of C-reactive protein in the sera of fish Labeo rohita induced by metal pollutants.

A group of C-reactive proteins (CRPs) have been purified to apparent homogeneity by calcium (Ca++) dependent affinity chromatography on phosphoryl choline sepharose column from the sera of L. rohita confined in normal and nonlethal doses of cadmium (Cd++) and mercury (Hg++) polluted water. The CRPs levels are elevated in the serum of both Cd++ and Hg++ polluted fishes. All three CRPs are glycoproteins having identical subunits of Mr 33,000. The total content of carbohydrate are 21.4%, 16.4% and 14.2% and that of sialic acid are 3.5%, 2.4% and 1.5% in CRPHg++, CRPCd++ and CRPN respectively. In native gel electrophoresis all three CRPs show differences in mobility. However they move to identical position after desialylation and deglycosylation. These CRPs react differentially with different lectins which indicates a variation in the carbohydrate moieties resulting their microheterogeneity.

Animals↗

PubMed1996

ABGEN: a knowledge-based automated approach for antibody structure modeling.

Immunoglobulin (Ig) amino acid sequences are highly conserved and often have sequence homology ranging from 70 to 95%. Antigen binding fragments (Fab), variable region fragments (Fv), and single chain Fv (scFv) of more than 50 myeloma proteins and monoclonal antibodies (mAb) have been crystallized and display a high degree of structural similarity. Based on this observation, several homology modeling approaches have been developed for the prediction of Fab and Fv structures prior to their experimental determination. We have extracted features from existing Ig sequences, 44 known Fab and Fv structures to create an automated AntiBody structure GENeration (ABGEN) algorithm for obtaining structural models of antibody fragments. ABGEN utilizes a homology based scaffolding technique, and includes the use of invariant and strictly conserved residues, structural motifs of known Fab, canonical features of hypervariable loops, torsional constraints for residue replacements and key inter-residue interactions. The validity of the ABGEN algorithm has been tested using a five-fold cross validation with the existing Fab structures. Molecular mechanics and dynamics methods have been implemented with ABGEN models to accurately predict two Fab structures of anti-sweetener antibodies prior to crystallographic determinations.

Algorithms↗

PubMed1995

The specificity of the binding site of AchatininH, a sialic acid-binding lectin from Achatina fulica.

A sialic acid-binding lectin, AchatininH (ATNH), having unique specificity towards 9-O-acetylneuraminic acid, has been purified and characterized. The specificity of this lectin for O-acetylsialic acids was studied in detail, using various sialic acid derivatives and sialoglycoproteins. The potent inhibition of hemagglutination by bovine submaxillary mucin (BSM), which contains 9(7,8)-O-acetylsialic acid and by free 9-O-acetylneuraminic acid confirms the preferential affinity towards this sugar. Further support for the role of O-acetylsialic acid was obtained by sialidase treatment of BSM. O-Deacetylation of the sialic acid residue abolished its inhibitory potency. Moreover, when the trihydroxypropyl side chain of the sialic acid molecule was modified by periodate-borohydride treatment, the truncated C7-sialic acid was unable to bind ATNH. This result suggests that the glycerol side chain of Neu5Ac, especially the C-8 and/or C-9 portion is an important determinant for ATNH. The hemagglutination-inhibition results using several mono-, di-, and tri-saccharides containing terminal sialic acid and various sialoglycoproteins reveals that ATNH preferentially binds the alpha-(2-->6)-linked sialic acid. Furthermore, beta-D-GlcNAc-(1-->3)-[alpha-NeuGc-(2-->6)]-GalNAc-ol was found to be the best ligand for ATNH.

Animals↗

PubMed1995

Structural predictions of the binding site architecture for monoclonal antibody NC6.8 using computer-aided molecular modeling, ligand binding, and spectroscopy.

Monoclonal antibody NC6.8 binds the superpotent sweetener ligand N-(p-cyanophenyl)-N'-(diphenylmethyl) guanidineacetic acid with high affinity (Kd = 53 nM). Using computer-aided molecular modeling and several experimental techniques, such as competitive ligand binding, absorbance spectroscopy, and fluorescence spectroscopy, we have predicted the structure of the variable domain fragment (Fv) and identified the key residues in the combining site of the antibody. We have identified nine specific amino acids as being involved in ligand recognition and complexation. Most notable are H:33W, which is responsible for ligand-induced tryptophan fluorescence quenching, H:56R, which forms a salt bridge with the carboxylate moiety of the ligand, and L:34H, which, deep in the binding site, interacts with the cyanophenyl portion of the ligand. Two residues located deep in the putative binding pocket, H:35E and H:50E, provide the negatively charged potential for interaction with the protonated aryl nitrogen and the positive guanidinium group. These modeling predictions were made before the solution of high-resolution structures of the native Fab (2.6 A) and the Fab-ligand complex (2.2 A). Comparisons between the theoretical model and experimental native and liganded Fab structures are made.

