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J H Freed

Publications and source records attributed to J H Freed.

At least 73 records · Page 4Linked to original sources
PubMed1985

Murine Ia-associated invariant chain's processing to complex oligosaccharide forms and its dissociation from the I-Ak complex.

The processing of murine invariant chain (Ii) to a cell surface form bearing complex N-linked oligosaccharides has been demonstrated in the B cell lymphoma, AKTB-1b. In addition, the rate of processing of pulse-labeled Ii has been determined relative to its rate of dissociation from the alpha/beta complex of I-Ak. Ii, alpha-, and beta-chains were immunoprecipitated with anti-I-Ak or anti-Ii monoclonal antibodies. The heretofore uncharacterized complex oligosaccharide form of Ii (Ii-c) was identified in gel-purified immunoprecipitates by peptide mapping with reverse-phase HPLC. Ii-c is resistant to deglycosylation by Endo H, which is specific for high-mannose N-linkages, but can be digested with Endo F, a glycosidase capable of cleaving both complex and high-mannose N-linked oligosaccharides. Immunoprecipitation of surface iodinated cells indicates that Ii-c is expressed on the plasma membrane. Pulse-chase metabolic labeling data show that the processing of Ii to Ii-c occurs with a t1/2 of about 120 min. In contrast, the processing of both alpha- and beta-chains of I-Ak to complex forms occurs with a t1/2 of 15 to 20 min. Our data show that Ii-hm begins to dissociate rapidly from the I-Ak complex after 100 to 120 min of chase. Only a small amount (less than 5% on a per mole basis) of Ii-c was found associated with the I-Ak complexes after 300 min of continuous metabolic labeling. These results are consistent with Ii serving as a carrier for Ia antigens as they are transported to the cell surface. In addition, they suggest that the processing of Ii to Ii-c, or a late processing event of the alpha- and beta-chains, such as their sialylation, may be a possible mechanism for inducing the dissociation of Ii from the I-Ak complex.

Animals↗
PubMed1984

Isolation and biochemical characterization of the H-2Kk and H-2Dk antigens from the RDM-4 lymphoma.

Milligram amounts of the H-2Kk and H-2Dk antigens from the RDM-4 lymphoma have been isolated in a one-step procedure employing affinity chromatography on monoclonal antibodies conjugated to Sepharose. The purified antigens have been characterized biochemically, including amino-terminal amino acid sequence and tryptic peptide analyses of the 45,000 mol. wt chains of the antigens. The amino acid sequence results for the H-2Kk antigen, when compared with previously reported data for this molecule, reveal a discrepancy in the sequence of this 45,000 mol. wt chain. We postulate that this discrepancy reflects a previously undescribed polymorphism for the Kk-gene. We also report the first amino-terminal amino acid sequence for the 45,000 mol. wt chain from the H-2Dk antigen and demonstrate that the sequence of the beta 2-microglobulin obtained from the H-2Kk antigen preparation is identical to that published previously. Finally, the structural integrity of the H-2Kk antigen is assessed using binding of beta 2-microglobulin, incorporation into liposomes and reaction with antibodies as criteria.

Amino Acid Sequence↗
PubMed1984

Immunological evaluation of a component isolated from Mycobacterium bovis BCG with a monoclonal antibody to M. bovis BCG.

A component of Mycobacterium bovis BCG referred to as BCG-a was isolated through the combined use of monoclonal antibody directed to BCG and affinity chromatography. Analysis of BCG-a by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single prominent band with a molecular weight of ca. 10,000. Structural characterization of BCG-a consisting of amino acid composition and amino-terminal sequence determination was carried out. The intact BCG-a antigen was bound by neither the lectin from common lentils nor concanavalin A, implying that BCG-a does not carry any asparagine-linked oligosaccharides. Immunoprecipitation of 125I-labeled BCG-a with polyclonal and monoclonal antibodies directed against BCG resulted in bands having the same mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as did free 125I-BCG-a. In radioimmunoassays 125I-BCG-a was bound by the monoclonal antibody and by polyclonal antibodies from rabbits that had been immunized to BCG and to Mycobacterium tuberculosis H37Rv. Antibodies to nontuberculous and to nonacid-fast bacteria bound BCG-a poorly or not at all. The binding of 125I-BCG-a by the monoclonal antibody was readily inhibited by extracts of BCG and H37Rv, but it was not as readily inhibited by extracts of nontuberculous mycobacteria and was not at all inhibited by extracts of nonacid-fast bacteria. Considerable inhibition was similarly observed by surface antigens of nonviable, intact BCG organisms. Delayed cutaneous hypersensitivity reactions to small concentrations of BCG-a were elicited in guinea pigs that had been immunized with BCG or H37Rv antigens, but such reactions were not elicited in unimmunized animals.

