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J J Marchalonis

Publications and source records attributed to J J Marchalonis.

At least 109 records · Page 6Linked to original sources
PubMed1987

The immunoglobulins of carcharhine sharks: a comparison of serological and biochemical properties.

The immunoglobulins of three carcharhine sharks were isolated from serum by means of salt precipitation and gel chromatography. The Galapagos shark (Carcharhinus galapagensis), the sandbar shark (Carcharhinus plumbeus) and the tiger shark (Galeocerdo cuvieri) each contained high molecular weight (18S) and low molecular weight (7S) IgM-like molecules as the major serum immunoglobulins. Both within and between species 18S and 7S immunoglobulins closely resemble each other in antigenic character, polypeptide chain composition, chain mass, amino acid composition, carbohydrate content and amino-terminal sequence. These results suggest that the immunoglobulins of carcharhine sharks have undergone little structural divergence during their evolution.

Amino Acid Sequence↗
PubMed1986

Binding and mitogenic properties of a galactosyl-specific lectin from the tunicate Didemnum candidum for murine thymocytes and splenocytes.

The affinity-purified major galactosyl-specific lectin (DCL-I) from the tunicate Didemnum candidum binds and induces blastogenesis in untreated and Vibrio cholerae neuraminidase (VCN)-treated murine splenocytes. However, it only binds and stimulates murine thymocytes when they have been pretreated with VCN. Binding and stimulation of VCN-treated splenocytes and thymocytes is specifically inhibited by D-galactose. Binding of lectin to splenocytes is highly increased by the VCN treatment, but the cells are stimulated to the same extent in both untreated and VCN-treated cells. This suggests that although additional DCL-I carbohydrate acceptors are uncovered by VCN treatment of the splenocytes, the ones responsible for triggering blastogenesis are already exposed on the untreated cells. On the contrary, cleavage of terminal sialic acids from the thymocyte cell surface is required to expose DCL-I carbohydrate acceptors in order to obtain a mitogenic response. Our results suggest that the distribution of cell surface galactosyl carbohydrate moieties to which DCL-I binds is different in thymocytes and splenocytes.

Animals↗
PubMed1986

Galactosyl-binding lectins from the tunicate Didemnum candidum. Purification and physicochemical characterization.

The plasma of the ascidian Didemnum candidum possesses lectin activity directed toward galactosyl moieties. We report the purification by affinity chromatography, the physicochemical properties, amino acid composition, and partial N-terminal amino acid sequence of two galactosyl-binding lectins D. candidum lectins I and II (DCL-I and DCL-II) from the plasma of this protochordate species. Both lectins were purified by affinity chromatography (on acid-treated Sepharose 4B and asialofetuin conjugated to Sepharose 4B) to homogeneity as judged by immunoelectrophoresis, size exclusion chromatography on high performance liquid chromatography, and polyacrylamide gel electrophoresis. Isoelectric focusing in polyacrylamide gels revealed that DCL-I focuses as a family of bands at pH 3.8-5.2, while DCL-II focuses at pH 9.2-10.2. Gas chromatography analyses of alditol acetate derivatives indicated that no carbohydrate components are associated with the lectins. Approximate subunit molecular weights estimated by polyacrylamide gel electrophoresis and size exclusion chromatography on high performance liquid chromatography in 6 M guanidine HCl under reducing conditions were 13,400-14,500 for DCL-I and 14,500-15,500 for DCL-II. Native molecular weights estimated by sedimentation equilibrium were 56,600 (DCL-I) and 57,500 (DCL-II), indicating that both species are constituted by four equal-sized subunits. Frictional ratios suggested that both lectins are globular proteins. Using rabbit antisera, the two molecules appeared serologically distinct. The extinction coefficient for DCL-I was E280 mg/ml = 2.52 ml mg-1 cm-1. Circular dichroism analyses of DCL-I suggested 29% alpha-helix and 37% beta-structure in the protein. Excitation/emission fluorescence spectra for DCL-I yielded maximum excitation and emission wavelengths at 288 and 330 nm, respectively. Amino acid compositions of DCL-I and DCL-II differed mainly in the proportions of aspartic and glutamic acids, serine, alanine, cysteine, valine, phenylalanine, and histidine. Amino acid compositions of DCL-I and DCL-II were compared to each other and to immunoglobulins and putative recognition molecules by the parameter S delta Q. DCL-I exhibited similarities in amino acid composition to lectins from the tunicate Halocynthia pyriformis, the lamprey Petromyzon marinus, and the horseshoe crab Carcinoscorpius rotundicauda, rabbit C-reactive protein, and lamprey and carp immunoglobulin mu chains. DCL-II showed amino acid composition and similarities with several fish immunoglobulin light chains, immunoglobulin-related molecules isolated from mouse and marmoset T cells, and carp and goldfish immunoglobulin heavy chains.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1986