Acetates↗

PubMed1995

Designing of peptides with immuno-modulatory properties using protein A as a probe.

A series of reports from our laboratory have described the multifarious properties of protein A of Staphylococcus aureus Cowan I, apart from its IgG binding affinity. Original reports regarding its anti-tumor, anti-toxic, anti-carcinogenic and immunomodulatory properties published earlier by the authors have implicated some uniqueness of this bacterial protein. It was conceived that such diversified properties must lie in its specific peptide sequences, rendering it to act and behave as a multipotent "Biological Response Modifier" (BRM). The high resolution X-ray structure of protein A-Fc complex has been delineated earlier, and has been the foundation of many protein engineering studies. This structure along with the amino acid sequence data of its four repetitive domains provided us the basis for designing an octapeptide. This octapeptide was synthesized by solid phase peptide synthesis considering it as the probable site through which PA binds IgG. This octapeptide (NH2-Gln-Asn-Ala-Phe-Tyr-Glu-Ile-Leu-COOH) is present in the first helical segment of B-domain of protein A, and also is a part of domain D, A and C. This octapeptide has been shown to bind IgG by the immunoblotting technique. The binding affinity of the octapeptide appears to be significantly higher than that of intact protein A, as was revealed by calculation of Ka (association constant) and Kd (dissociation constant) values. This octapeptide might serve as a good immunoadsorbant for IgG and/or immune complexes.

Adjuvants, Immunologic↗

PubMed1994

O-acetylated sialic acid as a distinct marker for differentiation between several leukemia erythrocytes.

AchatininH (ATNH) is a lectin, isolated from the hemolymph of Achatina fulica snail, which has been shown to have narrow specificity towards 9-O-acetyl sialic acid. Usually ATNH does not agglutinate normal human erythrocytes, however, it is capable of agglutinating erythrocytes of patients suffering from acute lymphocytic and acute myelogenous leukemia. Determination of binding constants, numbers of binding sites and lectin overlay experiments using patients' erythrocytes ghost, have suggested that some alterations in erythrocyte cell surface sialoglycoproteins or more precisely appearance of some O-acetylated sialoglycoprotein as a result of pathological transformations has caused this change in the binding of ATNH.

Acute Disease↗

PubMed1994

Computer-aided molecular modeling of the binding site architecture for eight monoclonal antibodies that bind a high potency guanidinium sweetener.

Computer-aided molecular modeling of the antibody binding site of eight different monoclonal antibodies (mAb) that bind the intense sweetener ligand (N-(p-cyanophenyl)-N'-diphenylmethyl) guanidine acetic acid was completed using canonical loop structures and framework regions from known immunoglobulins as "parent structures" for the molecular scaffoldings. The models of the fragment variable (Fv) region of the mAb were analyzed for the presence and location of residues predicted to be involved in ligand binding. Several binding site tryptophan residues in these models were located in positions that support previous flurospectroscopic observations of the mAb-ligand complexation. Computer-aided renderings of the electrostatic potential at the van der Waals surface of the Fv region were compared and found to be consistent with the ligand binding specificity profiles for the different mAb. The Fv model of mAb NC6.8 was consistent with the binding site features determined in the Fab structure recently solved by X-ray diffraction techniques. These Fv models should provide an adequate basis for site-directed mutagenesis experiments in order to characterize interactive motifs in the mAb binding site.

Acetates↗

PubMed1993

Modulation of sialic acid-binding proteins of rat uterus in response to changing hormonal milieu.

A group of sialic acid binding (SAS) agglutinins has been isolated from the rat uteri at different stages [Proestrus (P), estrus (E) and diestrus (D)] of estrous cycle. Studies of biochemical properties indicate that SAS agglutinins are glycoprotein in nature having molecular weights between 28-31 Kd and microheterogenous pI. Function-based characterization revealed that inspite of the fact that all three proteins exhibit sialic acid binding property, the sialic acid binding affinities, calculated from Scatchard analysis, using 4-methylumbelliferyl sialic acid as a ligand, varied in stage specific manner (Ka:D-SAS-9.03 x 10(5) M-1, P-SAS-2.33 x 10(5) M-1, E-SAS-2.13 x 10(5) M-1). Circular dichroism spectra of these three agglutinins suggested that differences exist in the secondary structures of the proteins isolated from different stages. Removal of carbohydrate moiety by trifluoromethane sulfonic acid treatment and CNBr cleavage studies showed some homology between these proteins, however, the variation in the carbohydrate moiety was apparent from the sugar analysis data. Functionally and immunologically these proteins can be grouped as estrogenic and progestogenic SAS agglutinins.

