Heterologous antisera to Lyt-1+, 2- -derived antigen-binding factor detect a subfactor of an antigen-specific suppressor factor and cell surface proteins on Lyt-1+, 2- and Lyt-1-, 2+ T cells

Rabbits were immunized with TNP-specific Lyt-1+, 2- T cell-derived, antigen-binding proteins (PCI-F) released by T cells sensitized by skin painting with picrylchloride. The resulting antiserum (anti-PCI-F) bound to PCI-F and TNP-specific factors that suppressed delayed hypersensitivity (TSF) known to be comprised of PCI-F and Lyt-2+ -derived polypeptides released by cells sensitized by injection of trinitrobenzenesulfonic acid (TNBSF). Anti-PCI-F bound to T lymphocytes and 68,000 to 72,000 m.w. T cell surface proteins but not B cells on their surface proteins. Anti-PCI-F bound to both Lyt-1+ and Lyt-2+ T cells and surface proteins. A comparison of anti-PCI-F with anti-TSF indicates that anti-TSF contains specificity for Ly-2+ T cell-derived components of TSF and T cells not present in anti-PCI-F. The possibility of multiple isotypes of T cell receptors and antigen-binding molecules is discussed.     

Properties of anti-Lyb-2-mediated B-cell activation and the relationship between Lyb-2 molecules and receptors for B-cell stimulatory factor-1 on murine B lymphocytes

The murine B-cell differentiation antigen Lyb-2 has been shown to be involved in B-lymphocyte activation and has been postulated by some to be related to a receptor for B-cell stimulatory factor I (BSF-1) (H. Yakura et al., J. Immunol. 137, 1475, 1986). Here we have demonstrated that monoclonal antibody (mAB) to Lyb-2 resembles BSF-1 in its ability to activate small resting B cells and enhancement of surface Ia. Anti-Lyb-2 antibodies bound B cells with very high avidity and were able to induce mobilization of cytosolic-free calcium. Anti-Lyb-2 mAB differs from BSF-1 in that BSF-1 but not anti-Lyb-2 is able to synergize with anti-mu in induction of B-cell proliferation. The relation between Lyb-2 molecules and BSF-1 receptors was tested in assays that measure binding of anti-Lyb-2 or BSF-1 in B cells and were found not to compete with each other. It appears that the two B-cell agonists anti-Lyb-2 and BSF-1 may exert their effects on B cells through different cell surface moieties as well as different intracellular pathways.     

Intermolecular cross-linking of vitelline envelope polypeptides predominates in the hardened sea urchin fertilization envelope

At fertilization, the sea urchin egg vitelline envelope (VE) elevates, and a subset of released cortical granule proteins, paracrystalline protein fraction (PCF), associates with the VE to form the fertilization envelope (FE). Cortical granule peroxidase cross-links FE polypeptides by phenolic coupling of tyrosyl residues. We have used an immunological approach to determine which polypeptides are linked together in the hardened FE of Strongylocentrotus purpuratus. Soluble polypeptides were extracted from hardened FEs, and antibodies were prepared in rabbits against the insoluble envelope matrix (FE ghost). Whole immune serum and purified IgGs each reacted with FE ghosts when using an enzyme-linked immunosorbent assay. VEs isolated by means of three published procedures cross-reacted with the immune serum and purified IgGs. Soluble FE polypeptides also cross-reacted with whole immune serum and IgGs owing to the presence of VE polypeptides. Hyalin, a protein not found in FEs, and PCF did not cross-react with antiserum against FE ghosts. To determine which VE polypeptides were cross-linked in the hardened FE, VE polypeptides were immunoblotted by using antiserum against FE ghosts. Most of the VE polypeptides that ranged from 68,000 to 283,000 molecular weight cross-reacted with the antibody.     

Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.     

Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice.

The outer membrane of the hepatitis B virus consists of host lipid and the hepatitis B virus major (p25, gp28), middle (gp33, gp36), and large (p39, gp42) envelope polypeptides. These polypeptides are encoded by a large open reading frame that contains three in-phase translation start codons and a shared termination signal. The influence of the large envelope polypeptide on the secretion of hepatitis B surface antigen (HBsAg) subviral particles in transgenic mice was examined. The major polypeptide is the dominant structural component of the HBsAg particles, which are readily secreted into the blood. A relative increase in production of the large envelope polypeptide compared with that of the major envelope polypeptide led to profound reduction of the HBsAg concentration in serum as a result of accumulation of both envelope polypeptides in a relatively insoluble compartment within the cell. We conclude that inhibition of HBsAg secretion is related to a hitherto unknown property of the pre-S-containing domain of the large envelope polypeptide.     

