Nucleotide sequence of the rat Bmyc gene.

We have cloned and sequenced the rat Bmyc gene. The rat Bmyc gene contains sequences related to the central part of c-myc, namely the first intron, the second exon, and the noncoding part of the third exon. The homology drops in the 3' part of the c-myc second exon, but continues in the noncoding part of the third exon. We have sequenced the total predicted coding region of the Bmyc. The longest open reading frame in Bmyc suggests a protein of 178 amino acids, which is only 41% of the c-myc protein size. To confirm the putative open reading frame, we have produced a trpE-Bmyc protein that is detected with a pan-myc antibody. We discuss these findings in the context of potential functional domains and the possibility of overlapping and distinct activities of myc-family proteins.

Peptides derived from HIV-1 Vif: a non-substrate based novel type of HIV-1 protease inhibitors.

The retroviral protease (PR) is absolutely essential for completion of human immunodeficiency virus multiplication cycle, and cannot be replaced by any cellular function. Thus PR, like reverse transcriptase, is an ideal target for the development of anti-AIDS therapy. A large number of human immunodeficiency virus type-1 (HIV-1) PR inhibitors have been developed, and several are currently used as anti-AIDS drugs. These inhibitors are mainly based on the natural PR cleavage sites within the viral Gag and Gag-Pol precursors. The major difficulty encountered while using anti-HIV therapeutic agents in patients has been the rapid emergence of drug-resistant viral strains. Most of the mutations which convert the PR into inhibitor-resistant are located within the substrate binding subsites of the enzyme. Recently, it has been shown that the HIV-1 auxiliary protein Vif, and especially the N-terminal half of Vif (N'-Vif) specifically interacts with the viral PR and inhibits its activity. We now show that efficient inhibition of HIV-1 PR activity can be achieved using Vif-derived peptides. Based on the above model we have performed peptide mapping of N'-Vif in order to find a small peptidic lead compound which inhibits PR activity. The screening revealed that peptides derived from two regions in Vif spanning from residues 30-65 and 78-98 inhibit PR activity in vitro, specifically bind HIV-PR and inhibit HIV-1 production in vivo. Further mapping of these regions revealed the lead compounds Vif81-88 and Vif88-98. These peptides specifically inhibit and bind HIV-1 PR, but do not affect pepsin and rous sarcoma virus protease. In contrast to other known PR inhibitors, these peptides are not substrate-based and their sequences do not resemble the sequences of the natural PR substrates (cleavage sites). Moreover, the Vif-derived peptides themselves are not cleaved by HIV-1 PR. Conversion of the lead peptides into small backbone cyclic peptidomimetics is taking place nowadays in order to turn these lead compounds into metabolically stable selective novel type of HIV-PR non-substrate-based inhibitors.

Characterization of vascular leak syndrome induced by the toxin component of Pseudomonas exotoxin-based immunotoxins and its potential inhibition with nonsteroidal anti-inflammatory drugs.

Clinical trials of immunotoxins in cancer patients have been limited in many cases by vascular leak syndrome (VLS). Recently, rats were identified as a model for VLS induced by BR96 sFv-PE40, a carcinoma-reactive single-chain immunotoxin. In this study, the toxin component of this immunotoxin, PE40, was found to be responsible for inducing hydrothorax in rats, thereby demonstrating that direct binding to the BR96 antigen was not essential to the onset of VLS. Mutational analysis of PE40 determined that both ADP ribosylation and proteolytic processing functions innate to Pseudomonas exotoxin A (PE) were necessary for PE40 to induce hydrothorax in rats; however, neither function by itself was sufficient for VLS induction. Additionally, nonsteroidal anti-inflammatory agents were found to block VLS in rats receiving BR96 sFv-PE40. These results demonstrate that the toxin component of PE-based immunotoxins induce VLS and suggest agents for clinical management of the toxicity.

Human immunodeficiency virus type 1 Vif-derived peptides inhibit the viral protease and arrest virus production.

Human immunodeficiency virus type 1 (HIV-1) Vif protein is required for productive HIV-1 infection of peripheral blood lymphocytes and macrophages in cell culture and for pathogenesis in the SCID-hu mouse model of HIV-1 infection. Vif inhibits the viral protease (PR)-dependent autoprocessing of truncated HIV-1 Gag-Pol precursors expressed in bacterial cells and efficiently inhibits the PR-mediated hydrolysis of peptides in cell-free systems. The obstructive activity of Vif has been assigned to the 92 amino acids residing at its N'-terminus (N-Vif). To determine the minimal Vif sequence required to inhibit PR, we synthesized overlapping peptides derived from N-Vif. These peptides were then assessed, using two in vitro and two in vivo systems: (i) inhibition of purified PR, (ii) binding of PR, (iii) inhibition of the autoprocessing of the Gag-Pol polyprotein expressed by a vaccinia virus vector, and (iv) inhibition of mature virus production in human cells. The peptides derived from two regions of N-Vif encompassing residues Tyr-30-Val-65 and Asp-78-Val-98, inhibited PR activity in both the in vitro and the in vivo assays. Thus, these peptides can be used as lead compounds to design new PR inhibitors.