Agglutinins↗

PubMed1993

PROGEN: an automated modelling algorithm for the generation of complete protein structures from the alpha-carbon atomic coordinates.

A modelling algorithm (PROGEN) for the generation of complete protein atomic coordinates from only the alpha-carbon coordinates is described. PROGEN utilizes an optimal geometry parameter (OGP) database for the positioning of atoms for each amino acid of the polypeptide model. The OGP database was established by examining the statistical correlations between 23 different intra-peptide and inter-peptide geometric parameters relative to the alpha-carbon distances for each amino acid in a library of 19 known proteins from the Brookhaven Protein Database (BPDB). The OGP files for specific amino acids and peptides were used to generate the atomic positions, with respect to alpha-carbons, for main-chain and side-chain atoms in the modelled structure. Refinement of the initial model was accomplished using energy minimization (EM) and molecular dynamics techniques. PROGEN was tested using 60 known proteins in the BPDB, representing a wide spectrum of primary and secondary structures. Comparison between PROGEN models and BPDB crystal reference structures gave r.m.s.d. values for peptide main-chain atoms between 0.29 and 0.76 A, with a grand average of 0.53 A for all 60 models. The r.m.s.d. for all non-hydrogen atoms ranged between 1.44 and 1.93 A for the 60 polypeptide models. PROGEN was also able to make the correct assignment of cis- or trans-proline configurations in the protein structures examined. PROGEN offers a fully automatic building and refinement procedure and requires no special or specific structural considerations for the protein to be modelled.

Algorithms↗

PubMed1993

Immuno-suppressive effect of human alphafetoprotein: a cross species study.

Alphafetoprotein (AFP) is the major serum protein of fetal life in human and other mammalian species. The phylogenetical conservatism of AFP demonstrated by extensive immunological cross reaction between human AFP and AFP of a number of species, suggest that AFP plays a general role in the successful pregnancy of all mammalian species. The present work clearly demonstrates the antiproliferative effect of human AFP on lymphocytes, harvested from normal human donors. The inhibitory effect of human AFP is quite significant in the same dose during blastic transformations of the lymphocytes. In this study, peripheral blood mononuclear cells were induced to blastic transformations with phytohaemagglutinin (PHA-M) and the effect of AFP was quantified by the incorporation of [3H]-thymidine into newly synthesized DNA during 24 hrs pulse. Moreover, human AFP shows similar immuno-suppressive effect to other species of lymphocytes also. In all the three species (mouse, rat and hamster) studied, a parallelism was noted in their respective percentage of thymidine incorporation values at the comparable doses. These results establish a cross species inhibitory effect of human AFP and it may be stated that this effect is directly targeted on T-helper cells and has no interaction with interleukin-2 (IL-2).

Animals↗

PubMed1992

Albumen gland of the snail Achatina fulica is the site for synthesis of AchatininH, a sialic acid binding lectin.

A sialic acid binding lectin, AchatininH was purified from the hemolymph of Achatina fulica snail. To identify the site of synthesis of AchatininH, in vitro incubation studies in presence of labelled amino acid precursor were performed. Different organs from the snail were sliced and incubated in methionine-deficient Eagle's minimum essential medium containing [35S]-methionine at 25 degrees C for 5 h. After termination of incubation, tissues were homogenized, centrifuged and the de novo synthesized protein was immunoprecipitated with specific AchatininH antibody, followed by protein-A. The precipitated antigen-antibody complex was analysed by SDS-PAGE. Data obtained from native gel electrophoresis and SDS-PAGE radioautographic analysis indicates that AchatininH is synthesized in the albumen gland.

Animals↗

PubMed1992

Kinetics of haloperidol binding to monoclonal antibodies as measured by direct fluorescence quenching.

Monoclonal antibodies which bind small neurogenic ligands may mimic certain aspects of the stereospecific binding present in the natural biological receptor, and may prove useful in designing engineered protein receptors, provided their interactions are correctly understood. We report here the kinetic and equilibrium characteristics of ligand-antibody complexes of haloperidol, a dopaminergic antagonist, with three of its monoclonal antibodies as studied by fluorescence quenching techniques. These antibodies possessed moderate to high affinity constants, ranging from 10(5) to 10(9) M-1, and caused fluorescence quenching of a fluorescein-labeled haloperidol as well as quenching (40-60%) of the internal tryptophan fluorescence. The dissociation rates of the ligand from the complexes were measured at different conditions of temperature, pH and ionic strength. The results provide important information regarding the kinetic and thermodynamic parameters of the binding pockets of these antibodies.

