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C Mandal

Publications and source records attributed to C Mandal.

At least 55 records · Page 3Linked to original sources
PubMed1991

Alpha-fetoprotein: a sensitive avidine-biotin assay.

A conventional enzyme-linked immunosorbent assay (ELISA) for alpha-fetoprotein was modified to incorporate the avidine-biotin complex, in order to enhance sensitivity. Five modifications of this assay were compared, of which the 2-step noncompetitive avidine-biotin assay (NABA) was found to be the most rapid and sensitive (detection limit 0.05 ng/ml). Levels of AFP in human cord serum measured by the 2-step NABA method showed good correlation (r = 0.965) with measurements by radio-immuno assay, and the assay can be performed within one hour. The simplicity and sensitivity of this assay should make it a reliable method of choice for determination of alpha-fetoprotein.

Avidin↗
PubMed1990

Sialic acid binding lectins.

The literature contains several reviews on lectins in general, covering mainly those from plants and invertebrates. However, the sialic acid binding lectins have not been reviewed so far. Considering the importance of sialic acids in cell sociology, lectins which specifically recognize terminal sialic acid residues are potentially useful as analytical tools in studying the biological functions of sialoglycoconjugates. These lectins, along with monoclonal antibodies raised against sialoglycoconjugates, have been used in the detection, affinity purification, cytochemical localization and quantitation of such glycoconjugates. In this review the main emphasis has been placed on the occurrence, general purification procedures, macromolecular properties, sugar specificities and applications of these lectins.

Animals↗
PubMed1989

Physiochemical studies on achatininH, a novel sialic acid-binding lectin.

We have purified a sialic acid-binding lectin, achatininH, in a single step by affinity chromatography, having high affinity for 9-O-acetylneuraminic acid. The physicochemical characterization of the interaction of achatininH with bivalent metal ions and sialic acid derivatives by the use of spectrofluorimetry, spectropolarimetry and precipitin reaction is reported. From fluorescence quenching studies the binding of Ca2+ (Ka = 251 +/- 9 M-1) and of Mn2+ (Ka = 86 +/- 5 M-1) was found to be weak, but their presence is absolutely necessary for sugar binding as well as biological activity. The nature and position of the substituent group play a very important role in the binding affinity. AchatininH shows a high affinity for 9-O-acetylneuraminic acid (Ka = 1.20 x 10(3) +/- 0.07 x 10(3) M-1) compared with that for the 4-O-acetyl derivative. In oligomers the binding strength increases in the order monosaccharide less than disaccharide less than trisaccharide. The binding affinity of achatininH for the disaccharide was found to reach a peak around pH 8. From c.d. spectral studies achatininH was found to have a high beta-sheet content (46%) and a low alpha-helix content (24%). From precipitin analysis at least one sugar-binding site on each of the 16 monomer subunits of the protein is indicated.

Animals↗
PubMed1989

Functional heterogeneity of sialic acid binding agglutinins of rat uteri towards in vitro lymphocyte transformation.

The sialic acid binding agglutinins (SAS) are purified from three different stages (proestrus [P], estrus [E], diestrus [D]) of rat uterine homogenate. All three proteins are capable of inhibiting the in vitro phytohemagglutinin (PHA)-induced blastogenic response of lymphocytes and thymocytes as manifested by inhibition of 3H-thymidine incorporation into cells. The lymphocytes isolated from pregnant female rats were found to be more susceptible to inhibition. In resting lymphocyte cultures (RLC), however, the P- and E-SAS proteins acted differently from the D-SAS by stimulating lymphocyte proliferation in the cultures initiated from normal rat lymphocytes. At the same dose these proteins were inhibitory to the RLC, where cultured lymphocytes were isolated from the pregnant female. Reverse hemolytic plaque formation assay results also indicated functional heterogeneity between these proteins. P-SAS induced a potent polyclonal activation of the B cell, whereas E- and D-SAS did not show much activation. SAS proteins do not have any complement-like activity. These results, corroborated by cell viability count and 51Cr uptake tests, suggest that D-SAS has genuine lymphoblastogenesis-suppressive properties whereas P- and E-SAS-induced suppression in the presence of PHA is probably due to an increased cell-density-dependent cytotoxicity.

