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J Luka

Publications and source records attributed to J Luka.

At least 55 records · Page 3Linked to original sources
PubMed1981

Membranes of EBV-carrying virus nonproducer cells inhibit leukocyte migration of EBV-seropositive but not seronegative donors.

We have previously shown that the leukocytes of healthy EBV-seropositive (but not seronegative) donors respond with migration inhibition (LMI) when confronted with extracts of EBV-carrying (but not EBV negative) cells. In the present study, we have examined whether this EBV-specific LMI response is capable of detecting a membrane antigen on the surface of EBV-carrying virus nonproducer cells. Crude membranes from EBV-genome carrying and EBV-negative cell lines were used as antigen. Contamination with the EBV-determined nuclear antigen (EBNA) was ruled out. Membranes from EBV-genome carrying nonproducer cells inhibited the migration of leukocytes from healthy seropositive donors, whereas membranes from EBV-negative lines had no such effect. Seronegative donors did not show any LMI. The clear difference between the EBV-negative Ramos line and its EBV-converted sublines was particularly conclusive in showing that the membrane component is determined or induced by the viral genome.

Antigens↗
PubMed1980

Purification and biochemical characterization of the Epstein-Barr virus-determined nuclear antigen and an associated protein with a 53,000-dalton subunit.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified 700-fold to apparent homogeneity from Raji and Namalwa cell extracts by a three-step procedure involving heat treatment, DNA-cellulose chromatography, and hydroxyapatite chromatography. Acid-fixed nuclear binding and complement fixation were used to monitor antigenic specificity. Purified EBNA was also capable of specifically inhibiting the regular anticomplement immunofluorescence reaction for EBNA against Raji target cells. The purified antigen had a molecular weight of 170,000 to 200,000. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it yielded a single 48,000-dalton (48K) monomer. An EBNA-associated protein was also purified from the same cell extract. It had a molecular weight of about 200,000 and yielded a single 53K protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The same protein was also found in Epstein-Barr virus negative B-cell lymphoma lines. The two types of protein were characterized by amino acid composition and peptide mapping. The results showed that the 53K and 48K protein components have no long regions in common; this excludes that the smaller product arises by breakdown of the larger product. Residue distributions were different, but an excess of hydrophilic residues was found in both proteins, suggesting a certain overall similarity in properties. 53K components from different cell lines appeared to differ somewhat. Epstein-Barr virus-positive lines carry two 53K components, one of which may be a slightly modified 53K product. Immunocomplexing assay showed that the 48K, but not the 53K, protein carries EBNA specificity. In mixtures, the 53K protein is co-precipitated with the 48K protein. The data suggest that EBNA may form a complex with the 53K proten within the cell.

Antigens, Viral↗
PubMed1979

Evidence for antigenic distinctness of the Epstein--Barr virus-determined nuclear antigen and the Herpesvirus papio-determined nuclear antigen.

Previous serological evidence indicated that Epstein--Barr virus-determined nuclear antigen (EBNA) has cross reactive components with the herpesvirus papio-determined nuclear antigen (HUPNA) and, in addition, it has distinct components of its own, not present in HUPNA. In the present study, this hypothesis was studied by absorption experiments. Absorption of anti-EBNA positive human sera with EBNA abolished both anti-EBNA and anti-HUPNA reactivity. Absorption with HUPNA removed all anti-HUPNA reactivity but did not reduce anti-EBNA to any detectable degree, thus confirming the hypothesis.

Animals↗
PubMed1979

Detection of a nuclear antigen in Herpesvirus ateles-carrying marmoset lines by the acid-fixed nuclear binding technique.

In vitro binding of a Herpesvirus ateles (HVA)-associated soluble antigen to amphibian erythrocyte nuclei was demonstrated by the acid-fixed nuclear binding technique in combination with anticomplement immunofluorescence. Incubation of concentrated salt-extracted soluble antigens derived from HVA-carrying marmoset lines with methanol/acetic acid-fixed erythrocytes of frogs and salamanders resulted in a brilliant nuclear fluorescence after exposure to a live virus-boostered, anti-HVS-positive squirrel monkey serum. Anti-HVS-negative sera did not stain. The activity of the positive serum could be abosrbed completely with extracts of HVA-carrying cells but not with Epstein-Barr virus-carrying or Herpesvirus papio-carrying cells. The HVA-associated antigen was also present in lytically HVA-infected marmoset kidney cells.

Animals↗
PubMed1979

Immunochemical characterization of Epstein-Barr virus-associated early and late antigens in n-butyrate-treated P3HR-1 cells.

Sodium butyrate induces the Epstein-Barr virus cycle in latently infected P3HR-1 cells with a high efficiency. This fact was utilized for the metabolic labeling of the Epstein-Barr virus antigens. Nonproducer Raji cells, lacking both early antigen and viral capsid antigen, were used as controls. Immunoprecipitation patterns were compared with 13 anti-Epstein-Barr virus (viral capsid antigen) - positive and 3 negative sera. Sixteen polypeptides were identified as being associated with the lytic Epstein-Barr virus cycle. Their molecular weights ranged from 31,000 (31K) to 275K on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two polypeptides, 158K and 165K, could be classified as late viral products on the basis of their sensitivity to cytosine arabinoside. Six of the polypeptides, i.e., 90K, 95K, 134K, 165K, 236K, and 275K, were detected by [(3)H]glucosamine labeling. Among the early, cytosine arabinoside-insensitive polypeptides detected by [(35)S]methionine labeling, a 152K component appears to be a major constituent of early antigen. This polypeptide was precipitated by all anti-Epstein-Barr virus-positive sera tested. As a rule, together with the 103K and 134K polypeptides, the 152K component is precipitated by anti-early antigen, R (restricted) antibodies. In addition, anti-early antigen D (diffuse) antibodies precipitate 31K, 51K, 65K, and 90K components.