Amino Acid Sequence↗
PubMed1983

Stable oligosaccharide microheterogeneity at individual glycosylation sites of a murine major histocompatibility antigen derived from a B-cell lymphoma.

The H-2Kk glycoprotein has been isolated by monoclonal antibody affinity chromatography, and an analysis of the asparagine-linked oligosaccharides present at the two major glycosylation sites has been performed. Antigen obtained from the AKTB-1b B-cell lymphoma that had been labeled with [2,6-3H]mannose for 5 or 21 h or for 5 h followed by a 5-h chase was digested exhaustively with trypsin. Each glycosylation site was then isolated by reverse phase high performance liquid chromatography using a C18 column. After removal from the peptide backbone by the almond emulsin peptide: N-glycosidase, the oligosaccharides from each isolated site were analyzed by gel filtration, ion exchange chromatography, concanavalin A affinity chromatography, and glycosidase treatment to assess the contribution of sialic acid and branching patterns of the oligosaccharide backbones to the overall microheterogeneity. The glycosylation of the H-2Kk antigen derived from several different AKTB-1b tumor preparations was examined during a period covering 1 year, during which time the tumor was passaged continuously in vivo in 2-week cycles. Our results conclusively demonstrate that the pattern of oligosaccharide microheterogeneity at the two glycosylation sites of the H-2Kk antigen derived from AKTB-1b cells is stable and that each site differs as to the specific array of oligosaccharide types found on the fully processed glycoprotein. In addition, this report describes an analytical scheme employing reverse phase high performance liquid chromatography to follow oligosaccharide processing and hydrolysis of the N-glycosidic bond by the peptide: N-glycosidase.

Animals↗
PubMed1983

Assignment of antigenic determinants to separated I-A kappa chains.

The alpha- and beta-chains of the I-A kappa antigen from the AKTB-1b B cell lymphoma were separated by ion-exchange chromatography on CM-Sephadex in the presence of propionic acid and urea. Removal of the denaturants by dialysis produced isolated chains that regained a significant amount of their native configuration. These materials were used with a battery of monoclonal antibodies in a direct binding assay to localize specific alloantigenic determinants to the A alpha kappa or A beta kappa chains. This method allowed the assignment of the nominal specificity Ia. 17 and at least one epitope of the specificity Ia.2 to the A beta kappa chain. Finally, the I-A kappa antigen from the B cell lymphoma AKTB-1b was shown to be identical, by the criterion of tryptic peptide analysis, to that derived from normal B10.BR splenocytes. This constitutes the first demonstration that the polypeptide portion of a tumor-derived class II MHC antigen is identical to that derived from a normal tissue.

Animals↗
PubMed1983

Characterization of the oligosaccharides from the invariant chain associated with murine Ia antigens.

The oligosaccharide side chains of the murine invariant chain (Ii) glycoprotein associated with the Ia antigens have been characterized. Affinity chromatography using a monoclonal antibody column was employed to purify the I-Ak antigen from lysates of the AKTB-1b B cell lymphoma. The invariant chain isolated by this procedure was subsequently digested with proteases, and the resulting glycopeptides were fractionated by reverse-phase high-pressure liquid chromatography (HPLC). The invariant chain appears to contain two glycosylation sites, both of which carry high-mannose oligosaccharides, each with a restricted size distribution and an average composition of Man6GlcNAc2 as judged by gel filtration and alpha-mannosidase digestion. These data, together with the observation that the invariant chain cannot be labeled metabolically with 3H-glucosamine, 3H-fucose, or 3H-galactose, allow the conclusion that the murine invariant chain does not contain complex oligosaccharides and is not O-glycosylated during its association with the Ia antigens.

