Energy balance in subcutaneous melanoma-bearing mice

Energy balance of control and melanoma-bearing mice was determined after 15 days of controlled food intake. Body and tumor energy contents were evaluated after preparing the materials for bomb calorimetry. Neither energy intake nor expenditure was different between control and melanoma mice. Body energy gain was lower in melanoma mice, but including tumor energy both groups had similar energy gain during the experimental period. It is concluded that a two-week skin melanoma did not seriously affect the energy balance of mice, and that tumor growth was achieved by utilizing body energy without promoting any increase in energy intake.

Asn-linked oligosaccharide-dependent interaction between laminin and gp120/140. An alpha 6/beta 1 integrin.

Receptor-mediated recognition and adhesion to laminin, a specific glycoprotein from basement membranes, exert an important role in many biological phenomena. Studying cell surface proteins of B16-F10, a metastatic murine melanoma cell line, we identified a 120-140 kDa glycoprotein (gp120/140) that binds laminin. This glycoprotein was recognized by a polyclonal antibody raised against the human fibronectin receptor beta 1-integrin chain, as well as immunoprecipitated by an anti-alpha 6 chain (monoclonal antibody GoH3), characterizing it as an alpha 6/beta 1-integrin. Its binding to laminin was specific and displayed moderate affinity, as its apparent dissociation constant was 18 nM. To characterize the influence of carbohydrate moieties on the laminin-gp120/140 interaction, metaperiodate oxidation, metabolic inhibition of glycosylation, and enzymatic deglycosylation studies were performed. Our results indicate that gp120/140 Asn-linked oligosaccharides play a part in this interaction. Reciprocally, both metaperiodate and N-glycanase treatment of native laminin reduced its binding to gp120/140, characterizing the latter as a lectin-like molecule. These results point to glycosylation processes as a possible mechanism for variable binding specificity profiles among integrins.

Identification and characterization of highly conserved antigenic determinants in the laminin molecule.

1. Fragments P1 and E8, the result of two different enzymatic digestions of the laminin molecule, represent interaction sites of laminin with specific cell receptors. By using negative and positive affinity purification of a rabbit antiserum against mouse laminin we have generated antibodies to these two fragments. 2. Antibodies against P1 were able to immunoprecipitate fragment E8 from elastase-digested laminin. By liquid phase competition experiments we demonstrated that the epitopes shared by P1 and E8 are a minor portion of the antigenic determinants of P1. When we checked for the presence of these shared epitopes in the human laminin molecule, they were the major fraction of the interspecies antigenic conservation. 3. A similar approach using polyclonal antibodies against human laminin has confirmed these results. 4. The shared epitopes present in both mouse and human laminin molecules seem to be spatially determined, because antibodies against these sites did not bind to fully denatured laminin. 5. Since human and mouse laminin bind to cell receptors and to other extracellular matrix proteins from both species, we conclude that these antigenic determinants may represent the actual sites for at least some of these interactions.

Identification and characterization of highly conserved antigenic determinants in the laminin molecule

1. Fragments P1 and E8, the results of two different enzymatic digestions of the laminin molecule, represent interaction sites of laminin with specific. By using negative and positive affinity purification of a rabbit antiserum against mouse laminin we have generated antibodies to these two fragments. 2. Antibodies against P1 were able to immunoprecipitate fragment E8 from elastase-digested laminin. By liquid phase competition experiments we demonstrated that the epitopes shared by P1 and E8 are a minor portion of the antigenic determinants of P1. When we checked for the presence of these shared epitopes in the human laminin molecule, they were the major fraction of the interspecies antigenic conservation. 3. A similar approach usisng polyclonal antibodies against human laminin has confirmed these reults. 4. The shared epitopes present in both mouse and human laminin molecules seem to be spatially determined, because antibodies against these sites did not bind to fully denatured laminin. 5. Since human and mouse laminin bind to cell receptors and to other extracellular matrix proteins from both species, we conclude that these antigenic determinants may represent the actual sites for at least some of these interactions

A murine nephritogenic monoclonal anti-DNA autoantibody binds directly to mouse laminin, the major non-collagenous protein component of the glomerular basement membrane.

The interaction of the murine monoclonal anti-DNA antibody H241 with extracellular glomerular antigens was found to be due to the binding of this antibody to laminin, the major non-collagenous protein constituent of the glomerular basement membrane. This interaction is specific, since it is inhibited by laminin, double-stranded DNA and single-stranded DNA in solution. Furthermore, the binding of H241 to mouse laminin is mediated by conformational properties of the antigen because mild denaturation of laminin strongly decreases the binding capacity of H241, while exposure of laminin to sodium dodecyl sulfate, completely abolishes this interaction. H241 is able to bind to both, human and mouse laminin. These findings are in agreement with the ligand binding specificities of the autoantibodies spontaneously produced, that differ from those generated by artificial immunization. We conclude that the polyreactivity of H241 that confers to it the capacity to bind laminin, may account for its ability to form immune deposits by binding directly to non-DNA glomerular antigens.