Agglutinins↗
PubMed1988

Chemical-modification studies of a unique sialic acid-binding lectin from the snail Achatina fulica. Involvement of tryptophan and histidine residues in biological activity.

A unique sialic acid-binding lectin, achatininH (ATNH) was purified in single step from the haemolymph of the snail Achatina fulica by affinity chromatography on sheep submaxillary-gland mucin coupled to Sepharose 4B. The homogeneity was checked by alkaline gel electrophoresis, immunodiffusion and immunoelectrophoresis. Amino acid analysis showed that the lectin has a fairly high content of acidic amino acid residues (22% of the total). About 1.3% of the residues are half-cystine. The glycoprotein contains 21% carbohydrate. The unusually high content of xylose (6%) and fucose (2.7%) in this snail lectin is quite interesting. The protein was subjected to various chemical modifications in order to detect the amino acid residues and carbohydrate residues present in its binding sites. Modification of tyrosine and arginine residues did not affect the binding activity of ATNH; however, modification of tryptophan and histidine residues led to a complete loss of its biological activity. A marked decrease in the fluorescence emission was found as the tryptophan residues of ATNH were modified. The c.d. data showed the presence of an identical type of conformation in the native and modified agglutinin. The modification of lysine and carboxy residues partially diminished the biological activity. The activity was completely lost after a beta-elimination reaction, indicating that the sugars are O-glycosidically linked to the glycoprotein's protein moiety. This result confirms that the carbohydrate moiety also plays an important role in the agglutination property of this lectin.

Amino Acids↗
PubMed1987

An unique specificity of a sialic acid binding lectin AchatininH, from the hemolymph of Achatina fulica snail.

A sialic acid-binding lectin, AchatininH, from the hemolymph of Achatina fulica snail is found to be highly specific for 9-0-acetyl sialic acid. The binding specificity of AchatininH distinguishes it from other known sialic-acid specific lectins which usually show a broader range of specificity for sialic acid. It is even better than crab lectin which shows specificity for both 4- and 9-0-acetylated derivatives of sialic acid. This limited specificity of AchatininH appear to account for the fact that it agglutinates only rabbit, rat and guinea pig erythrocytes which contain 9-0-acetylated sialic acid but not horse (mainly contain 4-0-acetylated sialic acid), human, monkey, sheep, goat and chicken erythrocytes which contain either N-acetyl or N-glycolyl neuraminic acid but no 0-acetylated derivatives. This finding was further supported by the potent inhibition of hemagglutination by free 9-0-acetylated neuraminic acid and by several glyco shingolipids of human origin having 0-acetylated sialic acid.

ABO Blood-Group System↗
PubMed1987

Liposome as an adjuvant for the production of estradiol antibodies.

Mice immunized with liposome associated estradiol-bovine serum albumin conjugate (E2-BSA) showed a significantly higher estradiol-specific immune response than did mice injected with the free conjugate. The conjugation of estradiol with BSA is imperative, since injection of liposomes with estradiol in the lipid bilayer was found to be ineffective in inducing an immune response. Entrapment of E2-BSA in neutral and positively charged liposomes was found to be a potent mode of immunostimulation, even better than emulsification in complete Freund's adjuvant (CFA). Covalent liposome surface association of E2-BSA induced a good immunopotentiation. However, the response was lower than that observed with the entrapped conjugate. The affinity and the specificity of antisera, raised in different ways, were determined.

Adjuvants, Immunologic↗
PubMed1987

Regulation of alpha- and beta-tubulin mRNAs in rat brain during synaptogenesis.

Developmental alterations in alpha- and beta-tubulin mRNA in polysomes from brains of -3 days (fetal) to 30-day-old rats were quantitated by using well-characterized chicken alpha- and beta-tubulin cDNA probes, pT1 and pT2 having 80-90% homology with rat sequences. Northern blot analysis revealed a single major (greater than 95%) 1.8 kb mRNA for both alpha- and beta-tubulin. Quantitation by slot hybridization indicates a virtually coordinate expression of alpha- and beta-tubulin mRNA with a maximal level around day 5 after birth, which represents the mid-phase of synaptogenesis.

