Biomedical subjects

J Luka

Publications and source records attributed to J Luka.

At least 37 records · Page 2Linked to original sources

PubMed1987

Identification of an Epstein-Barr virus glycoprotein which is antigenically homologous to the varicella-zoster virus glycoprotein II and the herpes simplex virus glycoprotein B.

The Epstein-Barr virus (EBV) antigenic homologue of the varicella-zoster virus glycoprotein II and the herpes simplex virus (HSV) glycoprotein B (gB) was identified through cross-reactivity with anti-glycoprotein II and anti-glycoprotein B peptide sera. The homologue is the previously characterized EBV glycoprotein, with an apparent molecular weight of 125,000 Da, which is synthesized late during productive EBV infection and appears to be encoded by the BamHI A EBV fragment. This glycoprotein, but not other EBV proteins, reacted with the antisera in immunoprecipitation experiments and by ELISA. In addition, absorption of the sera with the purified EBV 125-kDa glycoprotein removed the cross-reacting antibody. Whether the EBV gB homologue has the same biological functions associated with HSV gB has yet to be determined.

Antigens, Viral↗

PubMed1987

A restricted component of the Epstein-Barr virus early antigen complex is structurally related to ribonucleotide reductase.

The 85-kDa polypeptide previously shown to be associated with the restricted (R) component of the EBV-induced early antigen (EA) complex was subjected to amino acid sequencing analysis. This was accomplished by cyanogen bromide cleavage and by separation of individual peptides by high-pressure liquid chromatography employing reversed-phase C18 column techniques. Two of the isolated peptides, F11 and F13, were subjected to amino acid sequencing and both were found to have significant homology to the postulated protein encoded by the BamHI O right reading frame 2 (BamHI ORF2) of the B95-8 strain Epstein-Barr virus. Computer analysis revealed significant homology between the amino acid sequence of this polypeptide and ribonucleotide reductase, an enzyme previously mapped to this genomic fragment. Amino acid composition analysis also revealed a similar association. These results indicate that the 85-kDa EA(R) polypeptide is associated with a component of the EBV-induced ribonucleotide reductase.

Amino Acid Sequence↗

PubMed1986

Establishment and characterization of a chronic infectious mononucleosislike syndrome in common marmosets.

Epstein-Barr virus (EBV) was inoculated into two species of marmosets. Successful infection was established in the majority of the animals of one species, Callithrix jacchus, as evidenced by the development of high, persistent levels of antibody against virus-specific capsid and early nonstructural proteins. Antibodies also were produced against the major membrane antigen and, in some animals, against EBV nuclear antigen (EBNA) 2 but not against EBNA 1. This is the antibody profile normally noted in individuals with chronic infectious mononucleosis (IM). EBV-induced lymphoproliferation was not seen, and EBV-specific proteins were not detected in the peripheral blood lymphocytes of infected animals. Hence, EBV infection in C. jacchus apparently does not generally include extensive B-cell involvement. However, the marmosets clearly are useful as a model for EBV primary infection and also possibly for chronic IM.

Animals↗

PubMed1986

Characterization of the restricted component of Epstein-Barr virus early antigens as a cytoplasmic filamentous protein.

Four monoclonal antibodies produced against the restricted component of the Epstein-Barr virus (EBV) early antigen (EA-R) precipitated a polypeptide with an approximate molecular weight of 85,000. Three of these antibodies prepared against the native 85,000-molecular-weight protein (85K protein) reacted by immunofluorescence with acetone-fixed smears but not methanol-fixed smears of EBV-producing cells activated with tumor-promoting agent and sodium butyrate. The fourth monoclonal antibody which was produced against the denatured 85K protein reacted with both acetone-fixed cells and methanol-fixed cells. Blocking of direct immunofluorescence by the different monoclonal antibodies established that these monoclonal antibodies were directed against three different epitopes expressed on the 85K protein. The cytoplasmic staining pattern produced by each antibody was granular during the first 24 to 28 h after induction, developed into filamentous structures about 36 h after induction, and then began to aggregate after 48 h. Similar structures were observed in human placental cells transfected by EBV DNA and stained with three of the monoclonal antibodies. These results suggest that the EA-R polypeptide is assembled into filaments during the EBV lytic cycle. The significance of this in regards to replication has yet to be determined. Biochemical characterization of this major EA-R component did not reveal any major differences in this protein isolated from different cell lines.