Antigens, Viral↗
PubMed1979

Relationship between Epstein-Barr virus genome number, amount of nuclear antigen, and early antigen inducibility in diploid and tetraploid lymphoma cells of related origin.

In a collection of near-diploid and near-tetraploid Raji sublines and somatic Raji/Raji hybrids a linear relationship was found between the number of Epstein-Barr virus (EBV) genome copies and the relative amount of EBV-determined nuclear antigen per cell. Inducibility of the viral cycle by iododeoxyuridine and by P3HR-1 virus superinfection coulb be related to the number of resident EBV genome copies per haploid target cell. The implications of these findings are discussed.

Antigens, Viral↗
PubMed1978

Detection of the EBV-determined nuclear antigen (EBNA) in Burkitt's lymphoma and nasopharyngeal carcinoma biopsies by the acid fixed nuclear binding (AFNB) technique.

Epstein-Barr virus (EBV) carrying biopsies of Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) were used to examine the question whether the EBV-associated nuclear antigen (EBNA) can be demonstrated by the acid fixed nuclear binding (AFNB) technique, developed previously for the demonstration of EBNA in cultured cell lines [11]. Extracts of 5 BL and 5 NPC biopsies gave a brilliant, EBNA specific fluorescence after binding to acid fixed chicken red cells. Similar extracts of 3 other African tumors that are not known to carry the EBV-genome were negative, in spite of the fact that they were derived from EBV-seropositive patients with relatively high anti-EBV (VCA) antibody titers. Crude extraction and DNA-cellulose purification gave equally active extracts, provided that incubation was carried out at 4 degrees C. These results show that the acid fixed nuclear binding technique can be applied to biopsy material. This may be helpful in searching for EBNA carrying cells in heterogeneous normal and tumor tissues in vivo where the direct in situ ACIF staining for EBNA is known to meet great difficulties.

Acids↗
PubMed1978

Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.

Antigens, Viral↗
PubMed1977

Detection of a nuclear, EBNA-type antigen in apparently EBNA-negative Herpesvirus papio (HVP)-transformed lymphoid lines by the acid-fixed nuclear binding technique.

In agreement with the findings of previous authors, we could not detect a virally determined nuclear antigen in Herpesvirus papio (HVP)-transformed baboon lymphoid lines by anticomplementary staining in situ, as for EBNA. However, by means of our recently developed acid-fixed nuclear binding technique an EBNA-like antigen could be readily demonstrated, after extraction from both producer and non-producer lines. We propose to designate the antigen as HUPNA. It can be detected by a human anti-EBNA antibody, suggesting cross-reactivity, if not identity, between EBNA and HUPNA. HVP-DNA carrying non-producer lines, negative for in situ ACIF stainability but capable of yielding HUPNA by the nuclear binding technique, can be superinfected with EBV, with brilliant EBNA expression as the result, suggesting that the defective in situ staining is a property associated with the baboon HVP, rather than the baboon lymphoid cell per se.

Acetates↗
PubMed1977

Identification of a purified complement-fixing antigen as the Epstein-Barr-virus determined nuclear antigen (EBNA) by its binding to metaphase chromosomes.

A soluble complement-fixing antigen carried by Epstein-Barr virus (EBV)-transformed human cells has been previously extracted from cell nuclei and purified by DNA-cellulose chromatography [Luka, J., Siegert, W. & Klein, G. (1977) J. Virol., in press]. On addition of this antigen to methanol/acetic acid-fixed metaphase chrmosomes, followed by exposure to human sera containing antibodies against the EBV-determined nuclear antigen (EBNA), brilliant positive staining was obtained by anti-complement immunofluorescence. There was no staining after exposure to EBV-negative sera. Moreover, a nuclear protein fraction, prepared from an EBV-negative cell line in an analogous fashion, failed to induce the staining reaction. These data identify the soluble purified antigen as the EBV-determined nuclear antigen. The purified antigen has a molecular weight of 174,000 +/- 15,000, as determined by sucrose gradient centrifugation and gel filtration experiments. In neutral buffers containing 0.5-1.0 M NaCl, the antigen dissociates into a form of approximately one-half the original molecular weight with retained complement-fixing activity. This "monomer" has a molecular weight of 98,000 +/- 8,000.

Antigens, Viral↗
PubMed1977

Solubilization of the Epstein-Barr virus-determined nuclear antigen and its characterization as a DNA-binding protein.

The Eptstein-Barr virus (EBV)-determined nuclear antigen (EBNA) was solubilized from isolate nuclei of two EBV-transformed cell lines- Raji and AW-Ramos, by high-salt treatment. Its DNA-binding properties were studied by DNA-cellulose chromatography and a 51Cr release complement fixation assay. EBNA binds to both double-stranded and single-stranded calf thymus DNA, showing a higher affinity to double-stranded DNA. There was no detectable difference in the DNA binding of EBNA prepared from Raji and AW-Ramos cells.

Antigens, Viral↗