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C Milstein

Publications and source records attributed to C Milstein.

At least 127 records · Page 7Linked to original sources
PubMed1985

Sequence and evolution of the human T-cell antigen receptor beta-chain genes.

We present the nucleotide sequences of the two genomic constant (C)-region gene segments, C beta 1 and C beta 2, encoding the beta chain of the human T-cell antigen receptor. The two C beta genes are organized identically to each other and to the corresponding mouse genes, both having four exons, whose boundaries were confirmed from the sequence of a C beta 2 cDNA clone from the T-cell line MOLT-4. The predicted amino acid sequences of human C beta 1 and C beta 2 differ at only five positions, which suggests that the proteins have very similar functions. This similarity is the result of strong nucleotide-sequence conservation in protein-coding regions, which extends to silent positions. A quantitative analysis of an alignment of the nucleotide sequences of the two human genes shows that whereas the 5' ends (including the first exon) are extremely homologous, the 3' ends are widely divergent, with other regions having intermediate levels of homology. Analysis of published data [Gascoigne, N.R.J., Chien, Y., Becker, D.M., Kavaler, J. & Davis, M.M. (1984) Nature (London) 310, 387-391] shows that the mouse C beta 1 and C beta 2 genes are also virtually identical in their first exons but more divergent in the remaining coding regions. Therefore, partial gene conversion events may have occurred during the evolution of both human and mouse C beta genes.

Amino Acid Sequence↗
PubMed1984

Development and application of a monoclonal rat peroxidase antiperoxidase (PAP) immunocytochemical reagent.

Monoclonal antibodies are being increasingly used in immunocytochemistry but their localisation by the peroxidase antiperoxidase (PAP) procedure requires the use of rat or mouse PAP. In this paper we describe the development and application of a monoclonal rat PAP. This reagent has been used successfully for immunocytochemistry at light and electron microscopy level in combination with rat monoclonal antibodies against serotonin (5-HT), substance P and somatostatin. The monoclonal rat PAP has several advantages over conventional polyclonal rat PAP and is likely to be a valuable developing reagent in immunocytochemistry using rat monoclonal antibodies.

Animals↗
PubMed1984

Characterization and immunocytochemical application of monoclonal antibodies against enkephalins.

Monoclonal antibodies were produced following immunization of mice with either [Leu5]enkephalin-bovine serum albumin or [Met5]enkephalin-keyhold limpet hemocyanin conjugates. Two monoclonal antibodies coded NOC1 and NOC2, respectively, were derived. These monoclonal antibodies did not discriminate between Leu- and Met-enkephalin in either radioimmunoassay or immunocytochemistry. NOC1 was characterized in detail. In radioimmunoassay NOC1 displayed about 40% crossreactivity with C-terminal extended Met-enkephalin hexapeptides and 7% with the extended heptapeptide (-Arg-Phe-OH), but did not recognize other endogenous peptides. In immunocytochemistry the NOC1 and NOC2 recognized all well-established "enkephalin immunoreactive sites," but they did not bind to areas known to contain beta-endorphin or high levels of pro-enkephalin. NOC1 was shown to be a suitable tool to demonstrate enkephalin immunoreactive sites by radioimmunocytochemistry utilizing both internally and externally labeled monoclonal antibodies.

Amino Acid Sequence↗
PubMed1984

Intracellular processing of membrane and secreted immunoglobulin delta-chains.