Animals↗
PubMed1986

A single step purification of a sialic acid binding lectin (AchatininH) from Achatina fulica snail.

A sialic acid binding lectin, AchatininH, was purified in single step from the hemolymph of the land snail, Achatina fulica, by the affinity chromatography on sheep submaxillary mucin coupled to Sepharose 4B. The yield of the lectin was found to be 3 mg from 100 ml of hemolymph. The homogeneity of the lectin was established by alkaline gel electrophoresis, immunodiffusion, immunoelectrophoresis and analytical isoelectrophoresis. The molecular weight of the native protein was 242,000, having identical subunits of Mr 15,000. The lectin agglutinated rabbit erythrocytes in the presence of Ca2+. The inhibition study clearly suggests that the binding site of the lectin recognizes sialic acid as the immunodominant sugar. This was further confirmed by the observation that there was a marked decrease of agglutinating activity of the lectin with neuraminidase treated rabbit erythrocytes and asialofetuin was unable to inhibit the activity of AchatininH. Among the inhibitors used the glycoconjugate containing alpha 2----6 linkages of N-acetylneuraminic acid with subterminal galactopyranose or 2-acetamido-2-deoxy-galactopyranose residue was found to be better inhibitor than that containing alpha 2----3 linkages of N-acetyl neuraminic acid. Besides that sialoglycoprotein containing both N and O type of glycosidic linkages plays an important role in binding with the lectin. Fetuin was found to be the best inhibitor.

Animals↗
PubMed1985

Immobilization of antibodies on a new solid phase for use in ELISA.

Antibodies to myoglobin were immobilized by covalent linkage to polyester film for use in a solid-phase ELISA. The covalent linkage of antibody to this new solid phase was accomplished by partial acid hydrolysis of the film followed by periodate oxidation. About 60 ng of protein could be immobilized per cm2 of film and the binding was stable. Antimyoglobin IgM immobilized on the films was used in the ELISA technique to detect myoglobin within the range 0.25-10 ng. The assay procedure was found to be very accurate and the coefficient of variation of each concentration ranged from 0.63 to 2.1%. Furthermore the immobilized film could be re-used after dissociating the antigen antibody complexes.

Animals↗
PubMed1985

Identification and isolation of an agglutinin from uterus of rats.

A sialic acid-binding agglutinin was purified to apparent homogeneity by affinity chromatography on fetuin-sepharose column from the rat uterine homogenate in estrus. The agglutinin is Ca++ dependent, a glycoprotein, and composed of two very closely associated bands of molecular weights 28,000 and 30,000 and pIs of 4 and 4.1. Several sialoglycoproteins, sialic acid, EDTA, glucuronic acid and heparin acted as an inhibitor of the agglutinin.

Animals↗
PubMed1984

A new cold agglutinin from Achatina fulica snails.

An electrophoretically homogeneous agglutinin was purified from the albumin gland of Achatina fulica snails using asialofetunin-Sepharose 4B as an affinity column. The agglutinin was found to be temperature sensitive; it agglutinated rabbit and human umbilical cord erythrocytes only at low temperature. It was found to be specific for methyl-beta-D-galactoside, and the best inhibitor was N-acetyllactosamine.

Animals↗
PubMed1984

Restriction in IgM expression--V. Fine structure analysis in the anti-lactose system.

A methodology for the analysis of the fine specificity of monoclonal anti-lactose IgM and IgG antibodies is described using structural variants of the homologous lactoside epitope. These variants are used as inhibitors of the binding of a reference ligand N-(5-dimethylaminonaphthalene-1-sulfonyl)-p-aminophenyl-beta-lactoside. Excitation of the antibody with bound ligand at 295 nm leads to resonance energy transfer to and fluorescence emission by the ligand. Titration of the antibody-ligand mixture with the inhibitor and measurement of the emission at 550 nm provide the data for the calculation of the binding constants of the inhibitors. A comparison of two IgM and two IgG antibodies showed that the higher affinity of the IgG antibodies arises from their specific interaction with both hexosides of the lactoside in contrast to IgM antibodies which do not engage the non-terminal hexoside as effectively. The quantitative significance of this difference is a differential free energy contribution of about -3 kcal/mole to the binding of lactoside by IgG. A finer discrimination between homologous and several cross-reactive molecules is evident with IgG antibody compared to IgM. The former exhibits about 100-fold greater difference in their binding constants than does IgM. These differences applied to biologically relevant multivalent interactions, where functional affinity governs complex formation, suggest a possible explanation for the IgM to IgG conversion characteristic of the humoral immune response.