Antibodies, Monoclonal↗

PubMed1992

Kinetic and energetic parameters of imipramine binding to monoclonal antibodies as measured by fluorescence spectroscopy.

Monoclonal antibodies which bind small drugs are useful for the study of the interactive forces involved in antibody-ligand complexation. Detailed understanding of these supramolecular forces requires a careful examination of structural and thermodynamic parameters of the interacting molecules. Fluorescence spectroscopy techniques are very useful in this regard. We report here, the kinetic and energetic parameters of four monoclonal antibodies made against the tricyclic antidepressant imipramine. These monoclonal antibodies were found to possess high to very high binding affinity constants, ranging from 10(7) to 10(10) M-1, and caused fluorescence quenching or enhancement of a fluorescein labelled imipramine. The dissociation rates of the fluorescent ligand from the complexes were measured at different temperatures in order to provide some insight regarding the kinetic and energetic (thermodynamic) parameters of the antibody-ligand binding interactions.

Animals↗

PubMed1991

Isolation of a phosphoryl choline-binding protein from the hemolymph of the snail, Achatina fulica.

A phosphorylcholine-binding protein from the hemolymph of the snail Achatina fulica was purified to near homogeneity using a Sepharose phenylphosphorylcholine affinity column. The protein bound to the affinity column was eluted with 5 mM phosphorylcholine as a single symmetrical peak. The purified protein (400 Kda) contained 35-40% carbohydrate. On SDS-PAGE the protein separated into two bands of 20 and 24 Kda, and had a pI of 5.9. On immunodiffusion, antiserum to the snail phosphorylcholine binding protein did not cross-react against other phosphorylcholine binding proteins, like rat serum phosphorylcholine-binding protein (PCBP), limulus C-reactive protein (CRP), or human CRP. On pretreatment of the snail hemolymph with this antiserum, the hemagglutination titer of the hemolymph was markedly decreased. The purified snail phosphorylcholine binding protein agglutinated rabbit erythrocytes in the absence of divalent cation (Ca+2) but trace amount of Ca+2 increased its binding. The strongest inhibitor of the agglutination reaction was lactose, followed by melibiose and 2-deoxygalactose. The relationships of the snail phosphorylcholine binding protein to other hemolymph agglutinins and to CRPs are discussed in light of common phylogeny.

Animals↗

PubMed1991

Epitope analysis of the oncofetal antigen alphafetoprotein using monoclonal antibodies.

Alphafetoprotein (AFP), an oncofetal antigen, plays very important roles in the early embryonic life and oncogenesis. Under various physiological and pathological conditions AFP exhibits microheterogeneity, probably as a result of differential expression of its epitopes. To analyse the epitopes we have developed a panel of monoclonal antibodies against human AFP purified by a new and efficient method using an immunoadsorbent consisting of polyclonal antibodies immobilized on cyanogen bromide activated Sepharose. Clones producing antibodies of various isotypes, e.g. IgG1, IgG2a, IgG2b, IgA and IgM have been subcloned and characterized. The antibodies showed high avidity for AFP (with half-maximal binding concentrations between 0.012 and 3.87 nM). Mutual inhibition efficiencies of a panel of 14 monoclonal antibodies were determined by RIA. Based on these inhibition data a computer program was used to group these antibodies with respect to their "epitope specificity distance". As a result of this grouping, clones have been identified which can recognize at least five different epitopes on AFP. This panel of antibodies may be very useful for analysis of the epitopic variation of AFP under various physiological and pathological conditions.

Adult↗

PubMed1991

Monoclonal antibodies to sweet taste proteins. I. Analysis of antigenic epitopes on thaumatin by competitive inhibition assays.

Using a competitive inhibition binding immunoassay, we have examined some of the antigenic epitopes present on thaumatin, an intense sweet tasting protein from the African fruit katemfe. We have developed a library of monoclonal antibodies which react with different surface antigenic epitopes on thaumatin. Some of these monoclonal antibodies also cross-react with monellin, another unrelated sweet tasting protein. The competitive binding immunoassay examines the immunoreactivity of both solid-phase and liquid-phase monoclonal antibodies. At least six major antigenic epitopes on thaumatin were identified by our library of monoclonal antibodies. This type of competitive binding analysis may prove useful in the discovery of "sweet taste determinants" on plant proteins and in the development of tandem immunoassays for quantitation of sweet tasting proteins in plant extracts.

Antibodies, Monoclonal↗