Biosynthesis of alpha-fetoprotein in cultured hepatoma cells

Pulse and pulse-chase experiments demonstrated that a heterogeneous polypeptide with an apparent Mr = 68,000 was the first intracellular anti-alpha-fetoprotein (AFP)-precipitable polypeptide synthesized by rat Mc-A-RH-7777 hepatoma cells. The 68,000-dalton polypeptide may consist of polypeptides with apparent molecular weights ranging from 68,000 to 70,000. It was the precursor of two intracellular anti-AFP-precipitable polypeptides of 69,000 and 73,000 apparent molecular weight. The latter were secreted into the medium without further processing. The anti-AFP-precipitable polypeptides in both cells and medium incorporated [3H]glucosamine, indicating that these polypeptides are at least partially glycosylated. The 68,000-dalton polypeptide in cells was bound mostly to concanavalin A-Sepharose, whereas the 69,000-dalton polypeptide was entirely unbound. The 73,000-dalton polypeptide consisted of concanavalin A-bound and -unbound variants. Tunicamycin completely abolished the uptake of [3H]glucosamine into anti-AFT-precipitable polypeptides in both cells and medium, and the resulting polypeptide of apparent Mr = 66,000 did not bind to concanavalin A-Sepharose. Tunicamycin did not affect the synthesis or secretion of AFP by hepatoma cells.     

Immunological Studies on Viral Polypeptide Synthesis in Cells Replicating Murine Sarcoma-Leukemia Virus

Antibodies to disrupted murine sarcoma-leukemia virus (MSV[MLV]) were used to study the synthesis of viral polypeptides in the transformed, virus-producing rat cell line 78A1. When cultures were labeled for 10 min with radioactive amino acids, about 9% of the total labeled proteins were precipitated with antiserum against purified MSV(MLV), and 3 to 4% were precipitated with the same antiserum after it had been absorbed with an extract from uninfected rat cells. The difference is due to the presence in the unabsorbed antiserum of antibodies to cellular proteins that are present in purified virus preparations. Intracellular viral proteins labeled with radioactive amino acids were isolated by immunoprecipitation and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The mobilities of intracellular viral polypeptides were identical to those of the purified virion. However, labeled polypeptides having electrophoretic mobilities lower than that of the major virion polypeptide, the group-specific antigen of molecular weight 31,000, were present in higher proportion in the total cell extract and in the membrane fraction than in the virion. These polypeptides appear to be of cellular origin for they were present only in minute amounts in the immunoprecipitates obtained with the absorbed serum. After a 10-min labeling period, radioactive proteins were assembled into extracellular virions rapidly for the first 4 hr followed by a slower rate. More than 2% of the total proteins of the cell labeled in a 10-min pulse were assembled into virions at the completion of a 24-hr chase. The high-molecular-weight polypeptides with the same mobilities as those detected in the immunoprecipitate of intracellular proteins were found in virions released from cells after a 10-min pulse. A larger proportion of these high-molecular-weight proteins was detected in virions released after short chase periods (30-120 min) than after longer chase periods (6-24 hr). Two possible interpretations of these data are that the high-molecular-weight cell-derived polypeptides (i) have a turnover rate higher than that of the major virion polypeptides or (ii) are cleaved proteolytically from the virions during long incubation in the culture media.     

A human alloantiserum precipitates Ia-like molecules from chronic lymphocytic leukemia cells.

Alloantigens specific for human B lymphocytes can be identified with selected antisera. These antigens have similarities to murine Ia antigens in that they are found on human B lymphocytes and are controlled by genes linked to genes controlling HLA. Chronic lymphocytic leukemia cells bearing B cell antigens were labeled with 3H leucine and the membrane components reacting with the B cell antisera isolated by immunoprecipitation. These membrane components had m.w. of 33,000 and 24,000 daltons similar to the murine Ia antigens. The results complete the homology of murine Ia and human B cell alloantigens.     

Polypeptides of the Epstein-Barr virus membrane antigen complex.