Animals↗
PubMed1982

Intragenic recombination in an E beta gene for a murine Ia antigen.

The recombinant strain D2.GD was originally typed as I-Ad by serological methods. Indeed, the A alpha and A beta chains of the I-A antigens appear to exhibit normal behavior by the criteria of serology and two dimensional gel analysis. However, the E beta chain encoded by the I-A subregion of this strain, one of the two components of the plasma membrane located I-E antigens produced in D2.GD X A.TFR5)F1 animals, has been demonstrated to be the product of an intragenic recombinational event between E beta genes from the d and b haplotypes. Sequence analysis suggests that the amino-terminal portion of the Eg2 beta chain is derived from the d haplotype and, therefore, that the coding strand for this gene is oriented centromeric leads to telomeric (5' to 3' direction). Finally, these data combined with the data of Rose and Cullen (17) allow the ordering of the genes within the I-A subregion as (H-2K), A alpha, A beta, E beta ... (H-2D).

Amino Acid Sequence↗
PubMed1982

Biochemical documentation of allelic variation of the I-E antigens of the d, k, p, r, and u haplotypes.

The extent of allelic variation of the E alpha and E beta polypeptide chains of the I-E antigens from the H-2d, H-2k, H-2p, H-2r, and H-2u haplotypes is described. E alpha and E beta chains were individually labeled with arginine or lysine and compared by tryptic peptide analysis. The results indicate minimum variability among the E alpha polypeptides encoded by the d, k, p, and r haplotypes. However, the Eu alpha chain differed significantly from the other allelic E alpha gene products. On the other hand, the E beta alleles demonstrated substantial variability with the Ed beta being notably less similar to the other alleles than they are to each other. These findings are consistent with a number of observations regarding the serology and functions of the I-E antigens.

Alleles↗
PubMed1982

The use of the three-phase bone scan in the early diagnosis of heterotopic ossification (HO) and in the evaluation of Didronel therapy.

The purpose of this study is to investigate the use of a three-phase bone scan for early detection of HO formation and as a method of evaluating Didronel treatment. A marked vascular blush and blood pool was noted about the hips sometimes with a normal bone scan and normal X-ray of the hips. This appeared to represent the precursor phase of HO formation since, on repeat scans, the bone scan showed accumulation of the bone-seeking radionuclide usually in 2 to 4 weeks and the X-ray revealed ossification. Fifty-two patients treated with Didronel between October 1978 and December 1979 were reviewed to determine the value of Didronel treatment. There were 23 patients in the series who either showed HO by X-ray on admission or developed HO on follow-up X-rays before beginning Didronel therapy. A three-phase bone scan revealed increased vascularity and accumulation of radioactivity on the bone scan in all areas of ossification on the X-ray and in some areas that did not appear to be involved. The other 29 patients had serial three-phase bone scans, X-ray study, and an alkaline phosphatase determination at approximately 2-week intervals. Didronel treatment was started as soon as the precursor phase of HO was demonstrated on the three-phase bone scan in most of these patients. Nine have not developed ossification that could be seen in X-rays during 3 months of continuing study. Six patients seen at follow-Up during the past year had known HO of 4 to 7 years duration. The three-phase bone scan was used to predict the maturity of HO in these patients. Our study in indicates that increased vascularity precedes rather than being secondary to HO formation as is suggested in the literature. Didronel treatment appears to be most effective if initiated during this precursor phase.

Diphosphonates↗
PubMed1981

Biochemical evidence for trans complementation of structural genes in the expression of I-E antigens in F1 hybrids.

Analysis of the tryptic peptides obtained from the Ia.7-positive, "I-E" antigens produced by 4 F1 combinations derived from strain A.TFR5 has provided biochemical confirmation that I-A/I-E gene complementation can occur in the transconfiguration. The data obtained support the concept that structural genes, as opposed to regulatory genes, are the essential elements involved in this complementation and support the assignment of the I-E subregion of strain A.TFR5 as I-Ek. The data support the following assignments of the genes encoding the E beta chains: B10.A(4R) as E beta k, B10.A(5R) as E beta b, and B10.HTT as E beta s. Finally, data are presented demonstrating that the E beta chain derived from the recombinant D2.GD strain partially differs from the E beta d chain derived from a conventional H-2d haplotype strain, B10.D2.