Amino Acids↗

PubMed1986

Rheumatoid arthritis synovial membrane contains a 62,000-molecular-weight protein that shares an antigenic epitope with the Epstein-Barr virus-encoded associated nuclear antigen.

A monoclonal antibody, selected for reactivity with the Epstein-Barr virus (EBV)-encoded antigen EBNA-1, exhibited strong reactivity with the synovial lining cells in joint biopsies from 10 of 12 patients with rheumatoid arthritis (RA) and adherent cells eluted from these tissues. No staining of RA synovial membrane frozen tissue sections or eluted synovial-lining cells was obtained with monoclonal antibodies directed against other EBV-encoded antigens (anti-p160, anti-gp200/350) or with monoclonal antibodies directed against antigens encoded by cytomegalovirus, herpes simplex viruses, or human T cell leukemia virus type I. Among 12 osteoarthritis and normal synovial biopsies only rare reactive cells were noted. Characterization of the antigen(s) in RA synovium by the Western immunoblotting technique revealed a 62,000-molecular-weight (mol-wt) protein, in contrast to the 70,000-85,000-mol-wt EBNA-1 antigen found in EBV-transformed cells. The structural basis for the cross-reactivity of the RA synovial membrane 62,000-mol-wt protein and the EBNA-1 antigen appears to reside in the glycine-alanine rich region of these molecules. A rabbit antibody directed against a synthetic peptide (IR3-VI-2) derived from the glycine-alanine-rich region of EBNA-1 reacted with the 70,000-85,000-mol-wt EBNA-1 antigen in EBV-infected cells and with the 62,000-mol-wt molecule in RA synovial membrane extracts. Since strong antibody responses to EBNA-1 are known to exist in RA patients, these results suggest that immune responses to a cross-reactive antigen may play a role in the pathogenesis of RA.

Animals↗

PubMed1985

Characterization of a major protein with a molecular weight of 160,000 associated with the viral capsid of Epstein-Barr virus.

A monoclonal antibody designated V3 was produced against a late protein associated with the Epstein-Barr virus-induced viral capsid antigen complex. The antibody reacted with discrete patches in the nuclei of infected cells as well as with virus particles, as shown by immunofluorescence and ultrastructural immunoperoxidase staining. The molecular weight of the protein precipitated by this monoclonal antibody was ca. 160,000. All anti-viral capsid antigen antibody-positive sera tested in an enzyme-linked immunosorbent assay reacted with this purified protein. The synthesis of the antigen was inhibited by phosphonoacetic acid but was not affected by tunicamycin, indicating that this was a late nonglycosylated viral protein. No differences were noted between the protein isolated from the P3HR-1 and B-95-8 cell lines as determined by immunoprecipitation and peptide mapping. By isoelectric focusing, this protein had a pI on the basic side ranging from 7.5 to 9.0.

Antibodies, Monoclonal↗

PubMed1985

Purification of Epstein-Barr virus DNA polymerase from P3HR-1 cells.

The Epstein-Barr virus DNA polymerase was purified from extracts of P3HR-1 cells treated with n-butyrate for induction of the viral cycle. Sequential chromatography on DNA cellulose, phosphocellulose, and blue Sepharose yielded an enzyme preparation purified more than 1,300-fold. The purified enzyme was distinct from cellular enzymes but resembled the viral DNA polymerase in cells infected with herpes simplex virus type 1 or 2. The active enzyme had an apparent molecular weight of 185,000 as estimated by gel filtration on Sephacryl S-300. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a major polypeptide corresponding to a molecular weight of ca. 110,000. This polypeptide correlated with the catalytic function of the purified enzyme, whereas the other, less abundant polypeptides did not. By immunoblotting, the 110,000-molecular-weight polypeptide could be identified as a viral polypeptide. It could not be determined whether the native enzyme was composed of more than one polypeptide.