Epstein-Barr virus (EBV)-associated membrane antigens have been purified from the plasma membranes of the producer cell line P3HR-1 NONO. The antigens were assayed with a specific rabbit anti-ebv antiserum using an 125I-labeled staphylococcal protein A binding assay. The antigens have been shown to be present on purified plasma membranes. Treatment of the plasma membranes with Triton X-100 allows the separation of two antigenically distinct classes of antigens, one soluble and one insoluble in the detergent. Immunoprecipitates of [125I5- and [35S]methionine-labeled, detergent-soluble antigens contained three major polypeptides of molecular weights of 350,000, 140,000, and 75,003 (on 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and several minor components. These polypeptides were all specifically precipitated from four EBV-producer cell lines, P3HR-1, P3HR-1 NONO, B95-8, and 7744. They could not be precipitated from producer cell lines treated with phosphonoacetic acid, which inhibits late viral functions, nor could they be precipitated from nonproducer cell lines. The 350,000 and 75,000 molecular weight polypeptides bound to Ricin and lentil lectin columns; however, most of the 140,000 molecular weight material did not. A component of molecular weight 220,000 (prominent only in P3HR-1 NONO) was probably a degradation product of the 350,000 molecular weight polypeptide.     

Human cell membrane components bound to beta2-microglobulin in T cell-type cell lines.

Cell membrane components bound to beta2-microglobulin were isolated from Renex 30 (a nonionic detergent)-solubilized membrane materials of two human T cell-type cell lines, MOLT-4 and CCRF-CEM, by gel filtration and lectin affinity chromatography. The isolation was carried out by following the beta2-microglobulin activity by radioimmune inhibition assay. The T cell membrane components bound to beta2-microblogulin had a uniform molecular size of about 200,000 daltons and most of them showed an affinity to lentil lectin. The isolated membrane components were radioiodinated and examined for identity to HLA antigens by sequential precipitation with rabbit anti-HLA antiserum (specific to HLA large components) and with rabbit anti-beta2-microblogulin antiserum. In addition to HLA antigens, the beta2-microglobulin-bound components obtained from the MOLT-4 cells were found to contain certain membrane components that are the same in molecular size as the HLA large components but that are different antigenically from the HLA large components. On the other hand, the beta2-microglobulin-bound membrane components obtained from the CCRF-CEM cells were all HLA antigens. No other membrane components were involved in the binding.     

The preparation of xenoantiserum specific for a murine mastocytoma cell line

Summary A method is described whereby a xenoantiserum directed toward membrane components of DBA/2 murine mastocytoma P815 cells was raised. This antiserum was found to be specific for tumor cell extracts and had no reactivity with comparable extracts of normal cells when tested by complement fixation. The antiserum was capable of killing P815 cells in the presence of guinea-pig complement but had no reactivity with L1210 leukemia cells or a variety of normal DBA/2 cell preparations. When mixed with varying numbers of tumor cells and injected IP to either DBA/2 or B6D2 Fl mice, the antiserum demonstrated a protective effect by either prolonging survival time or, when low numbers of cells were injected, apparently facilitating complete removal from the body of tumor cells. When administered following resection of SC grown tumors in B6D2 Fl mice, the antiserum prevented tumor recurrence in 50% of treated mice in comparison to animals treated with normal rabbit serum, in which recurrence occurred in all test animals.     

Characterization of cross-reacting antigens on the Epstein-Barr virus envelope and plasma membranes of producer cells.

A rabbit antiserum has been prepared against the B95-8 transforming strain of EBV. The antiserum has a high virus neutralizing titer (approximately 1:1000) against both the marmoset B95-8 EBV and the human P3HR-1 EBV. The neutralizing antibodies may be absorbed completely with EBV producer cell lines, but not with nonproducer cell lines or producer cell lines treated with phosphonoacetic acid (PAA) so as to be nonproducer. After repeated absorption with PAA-treated B95-8, the serum remains reactive with the membranes of producer cell lines as judged by immunofluorescence or the 125I--Staphylococcal protein A radioimmunoassay. Thus the neutralizing antigens are expressed on the membranes of producer cell lines and may be purified from this source using the serum and 125I--Staph A binding as an assay. The ability of the serum to differentiate between producer and nonproducer cells by means of cell surface determinants has been exploited to achieve a separation of these two populations from the same culture. Immunoprecipitation by the protein A technique shows that the serum recognizes two polypeptides from producer cells of approximate molecular weights 150,000 and 75,000.     