Galactosyl-binding lectins from the tunicate Didemnum candidum. Carbohydrate specificity and characterization of the combining site.

The plasma of the ascidian Didemnum candidum possesses lectin activity directed toward galactosyl moieties. We report the characterization of the affinity chromatography-purified galactosyl-binding lectins from the plasma of this protochordate species in terms of their hemagglutination patterns, temperature stability, saccharide specificities, divalent cation requirements, and the comparison of the properties of their combining sites to those of other characterized lectins. The major galactosyl-specific lectin, termed DCL-I, has an apparent mass of 14,500 daltons and a minor lectin (DCL-II) has an apparent subunit mass of 15,500 daltons. The two molecules differed somewhat in their hemagglutination profiles with untreated and enzyme-treated erythrocytes: a 10-fold increase in DCL-II concentration is required to obtain agglutination titers comparable to those of DCL-I. Although both DCL-I and DCL-II will agglutinate neuraminidase-treated erythrocytes from all vertebrate species tested and most Pronase-treated erythrocytes, DCL-I will agglutinate some untreated erythrocytes which are not agglutinated by DCL-II. Both lectins required divalent cations, were inactivated by temperatures above 70 degrees C, and both exhibited optimal agglutinating activity over a wide range of pH (from 5 to 11). The DCL-I molecule was characterized for its saccharide specificity by binding and inhibition assays using characterized sugars and glycoproteins. Galactose and oligosaccharides bearing nonreducing terminal galactose were the best inhibitors. The inhibition analysis indicated that the DCL-I combining site is small, interacts only with hydroxyls on carbons 2, 3, and 4 of galactose, and exhibits moderate steric hindrance for voluminous groups on carbon 6 and the alpha-anomeric linkage. The data suggest that the combining site would be smaller than the peanut lectin combining site for galactose since DCL-I does not interact with the subterminal monosaccharide hydroxyls for C4 and C6 as does peanut agglutinin. To our knowledge, this is the first isolation and detailed characterization of a lectin from a protochordate species.

Binding Sites↗
PubMed1986

Conservation among the immunoglobulins of carcharhine sharks and phylogenetic conservation of variable region determinants.

To study primitive vertebrate recognition molecules we have purified the immunoglobulins of three species of carcharhine sharks. The tiger (Galeocerdo cuvieri), the sandbar (Carcharhinus plumbeus) and the galapagos sharks (Carcharhinus galapagensis) possess 18S high molecular weight and 7S low molecular weight immunoglobulin forms. Both within and between species these forms closely resemble each other in polypeptide chain composition, heavy chain mass, carbohydrate content, amino acid composition, antigenic character and amino terminal sequence. These carcharhine species are separated by at least 30 million years of evolutionary time and it is remarkable that so little diversification of immunoglobulin structure has occurred during their evolution. By several criteria carcharhine immunoglobulins most closely resemble mammalian IgM. Studies of serological cross-reactions indicate that some immunoglobulin determinants have been conserved over a broad phylogenetic range of vertebrate classes; most notably JH and VHa-related markers are shared between forms as diverse as sharks and mammals.

Amino Acids↗
PubMed1986

Heterogeneous humoral and hemocyte-associated lectins with N-acylaminosugar specificities from the blue crab, Callinectes sapidus Rathbun.