Characterization of the cellular receptor for fibronectin through a hydropathic complementarity approach.

It has been shown that a significant correlation is seen when the hydropathy scores of amino acids encoded by the coding strand of double-helical DNA are plotted against those of the noncoding strand. Thus, peptides encoded by complementary DNA strands might form amphiphilic structures and bind one another. We have used this approach to study the interaction between fibronectin (FN) and its cell receptor. Taking into consideration the nucleotide sequence from published rat cDNA clones that corresponds to the cell binding site (Arg-Gly-Asp-Ser) in the FN molecule, the deduced amino acid sequence found for the putative receptor binding site was Trp-Thr-Val-Pro-Thr-Ala. This peptide was chemically synthesized and coupled to an AH-Sepharose column. FN bound appreciably to this column and was eluted much more efficiently by a solution of Arg-Gly-Asp-Ser-containing peptide than by a solution of related but inactive Arg-Gly-Glu-Ser-containing peptide. Binding of labeled FN to receptor-rich MG63 human osteosarcoma cells was inhibited by the hexapeptide. The hexapeptide Gly-Ala-Val-Ser-Thr-Ala predicted similarly from the nucleotide sequence of human FN was equally efficient in such inhibition. Antibodies produced against Trp-Thr-Val-Pro-Thr-Ala recognized with equal efficiency Gly-Ala-Val-Ser-Thr-Ala in an ELISA assay. Furthermore, they were able to recognize a single 140-kDa band in whole-cell extracts from Chinese hamster ovary cells, attesting to their specificity. Identification of the recognized protein was provided by showing that this antibody was also able to bind to affinity-purified FN receptor from human osteosarcoma MG63 cells.

Inhibition of idiotype--anti-idiotype interaction for detection of a parasite antigen: a new immunoassay.

Described in this report is an immunoradiometric assay of general applicability that is based on a new principle: the inhibition of the interaction between monoclonal antibodies by an antigen. The advantages of this assay are that it measures concentrations of single epitopes, purified antigen is not required, and the reagents can be obtained in unlimited amounts and are homogeneous. Its features are particularly attractive when the antigen has not been purified and is a minor component of a complex mixture of molecule.

Biosynthesis of Pb44, the protective antigen of sporozoites of Plasmodium berghei.

In a previous paper (2) we identified a protective antigen (Pb44) of the surface membrane of sporozoites of Plasmodium berghei by means of a monoclonal antibody. Immunoprecipitation of extracts of mature salivary gland sporozoites, metabolically labeled with L[35S]methionine using the same monoclonal antibody, revealed three specific polypeptides: *Pb44, *Pb52, and *Pb54. Metabolically labeled *Pb44 is probably identical to the protective antigen previously identified by surface labeling. Both proteins have the same molecular weights and isoelectric points under denaturing conditions, and they share an epitope. Moreover, *Pb44 also seems to be located on the cell membrane. The results of pulse-chase experiments strongly suggest that *Pb52 is the precursor of *Pb44. The relationship between *Pb54 and the protective antigen is unknown. The three polypeptides seem to be strictly associated with only one of the developmental stage of the parasite. They were not detected in blood forms and were found in minute amounts in sporozoites from the midgut of mosquitoes. In contrast, in mature salivary gland sporozoites they constitute main products of protein synthesis.

Monovalent fragments (Fab) of monoclonal antibodies to a sporozoite surface antigen (Pb44) protect mice against malarial infection.

Monoclonal antibodies (IG1, k) directed against a surface component of Plasmodium berghei sporozoites (Pb-44) confer complete protection to mice against a lethal inoculum of parasites. The degree of protection is a function of the number of parasites used in the challenge and of the antibody concentration in serum. Passive transfer of 10 micrograms of antibody per mouse abolished or profoundly diminished the infectivity of 10(3) sporozoites, but much higher amounts of antibody were required for complete protection against challenge with 10(4) parasites. Fab fragments of the monoclonal antibodies were as effective as the intact antibodies in mediating protection as determined by the neutralizing assay. This observation suggests that the antibodies interfere with a parasite function necessary for its infectivity, such as, for example, the ability to penetrate into the target cell or to multiply in the hepatocytes. When sporozoites are incubated with the intact monoclonal antibodies at 37 degrees C, a long filament appears at its posterior end (circumsporzoite precipitation [CSP] reaction). Fab fragments are ineffective at high concentrations. However, if after treatment with Fab, the sporozoites are incubated with rabbit antibodies to mouse k-chains, a strong CSP reaction is observed. We conclude that the CSP reaction can result from the cross-linking of Pb44 and that it has the characteristics of a capping reaction followed by the shedding of the immune complexes.

Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite.

Hybrid cells secreting antibodies against sporozoites of Plasmodium berghei were obtained by fusion of plasmacytoma cells with immune murine spleen cells. The monoclonal antibodies bound to a protein with an apparent molecular weight of 44,000 (Pb44), which envelopes the surface membrane of sporozoites. Incubation of sporozoites in vitro with antibodies to Pb44 abolished their infectivity.