Membrane-bound and secreted immunoglobulin delta-chains are synthesized by the mouse hybridoma B1-8 delta.1 as two primary translation products (45,000 and 42,000, respectively) and are converted into three N-glycosylated forms. In addition to N-glycosylation, another modification, reflected in a size increase of 2000 to 3000, occurs within 8 min of synthesis and may be O-glycosylation. After these initial modifications, the N-linked carbohydrates of all three forms are partially trimmed, apparently in the endoplasmic reticulum. The secreted delta-chains acquire galactose and sialic acids less than 10 min before they are secreted. Monensin and CCCP, which are potent inhibitors of IgD secretion, inhibit the terminal glycosylation but not the other modifications of delta-chains. CCCP blocks the intracellular transport of IgD at an earlier stage of trimming than monensin. Within 10 min of the removal of CCCP, some of the accumulated IgD is terminally glycosylated and secreted. Membrane IgD is processed similarly to secreted IgD up to the stage blocked by CCCP. These observations suggest that final trimming and terminal glycosylation of IgD occurs in the Golgi complex, and that the other modifications occur in pre-Golgi compartments, where secreted IgD spends most of its transit time. We suggest that the rate-limiting step in the intracellular transport of both types of IgD is the passage from the ER to the Golgi complex.

Animals↗
PubMed1984

Junctional diversity is essential to antibody activity.

Many mechanisms of antibody diversification have been shown to exist, including combinatorial pairings of heavy and light chains, the use of multiple gene segments (combinatorial diversity), and the imprecise joining of these gene segments (junctional diversity). The contributions of each of these mechanisms to functional antibody activity has not been fully explored, especially in the case of junctional diversity. A chain recombination experiment between an anti-arsonate monoclonal antibody and an anti-oxazolone molecule in which light chains differ essentially only at the V/J junctional position show that junctional diversity may play an important role in antigen binding.

Animals↗
PubMed1983

Molecular characterization of a nonsecreting myeloma mutant.

The nonsecreting myeloma mutant, NSII/1, derived from MOPC 21 a mouse myeloma cell line, does not produce L chains and expresses a smaller than normal gamma 1 heavy chain, H* (Cowan, Secher and Milstein, J. Mol. Biol. 1974. 90: 691). The free H* is not secreted, but secretion can be rescued in hybrids expressing a light chain. By sequencing L and H chain-cloned cDNA derived from NSII/1 we find only one light chain mRNA expressed which belongs to an aberrantly rearranged kappa gene. We also find that the H* heavy chain is the result of a point mutation. H* mRNA contains an A instead of the G found in wild type at position 1125. The corresponding tryptophan 406 becomes a chain termination, giving rise to a 67 amino acid deletion at the C-terminus. The H* is core-glycosylated, but its state of assembly is abnormal: H* forms high molecular weight, S-S-bonded complexes. The results are discussed with reference to the putative toxic properties of free heavy chains and the failure of some chains to be secreted.

Animals↗
PubMed1983

Analysis of antibody diversity: V-D-J mRNA nucleotide sequence of four anti-GAT monoclonal antibodies. A paucigene system using alternate D-J recombinations to generate functionally similar hypervariable regions.

The nucleotide sequence of four anti-(Glu60-Ala30-Tyr10)n (GAT) monoclonal gamma 1 heavy chain mRNAs was determined from codon 10 to 120. This sequence overlaps with the NH2-terminal amino acid sequence, allowing elucidation of the complete protein sequence encompassing regions VH, D and JH. These sequences, which are highly conserved, indicate that anti-GAT antibodies expressing the same public idiotypic specificities represent a paucigene system, which uses at least two D-J combinations leading to functionally similar hypervariable regions involved in the recognition of the dominant Glu-Tyr determinant. D regions are encoded by D genes which are closely related either to the D-SP2 or the D.FL16 germ line gene cores.

Amino Acid Sequence↗
PubMed1983

Somatic variants of the level of expression of a cell surface antigen.