Identification and organization of the components in the isolated microvillus cytoskeleton

We have examined the effects of ATP and deoxycholate (DOC) on the cytoskeletal organization of Triton-demembranated microvilli (MV) isolated from chicken intestine brush borders. Isolated MV are composed of a core of tightly bundled microfilaments from which arms project laterally to the plasma membrane with a 33-nm periodicity. These lateral arms spiral around the core microfilaments as a helix with a 25 degrees pitch. Demembranated MV consist of four polypeptides with mol wt of 110,000, 95,000, 68,000, and 42,000, present in molar ratios of 1.1:1.6:1.3:10.0. After addition of 50 microM ATP and 0.1 mM Mg++, the cytoskeletons are organized as a tight bundle of microfilaments from which lateral arms are missing. In these ATP-treated cytoskeletons, the 110-kdalton polypeptide is reduced in amount and the 95,000, 68,000, and 42,000 polypeptides are present in a 1.3:1.2:10.0 ratio. In contrast, after incubation with 0.5% DOC, the core microfilaments are no longer tightly bundled yet the lateral arms remain attached with a distinct 33-nm periodicity. These DOC-treated cytoskeletons are depleted of the 95,000 and 68,000 polypeptides and are composed of the 110,000 and 42,000 polypeptides in a 2:10 molar ratio. These results suggest that the microfilaments are associated into a core bundle by the 95- and 68-kdalton polypeptides and from this core bundle project the lateral arms composed of the 110-kdalton polypeptide.     

Antigenicity of the major polypeptides of hepatitis B surface antigen (HBsAg).

The major polypeptides (P-1, P-2, and P-6) of HBsAg were isolated from purified preparations of 22-nm HBsAg particles, iodinated, and analyzed by double-antibody radioimmunoprecipitation assays for the presence of hepatitis B virus (HBV)-specific antigens. Each polypeptide fraction contained both group (a) and subtype (d) specific determinants in common by virtue of their immunoreaction with antiserum to native HBsAg and antisera to the other structural polypeptides. The antigenic and structural similarities of the HBsAg polypeptides establish that they are not each unique gene products of the HBV genome.     

Characterization of a monoclonal antibody directed against the murine B lymphocyte receptor for IgE

A rat hybridoma producing a high-affinity IgG2a monoclonal antibody (B3B4) directed against against the murine lymphocyte IgE receptor (Fc epsilon R) was established by using purified Fc epsilon R from Fc epsilon R+ murine hybridoma B cells as immunogen. The monoclonal and polyclonal anti-Fc epsilon R inhibited the binding of IgE to the murine lymphocyte Fc epsilon R and were also used to isolate the Fc epsilon R. B3B4 specifically recognized only the 49-Kd Fc epsilon R on murine B lymphocyte as determined by immunoprecipitation and SDS-PAGE analysis. In addition to its reaction with intact Fc epsilon R, B3B4 also recognized Fc epsilon R fragments that were present in the culture media of Fc epsilon R+ hybridoma cells. The predominant fragments isolated were 38 Kd and 28 Kd by SDS-PAGE analysis. When tested for reactivity with other cell types, B3B4 was highly specific for murine B lineage cells in that it did not significantly react with Fc epsilon R on macrophages and T cells and, in addition, did not react with the high affinity mast cell Fc epsilon R. B3B4 completely blocked IgE rosetting, and a reciprocal inhibition of binding was seen in a dose-dependent fashion between IgE and B3B4, indicating a close proximity of the IgE and B3B4 binding sites. Saturation binding analysis indicated that the Fab' fragment of B3B4 bound to twice as many sites/cell as IgE, suggesting that there are two identical B3B4 determinants per 49-Kd Fc epsilon R or that the IgE binding site is formed by the association of at least two 49-Kd Fc epsilon R. However, unlike IgE, neither B3B4 nor F(ab')2-B3B4 nor Fab'-B3B4 were very effective in causing Fc epsilon R upregulation on murine hybridoma B cells; in fact, B3B4 prevented this upregulation when added in combination with IgE. These results suggest that a site-specific interaction provided only by IgE may be essential for ligand-specific upregulation. Both polyclonal and monoclonal antibodies will be useful in further studies concerning the functional relationship between the membrane Fc epsilon R and the soluble Fc epsilon R fragments.     