Antibodies, Monoclonal↗
PubMed1981

Restriction in IgM expression. III. Affinity analysis of monoclonal anti-lactose antibodies.

A clonal analysis has been made of the murine BALB/C response to lactose-containing immunogens with respect to the affinity restriction in IgM expression. Monoclonal IgM and IgG antibodies were prepared from antilactosyl hybridomas generated from mice immunized with p-aminophenyl-beta-lactoside (PAPL) coupled to BGG or with a vaccine of Streptococcus faecalis (Strain N). Association constants for the binding of monovalent derivatives of PAPL were measured by quenching fluorescence. These derivatives carried a probe (2,4-dinitrophenyl or 1-dimethylaminonaphthalene-5-sulfonyl) that served to quench the protein fluorescence when the ligand was complexed with the protein. The central finding was that with both immunogens a restriction in the affinity of IgM was demonstrated since the highest values exhibited by IgG antibody exceeded by at least a factor of 50 the highest comparable values of the association constants for IgM antibody. It is suggested that the hypothesis of germ-line restriction, previously proposed as the basis for the affinity restriction of IgM, may also be applicable to the T cell receptor since its distinctive properties parallel those of IgM antibody.

Animals↗
PubMed1980

Hydrogen--deuterium exchange analysis of ligand--macromolecule interactions: ethidium--deoxyribonucleic acid system.

The interaction between DNA and the intercalating dye, ethidium bromide, was studied by use of a novel approach in which hydrogen--deuterium (H--D) exchange between ethidium amino groups and solvent was measured spectrophotometrically in a stopped-flow mode. The method depends on the fact that ethidium H exchange is greatly slowed on complexation with DNA and that H--D exchange kinetics of the chromophore can be monitored via an accompanying change in its spectral absorbance. The H--D exchange dependent spectral character of ethidium was characterized, and the catalyzed exchange behavior of the free dye and the dye--DNA complex was studied. From such measurements, one can obtain rate and equilibrium constants for the interaction and possibly also some stereochemical information. The constants obtained were checked in more conventional mixing experiments. At 20 degrees C in high salt, the equilibrium binding constant is approximately 5 x 10(4) M-1, and on and off rate constants are 1.6 x 10(6) M-1 s-1 and 30 s-1, respectively. The results independently confirm that one dye molecule is bound for each 2--2.5 base pairs. The method should be applicable to a range of binding interactions. Among other advantages, this approach can allow tight binding interactions to be studied at concentrations of the reactants far above the characteristic Kdiss value.

Animals↗
PubMed1980

Individual breathing reactions measured in hemoglobin by hydrogen exchange methods.

Protein hydrogen exchange is generally believed to register some aspects of internal protein dynamics, but the kind of motion at work is not clear. Experiments are being done to identify the determinants of protein hydrogen exchange and to distinguish between local unfolding and accessibility-penetration mechanisms. Results with small molecules, polynucleotides, and proteins demonstrate that solvent accessibility is by no means sufficient for fast exchange. H-exchange slowing is quite generally connected with intramolecular H-bonding, and the exchange process depends pivotally on transient H-bond cleavage. At least in alpha-helical structures, the cooperative aspect of H-bond cleavage must be expressed in local unfolding reactions. Results obtained by use of a difference hydrogen exchange method appear to provide a direct measurement of transient, cooperative, local unfolding reactions in hemoglobin. The reality of these supposed coherent breathing units is being tested by using the difference H-exchange approach to tritium label the units one at a time and then attempting to locate the tritium by fragmenting the protein, separating the fragments, and testing them for label. Early results demonstrate the feasibility of this approach.

Deuterium↗