Animals↗

PubMed1984

A sensitive enzyme-linked immunosorbent assay (ELISA) against the major EBV-associated antigens. I. Correlation between ELISA and immunofluorescence titers using purified antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed for the major Epstein-Barr virus (EBV) associated antigens. The ELISA assay was based on purified protein components of virus-capsid antigen (VCA), early antigen (EA), membrane antigen (MA) and the nuclear antigen (EBNA). These antigens, except EBNA, were purified on immunoaffinity columns containing monoclonal antibodies, while EBNA was purified by biochemical methods. Screening of 69 human sera including 26 sera from individuals with infectious mononucleosis against these antigens in ELISA showed a good correlation with immunofluorescence titers against the major groups of EBV associated antigens. The results indicated that the ELISA technique using purified EBV proteins could be useful for detecting and monitoring antibody responses to different EBV proteins in patients with EBV-associated diseases.

Antigens, Viral↗

PubMed1984

Production of monoclonal antibody to a late intracellular Epstein-Barr virus-induced antigen.

A monoclonal antibody designated L2 was produced against a late intracellular protein induced by Epstein-Barr virus (EBV). This protein was expressed in cells producing virus but not in EBV genome-positive nonproducer cell lines, EBV genome-negative cell lines, or producer cultures cultivated in the presence of phosphonoacetic acid as determined by immunofluorescence. In addition, the antibody did not react with the membranes of infected cells indicating that it was not directed against an EBV-induced membrane antigen component. The monoclonal antibody was shown to recognize a glycoprotein with a molecular weight of approximately 125K by SDS-polyacrylamide gel electrophoresis. This glycoprotein was consistently found to be slightly larger when isolated from the P3HR-1 cell line as opposed to the B-95-8 cell line. A similar difference was also noted by comparison of peptide maps of this protein isolated by immunoaffinity chromatography from the two cell lines. Serological studies indicated that this 125K glycoprotein was a major component of the viral capsid-antigen (VCA) complex as defined by immunofluorescence.

Antibodies, Monoclonal↗

PubMed1984

Interaction between Epstein-Barr virus-determined nuclear antigen (EBNA) and the viral DNA.

Epstein-Barr virus (EBV) nuclear antigen (EBNA) was purified from the Burkitt lymphoma line Raji and its EBV DNA-binding properties were characterized. EBNA binding protected fragments of about 30 bp of B95-8 cell-derived EBV DNA from an excess of DNase I. Human anti-EBNA antibodies prevented DNA binding. Purified extracts from EBNA-negative cells did not protect EBV DNA against DNase I digestion. Mapping of the EBV DNA fragments protected from endonuclease (EcoRI, HindIII, SalI) digestion revealed many binding sites. Similar results were obtained following mixing of crude cell extracts and HindIII-digested fragments of EBV DNA and subsequent immunoprecipitation of the EBNA-DNA complex. In experiments involving the analysis of EBV DNA, fragments were protected from DNase I digestion by purified EBNA.

Antigens, Viral↗

PubMed1984

Identification and characterization of a cellular protein that cross-reacts with the Epstein-Barr virus nuclear antigen.

A 62,000-dalton (62K) cell protein reacts with antisera to the 72K polypeptide of the Epstein-Barr virus nuclear antigen (EBNA) in immunoblots. This protein was initially detected in EBNA-negative as well as EBNA-positive cell lines with anti-EBNA-positive human sera. A monoclonal antibody raised against the 72K EBNA and an antiserum from a rabbit immunized with the glycine-alanine domain of EBNA also reacted with the cellular protein. The cellular protein was partially purified from Epstein-Barr virus genome-positive and -negative cell lines. Absorption experiments identified a shared antigenic determinant between the 72K EBNA and 62K cellular protein. A comparison of the 62K protein and EBNA by protease digestion did not reveal similar peptides.