Serological Cross-Reactivities of Murine and Human Class II Antigen Determined by Murine Xeno Anti-Human Class II Antibody

Murine anti-human class II antibodies were shown to cross-react with polymorphic determinants of murine class II antigens. The cross-reacting antibodies were raised in B10.S(9R) mice by immunizing with human nylon wool adherent cells (Ad cells) from peripheral blood leukocytes. The B10.S(9R) anti-human Ad cell antiserum bound to the molecules consisting of two chains with molecular weights of 35K and 28K dimers which were purified with a lentil-lectin column. The B10.S (9R) anti-human class II antiserum was also revealed to contain two distinct cross-reacting antibodies with polymorphic determinants of murine class II antigens coded for by the I-A subregion of the H-2. One is specific for a determinant of class II molecules coded for by I-A^b,d,q, and the other seems to be specific for class II molecules coded for by I-A^a,k,r.     

Inhibition of the biosynthesis of SRP polypeptides and secretory proteins by aflatoxin B1 can disrupt protein targeting

Cell culture and western blotting studies revealed that aflatoxin B(1) (AFB(1)) inhibits the biosynthesis of two of the constituent polypeptides of signal recognition particle (SRP) (SRP54 and 72). SRP escorts polyribosomes carrying signal peptides from free form in the cytosol to the bound form on endoplasmic reticulum (ER) membrane during protein targeting. These effects of AFB(1) on SRP biosynthesis may inhibit the formation of functional SRP. Our experiments have further shown that AFB(1) also inhibits the biosynthesis/translocation of a secretory protein, preprolactin, which fails to appear in the lumen of ER consequent to the treatment with this hepatocarcinogen. The results of the experiments presented in this article therefore enable us to infer for the first time that aflatoxin B(1) may inhibit the functioning of SRP as an escort and deplete the ER of polyribosomes for secretory protein synthesis. As these secretory proteins are important components of the plasma membrane, gap junctions and intercellular matrix, their absence from these locations could disturb cell to cell communication leading to tumorigenesis.     

Coxsackievirus B3 infection alters plasma membrane of neonatal skin fibroblasts.

Replication of coxsackievirus B3 occurred for days in cultures of murine neonatal skin fibroblasts in the absence of cytopathology and resulted in alteration of the plasma membrane. Dual immunofluorescence studies showed that the lectin Ulex europaeus agglutinin I bound only to cells producing viral capsid antigens. Cultures of coxsackievirus B3-inoculated murine neonatal skin fibroblasts showed maximum binding of this lectin at 72 h postinoculation. These data show that in a nonlytic infection a picornavirus can alter the surface of an infected cell.     

Two types of mu chain complexes are expressed during differentiation from pre-B to mature B cells.

Immunoglobulin mu chains synthesized in murine pre-B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre-B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross-linking of micron chains on the surface of pre-B cells resulted in an increase in intracellular free Ca2+, indicating that the micron chain complex on the surface of pre-B cell lines acted as a signal transduction molecule. However, the receptor cross-linkage of pre-B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre-B to mature B cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.     

Antigen-induced murine B cell lymphomas. II. Exploitation of the surface idiotype as tumor specific antigen.

In the accompanying report, we have described the characterization of two unusual murine B cell lymphomas, CH1 and CH2. A heterologous antiserum, which we refer to as "anti-idiotype" serum, has been raised to the detergent-solubilized surface immunoglobulin of CH1. The following criteria have established that this antiserum is specific for the CH1 tumor and that it reacts with V region determinants of the tumor surface IgM: 1) the antiserum reacts with CH1 tumor cells, but not normal mouse lymphoid cells or CH2 tumor cells, in indirect immunofluorescence and C-dependent cytotoxicity testing, 2) capping with the anti-idiotype serum removes all or most of the tumor surface Ig, 3) the antiserum forms a single band of precipitation against serum from CH1 tumor-bearing mice, when tested by double diffusion precipitin analysis, and 4) a single band of precipitation is formed in the electrophoretic migration position of IgM when the anti-idiotype antiserum is tested against serum from CH1 tumor-bearing mice in immunoelectrophoresis. Furthermore, we have demonstrated that this antiserum is useful in monitoring tumor growth and is a potent immunotherapeutic agent. Specifically, 50% of mice injected with a lethal tumor inoculum and given a small dose of anti-idiotype serum 2 days later remain tumor free, whereas all tumor-challenged control mice died within 30 days.