Somatic variants with constitutive changes in the expression of the cortical thymocyte differentiation antigen HTA 1 were derived from the T-cell leukemia line Molt 4 using monoclonal antibody NA1/34 and the fluorescent activated cell sorter. Cells with the highest and lowest fluorescence were sorted and expanded. After several cycles, high expressor variants, with 10- to 12-fold increased surface HTA 1 and low expressors, with a level of one third relative to the wild-type, were obtained. The stability of the high expressors was improved by cloning. In spite of the large differences in the level of HTA 1 between the various mutants and the wild-type, the expression of HLA-A,B,C or its increase following interferon-alpha stimulation, remained unchanged. Although in HTA 1 there was only a trace of beta2-microglobulin (beta2m) detected by lactoperoxidase labelling of the high expressor variants, significant levels of both beta2m and HTA 1 light chain beta(t) were observed in gels stained with Coomassie blue. The physiological association of beta(t) with HTA 1 was confirmed by the substantial increase of beta(t) which parallels the increase of HTA 1.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1983

The discrepancy between the cross-reactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity.

The rat monoclonal antibody YC5/45 has been shown to react specifically with serotonin containing neurones, and does not detect dopamine containing sites. On the other hand the monoclonal antibody recognizes dopamine, since it is capable of inhibiting the binding of 3H-YC5/45 to sheep red blood cells coated with serotonin albumin conjugate, or the direct agglutination of the same cells by the monoclonal antibody. Tryptamine and 5-methoxytryptamine are even better inhibitors, while derivatives with a modified-CH2-CH2-NH2 group are inactive. Treatment of the active compounds with paraformaldehyde increases their efficiency dramatically. The binding of antibody was also tested by cross linking with paraformaldehyde: dopamine, serotonin, tryptamine, 5-methoxytryptamine etc., to serum albumin. Only the serotonin derivative was active. The result shows that the discrimination specificity observed in immunohistochemistry is due to the paraformaldehyde fixation. It is concluded that for histochemical detection of serotonin by YC5/45, two reactive sites are necessary. The -CH2-CH2-NH2 group is probably modified by formaldehyde to produce a cyclic derivative to form the antibody recognition site. A different formaldehyde reactive site is required for cross linking to the tissue and this is provided by the presence of the 5-OH of serotonin which is absent in the others.

Animals↗
PubMed1983

Is beta t a component of HLA-A,B,C in thymus derived cells?

beta t, the 12,000 molecular weight polypeptide originally found in HTA 1, a thymus specific differentiation antigen, is a major labelled component of surface iodinated HLA-A,B,C purified by monoclonal antibody W6/32 from the T leukaemia cell line Molt 4. A correlation between the amount of beta t in HLA-A,B,C and the level of HTA 1 expression in Molt 4 and variants derived from it was established. The presence of beta t in the HLA-A,B,C complexes is not due to cross contamination with HTA 1. However, analysis of a large scale HLA-A,B,C preparation using Coomassie blue staining shows that, under conditions of surface iodination, beta t is over-represented by a factor of about 100 times relative to beta 2 microglobulin. The significance of beta t as a minor component of HLA-A,B,C in thymus derived cells remains uncertain.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1983

Anti-oxazolone hybridomas and the structure of the oxazolone idiotype.

Antibodies raised in several mouse and rat strains against the hapten 2-phenyloxazolone (phOx, "oxazolone") regularly contain a fraction recognized by antiidiotypic reagents. We have studied this response in BALB/c and DBA/2 mice by generating over fifty anti-phOx antibody-secreting hybridoma clones. The hybridization was performed either 7 or 14 days after a primary immunization with phOx-protein conjugate. Most of the hybrids secreted IgG1. Whereas over 80% (17/21) of IgG-producing hybrids from day-7 fusions secreted oxazolone-idiotype positive immunoglobulin, all hybridomas originating from day-14 fusions were idiotype negative. The mRNA for heavy (H) and light (L) chains of three idiotype-positive and one idiotype-negative IgG1 hybridomas were sequenced by a modification of Sanger's dideoxynucleotide method of DNA sequencing, using crude mRNA as template, synthetic oligonucleotides as primers, and reverse transcriptase to incorporate both dideoxynucleotides and labeled deoxynucleotides. The sequence of the mRNA coding for the whole variable region of each chain was established using primers complementary to the constant region near the V-C boundary and another two that coded for a framework segment in either VH or VL. This method not only provided more information than protein sequencing but was also faster and simpler. The mRNA preparation did not need fractionation beyond the poly A-containing fraction. The sequences of the H and L chain mRNA of the three idiotype-positive anti-oxazolone antibodies were extremely similar or identical, and from them a tentative oxazolone-idiotype basic sequence was derived. Only three nucleotide differences were detected; these occurred in the D segment of one H chain mRNA, in the V-J boundary of one of the light chain mRNA, and in the first hypervariable region of another. The idiotype-negative antibody had a totally different H chain mRNA and a light chain mRNA that differed by 21 bases, almost all affecting the amino acid sequence.