Callinectes sapidus serum and hemocyte microsomal fraction agglutinated a panel of untreated and enzyme treated vertebrate erythrocytes and cultured lymphoid cell lines. Crossed absorption experiments suggested the presence of multiple specific lectins in the serum. The microsomal fraction showed a 35-fold increase in specific activity when compared to the hemocyte lysate suggesting that hemocyte lectins are membrane-associated. Agglutination by serum and hemocyte lectins was inhibited by low concentrations of N-acylamino compounds including sialic, N-acetylmuramic and N-acetylglutamic acids, GalNAc, GlcNAc, ManNAc, and glycoproteins and polysaccharides which contain these carbohydrates: bovine submaxillary mucin, human orosomucoid, porcine stomach mucin and colominic acid. Hemagglutination by lectins of both serum and hemocyte microsomal fraction required divalent cations as suggested by the reduction in hemagglutination titer in the presence of the chelators EDTA, EGTA, CDTA and citrate.

Amino Sugars↗
PubMed1986

Antibodies to synthetic joining segment peptide of the T-cell receptor beta-chain: serological cross-reaction between products of T-cell receptor genes, antigen binding T-cell receptors, and immunoglobulins.

Immunoglobulin light and heavy chains show sequence homology to one another and to the polypeptide chains of putative T-cell receptors in the J (joining) segment of the variable region. Antibodies produced against synthetic peptides corresponding to the entire JH1 region and part of the diversity segment region cross-react serologically with products of normal T cells and monoclonal T-cell lines. In this study we generate immune affinity-purified rabbit antibodies to a synthetic 16-mer peptide consisting of the entire JT sequence and part of the T-cell diversity sequence corresponding to these segments of the human putative T-cell receptor beta gene YT35. Both free peptide and peptide coupled to bovine serum albumin as carrier were found to stimulate the production of antibody. The immune affinity-purified anti-JT peptide antibodies bound to intact immunoglobulin and to light and heavy chain as detected by enzyme-linked immunosorbent assay and by immunoblot transfer. The antibody reacted by these techniques with membrane components of the human monoclonal amplifier T-cell MOLT-3 and the murine suppressor T-cell WEHI-7. The component detected in the MOLT-3 cell corresponded to the beta-chain of the alpha/beta heterodimer putative T-cell receptor; whereas the molecule detected in the WEHI-7 line had properties corresponding to those of antigen-specific T-cell suppressor receptors. The molecular size of this component under reducing conditions was approximately 68 kDa and the intact form had an apparent mass of 140 kDa. These results provide direct proof of serological cross-reaction among products of putative T-cell receptor genes, antigen-binding T-cell receptors, and immunoglobulins, thereby supporting the concept that antigen receptors of T lymphocytes all represent new immunoglobulin translocons.

Animals↗
PubMed1986

Studies on the relationship of the B700 and B50 murine melanoma antigens.

The B50 and B700 proteins of B16 murine melanoma were studied; they were determined to be distinct, unrelated molecules. This was determined by V8 peptide mapping, N-terminal amino acid sequencing, absence of cross-reactivity with specific polyclonal antibodies, and monoclonal antibodies recognizing different epitopes.

Amino Acid Sequence↗
PubMed1985

Surface expression and partial characterization of an arsonate hapten-specific idiotype-bearing T-cell receptor.