Antibodies, Monoclonal↗

PubMed1983

Enzyme-linked immunosorbent assay for the detection of Epstein-Barr virus-induced antigens and antibodies.

Two Epstein-Barr virus (EBV)-specific ELISA tests were developed. One, based on the use of crude extracts from virus producer cells highly induced in the presence of Ara C (providing EA + VCA- cells) or in the absence of the drug (providing EA + VCA + cells) is suitable for the detection of antibodies directed against antigen complexes associated with the lytic virus cycle; i.e., EA, VCA and presumably also MA. The second, performed with purified EBNA, can be used for the detection of antibodies to the transformation-associated nuclear antigen. The tests are expected to find application in the dissection of antibody responses of patients to various antigenic subcomponents, the monitoring of EBV-coded antigens during biochemical purification, and the screening of spent media from hybridoma cultures for EBV-specific antibodies.

Antibodies, Viral↗

PubMed1983

Antibodies of predetermined specificity for the NH2 terminus of a cellular protein p53 react with the native molecule: evidence for the presence of different p53s.

Two synthetic peptides corresponding to residues 1-20 and 10-20, respectively, of one type of a cellular protein called "p53" have been linked to a carrier protein and injected into rabbits to raise antibodies. The antibodies obtained were capable of reacting with the native protein, as judged by an enzyme-linked immunosorbent assay, protein A-linked staining of immunoblots after NaDodSO4 gel electrophoresis, and immunoprecipitation. The immunoassay titers against the protein were lower for these antibodies than for antisera derived from immunization with purified p53. However, staining with the immunoblot method showed that the antipeptide antibodies against p53 were uniquely specific. The data suggest that at least two different types of p53 molecules occur. The cellular protein previously isolated from human cells transformed by Epstein-Barr virus and from murine tumors induced by methylcholanthrene appears to be larger than the p53 reported in relation to simian virus 40- or adenovirus-transformed cells and to some other tumors. Some interrelationships have not been excluded, but it is clear that the two protein molecules do not behave identically. The reactions of the antipeptide antibodies with the intact protein have implications in regard to protein conformations. The strict specificities of such antibodies allow the generation of distinct sets of reagents useful for quantitation, purification, and cloning.

Amino Acid Sequence↗

PubMed1983

The Epstein-Barr virus-determined nuclear antigen: a previously identified 48K component and higher-molecular-weight forms of the antigen are structurally related.

The Epstein-Barr virus(EBV)-determined nuclear antigen (EBNA) was purified from a variety of EBV-carrying nonproducer cells. Antigen was detected by Western blotting and subsequent visualization of complexes with antiserum/protein A. All cell lines contained several EBNA components, ranging in molecular weight from 48,000 (48K) to 73K. Even higher-molecular-weight forms of EBNA-positive immunoreactivity were detected, but in much lower yield and in only some preparations (81K in Raji; 120K in Namalva and Raji). Preparations in which the 48K, 65K, 70K, or 73K polypeptides predominated had amino acid compositions with largely similar distributions for most residues. The preparations also yielded similarly sized large fragments upon proteolytic treatments. These findings suggested that all structures are related and that the 48K component may be a part of, or a degradation product from, the larger 65-73K components.

Amino Acids↗

PubMed1982

Migration inhibition caused by EBV-specific 48K subcomponent of EBNA and the associated 53K cellular protein.