Determination of immunoglobulin classes of specific antibodies secreted by single cells. A cluster formation assay.

A method is described to identify specific antibody-forming cells and their immunoglobulin classes by a multilayer cluster formation. Cells producing specific antibodies are mixed with erythrocytes coated with the corresponding antigen in presence of rabbit anti-human immunoglobulins. Clusters are formed only if the mixtures contain rabbit antiserum against the immunoglobulin chains of the specific secreted antibody. Thus the immunoglobulin class of the specific antibody is assessed. For this study the cells of three EBV-transformed, antibody-producing human lymphoblastoid cell lines were used.

An improved method to create nitrocellulose particles suitable for the immobilization of antigen and antibody.

A method is described for producing 1-3 microns sized particles of nitrocellulose (NC) which are able to absorb protein. Protein is absorbed onto preformed particles made by first dissolving a sheet of nitrocellulose paper in DMSO, and then precipitating it with sodium carbonate/bicarbonate buffer. The efficiency of binding is the same as that of an equivalent sheet of non-processed NC filter paper. Antibodies absorbed onto preformed particles are not exposed to DMSO and carbonate buffer and therefore retain a high antigen binding capacity. Antigen and antibody-absorbed NC preformed particles were used to capture antibody and antigen, respectively. Using lysis buffer, the captured antibodies and antigens were readily released from the NC particles. This makes the latter an appropriate matrix for immunoprecipitation assays either for an antigen or for specific antibody. Antigen-coated NC particles were specifically aggregated ('agglutinated') by specific antibodies and thus can be used in semi-quantitative tests.

The coating of erythrocytes with detergent-solubilized molecules: a general method for improved coupling of antigens and antibodies.

A method is described by which different antigens and antibodies in solutions containing deoxycholate (DOC) may be coated to glutaraldehyde (GA)-fixed erythrocytes. This method, based on the use of CrCl3, yields erythrocytes which preserve the antigenicity and antibody activity of the coated molecules and can thus be applied to various assays in which indicator red cells are required. The sensitivity of the assays increases when these red cells are used. The cells form rosettes with the appropriate receptor positive cells. They sediment in Ficoll-Isopaque as do native erythrocytes and do not aggregate spontaneously even when coated at very high or low concentrations of CrCl3 or antigen. This coupling method, which is performed in the presence of DOC, and which requires only a small amount of antigen, is proposed for the coupling of cell membrane dissolved antigens.

A rapid method for estimating the binding of ligands to ELISA microwells.

This report presents a rapid and simple assay for estimating to what extent the surface of ELISA microwells is coated by a ligand of choice such as, for example, proteins, peptides, hormones, polysaccharides and nucleic acids. The method also provides a practical approach for defining the conditions required for optimal coating, such as ligand concentration, coating buffer, temperature and duration of coating and also for evaluating the efficiency of the reagents used to saturate the ELISA microwells. The important advantage of this procedure is that, in contrast to conventional ELISA procedure, the detection of the microwell-adhered ligand is not achieved by using an antibody. It is therefore the solution of choice when, as is often the case, no primary specific antibody is available. The test consists of three steps: first the ligand is allowed to adsorb to the microwells. Second, alkaline phosphatase is added to bind to any residual microwell surface not occupied by the ligand. Finally, substrate is added and the resulting color reaction is measured. Light absorbancy is inversely correlated with the level of ligand adherence. The results obtained by this method match those of direct ligand quantitation, as evaluated by a regular ELISA procedure.

An improved dot immunobinding assay for screening hybridoma supernatants. Non-purified antigen immobilized on nitrocellulose paper discs.

This report describes a modified dot immunobinding assay (DIA) in microplates using a crude mixture of non-purified antigen. Nitrocellulose filter paper discs exposed to the antigen mixture were inserted into the wells and kept in place by a specially constructed device. To test the efficiency of the modification a set of monoclonal antibodies from a mouse immunized with 58 kDa trpE-Bmyc fusion protein were screened. The advantage of this modified method over conventional ELISA is that it permits the use of non-purified antigen for screening large numbers of monoclonal antibodies.