Anti-idiotype antibodies raised against the arsonate hapten idiotype have been used to detect arsonate-binding receptors on the surface of peripheral T cells of A/J mice and to isolate this material after biosynthetic labeling for partial chemical characterization. It was found that 2-3% of splenic T cells from arsonate-immune mice specifically bound the hapten using immunofluorescent keyhole limpet hemocyanin as a carrier. In double-immunofluorescence labeling experiments, a high proportion (approximately equal to 70%) of these cells also bound the (Fab')2 fragment of rabbit anti-idiotype antibody in exactly the same patches on the cell as the arsonate hemocyanin antigen. In addition, the anti-idiotype antibody inhibited the binding of the hapten-carrier complex to T cells by approximately equal to 70%. In parallel experiments, fowl antibodies against mouse (Fab')2 fragments bound to 100% of arsonate-binding T cells in the same cell-surface patches as the hapten, and were capable of inhibiting 100% of the hapten-binding cells. Capping, shedding, and resynthesis experiments indicated that the T cells synthesized their antigen-binding idiotype-bearing receptors. Immunoblots of unreduced detergent extracts of purified splenic T cells developed with anti-idiotype antibodies showed bands at 150,000 and 94,000 Da. Equal amounts of protein extracted from liver and analyzed in the same gels as the T-cell material failed to show any reactivity with anti-idiotype antibodies. To confirm the biosynthetic origin of the idiotype-positive materials, detergent extracts from 75Se-methionine- or [3H]leucine-labeled Con A-treated splenic T cells were reacted with anti-idiotype antibodies and the bound material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of 2-mercaptoethanol the major band was at 68,000 Da, with variable minor levels of material at 45,000 Da, while when hapten was used to isolate the receptor a dominant 25,000- to 30,000-Da band was seen. We believe that the higher-molecular-weight materials are multimers of the 25,000-30,000 subunit.

Animals↗
PubMed1985

Humoral and cell membrane-associated lectins from invertebrates and lower chordates: specificity, molecular characterization and their structural relationships with putative recognition molecules from vertebrates.

Based on their carbohydrate-binding properties and their ubiquitous presence in pro- and eukaryotes, recognition functions have been hypothesized for many humoral and tissue lectins. In this review three main topics relevant to the possible biological roles and evolution of invertebrate and chordate lectins are discussed. They include the broad carbohydrate-binding spectrum of lectins from chelicerata, the distribution and specificity of cell membrane-associated lectins in mollusks and the serological and biochemical characterization of lectins from tunicates and their structural relationships with putative non-self recognition molecules from vertebrates.

Amino Acid Sequence↗
PubMed1985

Broad phylogenetic expression of heavy-chain determinants detected by rabbit antisera to VHa allotypes.

Immunoglobulin molecules from diverse vertebrate species were examined, using an enzyme-linked immunosorbent assay (ELISA), for the expression of determinants detectable by rabbit antisera to VHa allotypes. The data indicate that immunoglobulins of elasmobranchs, teleosts, amphibians and birds express determinants cross-reactive with those specified by the a1, a2 and a3 alleles in the domestic rabbit. We localize VHa cross-reactive specificity to the denatured heavy chain of a primitive vertebrate, the Galapagos shark (Carcharhinus galapagensis). Furthermore, the N-terminal amino acid sequence of the shark heavy chain shows significant homology with rabbit heavy chains of known VHa type at positions where allotype-correlated differences have been implicated. VHa-related determinants are shared by immunoglobulins of a wide range of vertebrates from sharks to man and thus seem to be epitopes which have been conserved during vertebrate evolution. The determinants detected on immunoglobulins of lower vertebrates by rabbit anti-VHa allotype sera most probably are VH-subgroup rather than allotypic markers. Their distribution demonstrates a strong phylogenetic conservation of VH-regions.

Amino Acid Sequence↗
PubMed1985

Segmental homology between T-cell receptors and immunoglobulin variable regions: evidence that antisera to synthetic JH1 peptide react with murine and human T-cell products.