Amino Acid Sequence↗
PubMed1982

Control of HLA-A,B,C synthesis and expression in interferon-treated cells.

Cytofluorimetric analyses with monoclonal antibodies were used to follow the specific effect of highly purified human leucocyte interferon (IFN-alpha) on the expression of transplantation antigens (HLA)1 in Molt 4, a thymus leukaemia cell line. After a lag period of approximately 10 h the amount of HLA steadily increases to reach a maximum at day six of approximately 10 times its original. The effect is reversible and due to an increase in the rate of synthesis of HLA and beta 2m molecules as demonstrated by analysis of [35S]methionine incorporation into specific proteins after a 3-h pulse of Molt 4 cells treated with IFN-alpha for one or six days. A dramatic increase in the amount of mRNA that hybridised with a [32P]cDNA probe containing HLA sequences has also been demonstrated. Surface iodination of IFN-treated Molt 4 cells showed an increase in the iodinated HLA and beta 2m chains and in addition another, apparently unrelated, component with an apparent mol. wt. of 16 000. This component, which appears after incubations with IFN-alpha for longer than 1 h at 37 degrees C, was also detected in the B lymphoid cell lines RAJI and DAUDI.

Antigens, Surface↗
PubMed1982

Immunocytochemistry with internally labeled monoclonal antibodies.

One of the advantages of the production of monoclonal antibodies by tissue culture methods is that they can be internally labeled by using appropriate radioactive amino acid in the culture fluid. Thus, radioactive immunological probes of high specific activity can be prepared. Here we report applications of these internally labeled monoclonal antibodies for the direct localization of immunoreactive sites in the central nervous system of the rat at both light and electron microscopic levels (radioimmunocytochemistry). We explored the combined use of radioimmunocytochemistry and immunoenzymatic methods for the simultaneous detection of two antigenic sites: substance P and serotonin or substance P and enkephalin. In neurobiology this procedure could help to clarify certain aspects of transmitter-specific synaptic interactions and the coexistence of neuroactive substances in single neuronal cell bodies or nerve terminals. We also describe the application of radioimmunocytochemistry with internally labeled monoclonal antibodies to quantify the immunoreactions in discrete microscopic areas.

Antibodies, Monoclonal↗
PubMed1982

Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line.

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.

Animals↗
PubMed1982

Chemical typing of human kappa light chain subgroups expressed by human hybrid myelomas.

Hybrids between human spleen cells and the non-secretor NSO mouse myeloma, and also between the rat non-secretor line YB2/O and human peripheral blood cells were prepared. After a month in culture very few hybrids retained the ability to secrete the human kappa light chain. From these, clones could be derived which remained stable over several months of continuous culture. On incorporating [3H]-Leu into the culture medium the cells secrete large amounts of radioactive light chain. It is shown that, even without dialysis, the purity of the preparation is sufficient for an automatic N-terminal sequence analysis at the radioactive level. From the pattern of distribution of leucine in the first twenty-two amino acid residues, it is possible to assign the synthesized light chains to one of the four established human subgroups. The method permits a fast and simple classification of human light chains secreted by hybrid myelomas. Although tested with rodent x human hybrids, we see no reason why the method could not equally apply to human x human hybrids.

Animals↗