Animals↗
PubMed1978

Structural studies of the protein portion of the H-2-linked Ia glycoprotein antigens of the mouse: tryptic peptide comparison of products from the I-A and I-C subregions of B10-HTT.

Ia antigens from the I-A8 and I-Ck subregions of the B10.HTT (H-2t3) strain of mice were isolated by indirect immunoprecipitation of arginine-labeled, nonionic detergent-solubilized materials. After biochemical purification the electrophoretically homogeneous 28,000 dalton glycoprotein beta chains from the Ia precipitates were digested with trypsin and the resultant radiolabeled tryptic peptides were compared by analytical ion exchange chromatography. These comparisons reveal that the beta chains of Ia antigens from the A (I-A8) and C (I-Ck) subregions of B10.HTT share only two out of 12 to 14 of their arginine tryptic peptides. Thus these noncross-reactive Ia antigens are structurally quite diverse, and would possess sufficient structural variability to account for their lack of antigenic cross-reactivity.

Amino Acids↗
PubMed1977

The private specificity H-2.4 and the public specificity H-2.28 of the D region are expressed on two independent polypeptide chains.

The antigenic specificities H-2.4 (private) and H-2.28 (public) in the H-2a haplotype are controlled by the D region of H-2 as defined by the available recombinants. In previous studies we have demonstrated by the antibody-induced redistribution method that the antisera against these specificities contain antibodies against at least two different polypeptide chains. We here report the results of the indirect immunoprecipitation of radiolabeled antigens after solubilization with Nonidet-P40. The antisera against the two specificities precipitated from such extracts two different and independent polypeptide chains, indicating that the products of the D region, as presently defined, comprise at least two different molecules. The molecular weight of both chains is approximately 45 000, which is similar to other molecules bearing private H-2 antigenic specificities. Consequently, the chromosomal segment presently defined by recombination studies as the D region, must contain another locus, controlling the second polypeptide chain which is detectable by anti-H-2.28 antisera, besides the H-2D locus which controls the polypeptide chain bearing the private specificity H-2.4 as well as most of the public specificities.

Animals↗
PubMed1977

Similarity of the carbohydrate structures of H-2 and Ia glycoproteins.

The glycopeptides produced by pronase digestion of two H-2K, two H-2D, and three Ia glycoprotein antigens were examined for size and charge. The glycopeptides derived from all of the antigens examined were found to have m.w. of 3250 +/- 200 daltons with a similar and variable composition of sialic acid residues. These data, when combined with the similarity in monosaccharide incorporation, suggest that the general parameters of the carbohydrate structure of the Ia glycoproteins from different I subregions and H-2 glycoproteins are highly similar if not identical.

Animals↗
PubMed1976

Amino acid sequence studies on the branched, synthetic polypeptide antigens of the immune response- 1 gene system.

Three immunogens with side chains of random amino acid sequence, poly (L Phe, l glu)-poly (DL Ala)--poly (L Lys) [(Phe, G)-A--L 223], poly (L Tyr, L Glu)-poly (DL Ala)--poly (L Lys) [(T, G)-A--L 509] and (T, G)-A-L 52, as well as two immunogens with side chains of defined amino acid sequences, GGT-A--L and TG-A--L, were sequenced using a Beckman automated sequenator. Despite the lack of a unique amino acid sequence for the amino terminus, reasonable results for the sequence studies were obtained using the Edman reaction. GGT-A--L and TG-A--L had 70% and 80% of their side chains respectively, with the desired sequence. The three compounds of random amino acid sequence were found to contain a large proportion of their A--L side chains unsubstituted. The side chains had a much greater probability of terminating in the aromatic amino acid than in the glutamic acid. The distribution of the length of side chains and their amino acid sequences was completely heterogeneous.

Alanine↗