To determine precisely the nature of serological determinants shared between T-cell surface molecules and immunoglobulin variable regions, the capacity of antisera directed against a synthetic peptide corresponding to the entire JH 1 region of classical immunoglobulin plus five residues of the D region were tested for their capacity to bind to T-cell membranes and isolated T-cell products. The anti-JH 1 antisera reacted with normal and monoclonal in vitro grown T-cell lines as judged by microhemagglutination and binding in enzyme-linked immunosorbent assays. Immunologically cross-reactive membrane components disclosed by immunoblot transfer analysis ("Western blots") consisted of major components in the molecular weight range 30-35,000 and minor components in the range 65-70,000. The major product of the human T-cell leukemia line MOLT-3 had an approximate mass of 34,000 Da, a value consistent with the predicted size of the molecule specified by the recently described putative T-cell receptor gene YT35. The 65 to 70,000-Da components are most probably tightly associated dimers of the 30 to 35,000-Da forms. It was possible to align the JH sequences of molecules reactive with the anti-JH 1 antisera and other characterized VH sequences of molecules known to be cross-reactive with T-cell products. This facilitated a comparison disclosing clear segmental homology between the protein sequence derived from the YT35 gene and immunoglobulin VH framework regions sharing approximately 50% of sequence identity. The identification of VH-related T-cell products (termed VT-bearing molecules) with products of putative T-cell receptor genes gained further support by N-terminal sequence of the 68,000-Da product of the 70-N2 T-cell line which showed homology to the predicted N-terminal region of the YT35 product. These serological and protein chemical data, coupled with the comparison to gene sequence, show that T-cell components that bear serological determinants cross-reactive with VH show segmental homology with products of putative T-cell receptor genes and immunoglobulin VH.

Animals↗
PubMed1984

A lectin on the hemocyte membrane of the oyster (Crassostrea virginica).

Using antisera produced against a serum lectin we have shown by employing immunocytofluorescence that hemocytes from the oyster, Crassostrea virginica, possess a lectin which is situated on the external surface of the cell membrane. The antisera block the binding of hemocyte microsomes to protease-treated vertebrate erythrocytes, thus confirming that the hemocyte membrane lectin is serologically related to the serum lectin. The major serum lectin has an apparent mass of 34,000. Flow cytometry has revealed that the distribution of the surface lectin on hemocytes represents a heterogeneous expression on a population basis, but no discrete cell subpopulations can be identified.

Animals↗
PubMed1984

Partial characterization of T cell components related to defined VH (VT) markers.

Certain antigen-binding surface molecules and factors of T cells possess serological determinants related to immunoglobulin (Ig)-heavy-chain-variable regions (VH). We obtained sufficient quantities (greater than 100 micrograms) of homogenous VH-related T-cell molecules (VTM) for biochemical studies from normal murine thymocytes and by growing large quantities of monoclonal T-cell leukemia lines expressing the determinants. A solid phase immune adsorbent prepared from the IgG fraction of rabbit anti-IgT serum was used to isolate VTM from formic acid-solubilized T cells. The VTM from murine thymocytes and T-cell lines had Mr of 65,000-68,000. The VTM from distinct cell lines differ by isoelectric focusing and resolution of tryptic peptides indicating clonal restriction. VTM lack conventional light- or heavy-chain-constant region determinants but cross-react with antisera directed against defined VHa allotypes and JH peptides. The detection of a cross-reaction with a synthetic JH peptide is consistent with recently published data identifying JH-related sequences in putative T-cell receptor genes. The amino acid compositions of the VTM were distinct from those of mammalian Ig, major histocompatibility complex (MHC) antigens, and viral glycoproteins, but significant similarities occur with Ig V regions or heavy chains of primitive vertebrates. The results indicate that the VH-bearing T-cell products are not classical Ig, but bear limited VH-cross-reactive determinants.

Amino Acids↗
PubMed1984

Amino-terminal variation in melanoma antigens.

Melanoma tumors express both common antigenic determinants and individually specific markers. A melanoma-specific glycoprotein antigen ( B700 ) with a molecular weight of approximately 65,000 daltons was detected on murine B16 melanoma cells but appears on other murine and human melanoma tumors. In order to determine the relationship between the B700 antigen and other melanoma antigens which have been described and to elucidate molecular changes that have taken place in the transformation from melanocyte to melanoma, we have purified the B700 glycoprotein to homogeneity. We have carried out amino acid composition analysis and partial sequence determinations and report that the B700 melanoma antigen shows similarities to serum albumin, but is not identical to this normal component. Moreover, amino-terminal variation occurs in the first 15 residues of the B700 antigen produced by separate B16 tumors.

Amino Acid Sequence↗