A sensitive method for detecting cells coated with antibodies with human monoclonal rheumatoid factor produced in vitro.

A human monoclonal rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus immortalized cell line has been used to detect and quantitate antibodies specifically bound to cells. The RF is purified from cell culture supernatant by a simple procedure and then radiolabeled. In this assay, the binding of 125I-labeled RF to cells coated with specific antibodies is determined. This RF method detects very low titers of antibodies directed against specific cellular antigens and also minorities of antibody coated tumor cells in mixtures with identical non-coated cells. The advantages of this unique human monoclonal antibody are discussed.

The detection of heat-aggregated IgG (as a model for immune complexes) by reverse passive haemagglutination using a human monoclonal rheumatoid factor coupled to erythrocytes.

A human monoclonal IgM rheumatoid factor (RF) produced in vitro by an Epstein-Barr virus (EBV)-immortalized cell line was purified by protein A-Sepharose adsorption and coupled by the chromic chloride method to human erythrocytes. The RF-coupled cells were incorporated in reverse passive haemagglutination (RPH) assays to detect immune complexes (IC) using heat-aggregated human IgG as a model system. The sensitivity of the RPH was comparable to an enzyme-linked immunosorbent assay (ELISA) using sheep C1q for the detection of ICs.

In vitro synthesis of antibodies to acetylcholine receptor by Epstein-Barr virus-stimulated B-lymphocytes derived from patients with myasthenia gravis.

Peripheral blood lymphocytes and B-cells were obtained from patients with myasthenia gravis and stimulated in vitro with either pokeweed mitogen or Epstein-Barr virus (EBV), respectively. EBV stimulation of B-cells caused a production of antibodies to acetylcholine receptor in 15 of the 25 myasthenia gravis patients: the EBV stimulation of B-cells was more effective in this regard than the pokeweed mitogen stimulation of peripheral blood lymphocytes. The in vitro synthesis of anti-acetylcholine receptor antibodies was found to be positively correlated with both the patients' sera antibody titers and with the disease severity.

The effects of digitalis-like compounds on rat lenses.

PURPOSE: Fundamental to the maintenance of ionic concentration gradients and transparency of the lens is the activity of Na+,K+-adenosine triphosphatase (ATPase) in the epithelial layer. Recent studies have identified endogenous digitalis-like compounds (DLCs) and 19-norbufalin and its peptide derivatives in human cataractous lenses. These compounds inhibit the activity of Na+,K+-ATPase and have been suggested to be involved in cataract formation. The present experiments were designed to test this hypothesis by determining the ability of digitalis and DLCs to induce changes in protein composition and leakage from rat lenses in organ culture. METHODS: DLCs were determined in rat lenses using three independent assays: interaction with ouabain antibodies, interaction with bufalin antibodies, and inhibition of [3H]-ouabain binding to red blood cells. Rat lenses were incubated in modified TC-199 medium in 5% CO2 atmosphere at 37 degrees C for the time of the experiment. The onset of cataractogenesis was assessed by measuring protein leakage from lenses and by crystallin composition in the lens and media. RESULTS: DLCs were present in rat lens with concentrations 7 to 30 times higher in the capsular-epithelial layer than in the lens fibers regions. Ouabain, bufalin, digoxin, and DLC induced dose- and time-dependent leakage of protein from rat lenses. Lenses incubated with these compounds showed alterations in crystallin content consistent with changes that initiate opacity. All the compounds caused a multilayering of epithelial cells in the region surrounding the mitotic area and, at the same time, cell death in the central anterior region. CONCLUSIONS: Digitalis and endogenous DLCs are cataractogenic factors. These results, together with the demonstration of DLCs in the normal lens and their increased levels in human cataractous lenses, strongly suggest their involvement in the molecular mechanisms responsible for cataract formation.

Human monoclonal antibodies produced by Epstein-Barr virus transformed cell lines bind protein A.

Epstein-Barr virus (EBV) is a polyclonal T-independent activator of viral receptor positive human B lymphocytes. Lymphocytes infected in vitro with the virus are transformed into immortalized cell lines [Nilsson, K, and Klein, G. (1982) Adv. Cancer Res. 37, 319]. In this way human cell lines that secrete specific IgM, IgG and IgA monoclonal antibodies are established. Protein A is also a polyclonal T-independent B cell activator [Langone, J. J. (1982) Adv. Immunol. 32, 157], the targets of which are surface immunoglobulin and C3d receptor positive cells, as are the targets of EBV. We found that almost all (16 out of 17) of the specific monoclonal antibodies (IgM, IgG and IgA) produced in vitro by EBV cell lines bind protein A. Unlike these in vitro produced antibodies, a substantial fraction of the immunoglobulins in human serum does not bind protein A. Thus, those plasma cells which in vivo secrete protein A nonbinding immunoglobulins originate from precursors of B cell that were EBV noninfective. Alternatively, during in vivo B differentiation some immunoglobulins undergo a change from protein A binding to protein A nonbinding molecules.