Leukocytes from EBV-seropositive but not seronegative healthy donors responded with significant migration inhibition to the 48K subcomponent of the Epstein-Barr virus determined nuclear antigen (EBNA), known to carry the virally determined antigenic specificity. A concentration of 10 micrograms/ml was still effective while 5 micrograms/ml had no detectable effect. EBNA-associated cellular 53K protein had no effect by itself, but it potentiated the effect of 48K, even if the latter was added at the subliminal concentration of 5 micrograms/ml. The related 53K protein, isolated from EBV-negative human lymphoma cells, was also effective, whereas the corresponding murine-tumor-associated 53K had no potentiating effect. Immunization of mice with an extract of DNA-binding proteins from EBV-carrying Raji cells, known to contain both 48K and 53K, induced a significant macrophage migration inhibition response, to both human 48K and 53K. Murine 53K was ineffective, however. Human but not murine 53K increased the migration inhibitory activity of subliminal concentrations of 48K in the murine macrophage system as well. These findings suggest that human but not murine 53K may reconstitute with 48K (EBNA) to form a highly immunogenic complex.

Animals↗

PubMed1982

Detection and characterization of EBV antigens by micro-ELISA and chromatofocusing.

A micro-ELISA technique was developed for the detection of Epstein-Barr virus (EBV)-determined antigens. The enzyme-linked immunosorbent assay (ELISA) was applied with peroxidase-protein A to detect the antigens adsorbed to micro-ELISA plates. Human and rabbit antisera containing antibodies to known EBV components were used as reagents. The early antigen (EA) complex, associated with the viral cycle, was readily detected in extracts of n-butyrate- or n-butyrate + TPA-induced cells. The nuclear antigen, EBNA, could be unequivocally detected only after the partial purification of the antigen by DNA cellulose chromatography. EA (and VCA) could be separated by chromatofocusing of induced cell extracts into several fractions detected by the micro-ELISA technique. This indicates that the purification of individual antigens of the EA complex can be monitored by ELISA.

Animals↗

PubMed1982

A 53-kilodalton protein common to chemically and virally transformed cells shows extensive sequence similarities between species.

A heat-stable DNA-binding protein with subunits of about 53 kilodaltons (kDal) was purified from two virally transformed human cell lines (Epstein-Barr virus-positive Raji and Namalwa) and two mouse tumor cell lines (methylcholanthrene-induced Meth A sarcoma and TA3 mammary carcinoma). All four 53kDal proteins showed closely related total amino acid compositions, similar peptide maps, and identical NH2-terminal amino acid sequences for 20 residues. These 53-kDal proteins are therefore evolutionarily highly conserved, independent of whether they originate from virally or chemically transformed cells. The NH2-terminal sequence and the protein chain as a whole are not hydrophobic; however, some unexpected residue distributions were observed. Comparisons with other proteins reveal no clear sequence similarity with known tumor antigen structures, homologous immunoglobulins, or some other proteins of known sequence. Epstein-Barr virus-determined nuclear antigen also appears to have a different NH2-terminal sequence. Thus, the results show that the 53-kDal proteins represent a unique protein type with little species variation; this finding suggests that these proteins must perform an important common function in different transformation systems.

Amino Acid Sequence↗

PubMed1982

Immunochemical characterization of EBV antigens detected by double immunodiffusion.

Double immunodiffusion (Ouchterlony) tests gave regularly positive reactions when extracts of [35S]-methionine-labeled, n-butyrate-induced P3HR-1 cells were allowed to react with serum pools with high Epstein-Barr virus (EBV) antibody titers from Burkitt lymphoma or nasopharyngeal carcinoma patients, but not with EBV antibody-negative sera. Similarly prepared extracts of noninduced P3HR-1 cells gave no precipitation lines with the antibody-positive sera. The composition of the precipitates was analyzed by SDS-PAGE and compared with immunoprecipitates obtained by indirect immunoprecipitation in solution. Three lines precipitated on the Ouchterlony plate were analyzed; these lines were located at close (a), intermediate (b), and distant (c) positions in relation to the antigen well. Precipitate (a) contained polypeptides with molecular weights of 165,000 (165K), 152K, 138K, 134K, 103K, and 55K. Precipitate (b) contained 152K and 138K as the major components, while 55K was relatively underrepresented. Precipitate (c) contained 90K and 55K as major components, while 152K was a minor component. The method is suitable for the study of possible subtype differences between defined antigenic components of the early and late EBV-determined antigen complexes.

Antigens, Viral↗