Continuous in vitro production of IgA by a human colostral immortalized cell line.

Breast milk samples from 8 postpartum mothers were collected and incubated with Epstein-Barr virus, immortalizing their B-lymphocytes and giving rise to lymphoblastoid cell lines. Three samples showed no growing lymphocytes and five samples gave rise to transformed cell lines. IgA positive cells were selected from one such cell line by rosetting with anti-IgA-coated sheep erythrocytes. The emerging cell line was similarly reselected giving rise to an IgA-surface positive lymphoblastoid cell line. Monoclonal IgA-producing cell lines were obtained by cloning in the soft agar method. IgA is continuously secreted by the cell lines which have been growing in vitro for more than 24 mth.

Immunogenic and antigenic epitopes of immunoglobulins. VIII. A human monoclonal rheumatoid factor having specificity for a discontinuous epitope determined by histidine/arginine interchange as residue 435 of immunoglobulin G.

A monoclonal human rheumatoid factor (RF-AN) reactive with human Fc gamma is shown to recognise a discontinuous epitope dependent on the presence of histidine (His) at residue 435. RF-AN is not reactive with IgG1 paraproteins having C gamma 2 or C gamma 3 domain deletions or IgG3m (5) or Ig3m (21) proteins having arginine (Arg) at residue 435. RF-AN is reactive with IgG1, 2, 4 and IgG3m (15,16) proteins having histidine at 435.

Synthesis of antibodies against measles virus and myelin by in vitro stimulated B-cells derived from patients with subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) patients carry persistent measles virus infection in the brain. Furthermore, the blood lymphocytes contain viral RNA. Lymphocytes derived from 6 SSPE patients were stimulated with Epstein-Barr virus (EBV). Production of antibodies against measles virus of the IgG isotype was detected in the supernatants of cell cultures of all patients, regardless of the disease's activity, duration or interferon therapy. In contrast, only some of these cell cultures also produced antibodies against myelin.

Linkage localization of the thoraco-abdominal syndrome (TAS) gene to Xq25-26.

The thoraco-abdominal syndrome (TAS) presents a closure defect confined to the ventral midline, manifested as ventral hernia of various degrees in all affected individuals and antero-lateral diaphragmatic defect manifested almost exclusively in affected males. The syndrome is inherited as an X-linked dominant trait affecting blastogenesis (XLB mutation). We studied 27 members of the TAS family for linkage on the X chromosome. The best lod score of 5.5 at theta 0.04 was found for the HPRT locus on Xq26.1. A multilocus lod score of 12.4 was observed when the linkage analysis utilized additional markers in Xq25-26.

Establishment of a human lymphoblastoid cell line with specific antibody production against group A streptococcal carbohydrate.

A human lymphoblastoid cell line, secreting specific antibody against Group A carbohydrate (A-CHO) was established by pre-selection of antigen binding normal human lymphocytes, followed by Epsetin Barr virus (EBV) induced immortalization. Culture supernatants were assayed for anti A-CHO antibodies by radioimmunoassay, N-acetyl-glucosamine-coupled T4-phage plaque inhibition tests and passive hemagglutination. As a rule, the supernatants contained about 10 micrograms/ml anti-A-CHO antibodies of the IgM-kappa type. The antibody was fractionated and partially purified on an N-acetyl glucosamine Sepharose 4B column with a recovery of about 3 micrograms/ml of supernatant.

Clonal analysis of a human lymphoblastoid cell line (B17) secreting antibody to N-acetyl-D-glucosamine.

In this paper we analyse the clonal composition of a human lymphoblastoid B-cell line secreting IgM/k antibody to N-acetyl-D-glucosamine, the immunodominant sugar of Group-A-streptococcal carbohydrate. Besides non-antibody secreting cells, the line consists of two clonotypes of antibody-secreting cells: B17 cells producing over 90% and F6 cells producing less than 10% of the antibody in the supernatant. The proportions of B17 and F6 cells in the cell line seem to be similar to the proportion of antibodies in the supernatant. F6 cells can be isolated by cloning and maintained as stable lines, whereas this is more difficult with B17 cells. The results suggest that upon establishment of the line, at least two N-acetyl-D-glucosamine-specific B cells were immortalized and coexist together as independent clonotypes. Although F6 cells seem to have a slight tissue culture advantage, they represent the minor clonotype in the B17 cell line.