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Biomedical subjects

G Klein

Publications and source records attributed to G Klein.

At least 649 records · Page 36Linked to original sources
PubMed1985

Characterization of a second Epstein-Barr virus-determined nuclear antigen associated with the BamHI WYH region of EBV DNA.

The Epstein-Barr virus-determined nuclear antigen (EBNA) is the only known virally-determined component that is regularly associated with EBV-transformed cells. A main component of EBNA, herein designated EBNA-1, has been conclusively localized to the BamHI K fragment of the viral genome. EBNA-1 is present in all EBV-carrying cell lines so far studied. Our current study deals with a second component. We have found that the EBNA reaction detected by anti-complement immunofluorescence (ACIF) in Burkitt lymphoma lines Daudi, Jijoye, and P3HR-1 could be completely removed by preabsorption of sera with any one of these 3 lines, when tested against any other of them. The same absorbed sera still gave a brilliant nuclear staining against other EBV-carrying lines, e.g. Raji or B95-8. The 3 lines in the first category carry EBV genomes that have deletions in the BamHI WYH region of the EBV genome. This region is intact in the second group of lines. This result is interpreted as showing the existence of 2 different ACIF-stainable EBV-determined nuclear antigens, one of which is associated with the BamHI WYH region. We designate this antigen as EBNA-2. We found that the two different EBNAs are different with regard to their association with metaphase chromosomes. In lines positive for both EBNA subtypes, metaphase chromosomes gave brilliant EBNA-1 staining, but could not be stained for EBNA-2, indicating differences in chromatin association of the two EBNAs. An 86 kd polypeptide was identified by immunoblotting of DNA-binding proteins from EBV-transformed lymphoid cell lines. EBV-specificity of the polypeptide was demonstrated by the presence of antibodies against this polypeptide in antisera from a population of EBV-seropositive donors, but not from seronegative donors, by the presence of the polypeptide itself in EBV-carrying but not in EBV-negative cell lines and by the appearance of antibodies against this polypeptide during the course of infectious mononucleosis (IM). The polypeptide was absent from the EBV-carrying P3HR-1, Daudi and Jijoye cell lines, which suggested that it may be encoded by the BamHI WYH region that is deleted from the viral substrains carried by these lines.

Antigens, Viral↗
PubMed1985

Thyroid function in epileptic mothers and their infants at birth.

It has been suggested that patients receiving anticonvulsant therapy have depressed thyroid function. Thyroid function was studied in 16 pregnant epileptic women who were receiving various anticonvulsants; 20 nonepileptic pregnant women served as controls. Maternal and umbilical cord blood was collected at delivery and serum thyrotropin, total thyroxine, triiodothyronine, triiodothyronine resin uptake, and free thyroxine levels were measured. The free thyroxine index was calculated from the thyroxine and triiodothyronine resin uptake data. There were no significant differences in any of the maternal parameters. In cord serum, the thyroxine level was significantly lower (p less than 0.001) in the infants of the epileptic mothers. The triiodothyronine resin uptake was slightly increased in the epileptic group (p less than 0.05) so that the free thyroxine index largely compensated for this. The thyrotropin, free thyroxine, and triiodothyronine levels did not differ between the two groups. Thus the low thyroxine values in cord blood of infants of epileptic mothers receiving anticonvulsants probably reflect an alteration in protein binding rather than a true alteration in thyroid function.

Anticonvulsants↗
PubMed1985

Capacity of B-lymphocytic lines of diverse tumor origin to produce and respond to B-cell growth factors: a progression model for B-cell lymphomagenesis.

Human cell lines established from cases of acute lymphoblastic leukemia, lymphosarcoma, Burkitt's lymphoma and multiple myeloma and representing stages of B-lymphocyte development ranging from pre-B through to plasma cells, were assessed for their ability to produce and respond to B-cell growth factors (BCGF). All B-cell lines studied were found to be constitutive producers of a growth activity which assisted the S-phase entry of normal activated B-cells and provided growth support for lymphoblastoid cells transformed by Epstein-Barr virus. Furthermore, all lines responded by enhanced proliferation to supernatants from a BCGF-producing T-cell hybridoma. Not all lines, however, displayed autostimulation to their own supernatants and no tumor B-cell line appeared totally dependent on soluble factors for its growth. Non-tumorigenic B-cell lines, by contrast, revealed a strict dependency on homologous growth factor for their continued proliferation in suspension culture. The findings support a progression model of lymphomagenesis based upon the utilization, production and, ultimately, emancipation from growth-promoting soluble factors.

B-Lymphocytes↗
PubMed1985

Antibodies against synthetic peptides react with the second Epstein-Barr virus-associated nuclear antigen.

Five peptides were synthesized on the basis of amino acid sequences predicted from the transformation-associated BamHI WYH region of the genome of the Epstein-Barr virus (EBV). Antisera to two peptides deduced from a 1.6-kb open reading frame in the BamHI H fragment identified an 87 000-dalton nuclear polypeptide that was present in EBV-carrying cell lines that expressed the second EBV-determined nuclear antigen (EBNA-2). This polypeptide was not detected in cell lines that carried EBV variants with a deleted BamHI WYH region or in EBV-negative cell lines. Three peptides deduced from the 1.6-kb open reading frame reacted with human EBNA-positive sera, but not with EBNA-negative sera. Following affinity purification with the peptides, two of the corresponding human antibodies also reacted with the 87 000-dalton polypeptide.

Amino Acid Sequence↗
PubMed1985

Acute effects of antiarrhythmic drugs on stable ventricular premature beats. Controlled comparison of lorcainide and lidocaine.

Nineteen patients with refractory but stable ventricular premature beats (VPB) received 3 medications intravenously at 24-h intervals. Lorcainide (2 mg/kg) and placebo were given double-blind in randomized sequence and the third treatment was 100 mg lidocaine, the standard reference drug. Continuous ECG recordings were made for the first 2 hours after administration to study the antiarrhythmic effect; the stability of the arrhythmia, the absence of residual and of period effects, and the interdrug differences in efficacy were statistically evaluated. Lorcainide significantly reduced the frequency of VPB during the 2-hour period, whereas the effect of lidocaine was more short-lived. The median individual peak reduction in VPB was 96% for lorcainide and 47% for lidocaine. No significant reduction in VPB was observed with placebo. Adverse effects were acceptable with either active treatment.

Adult↗
PubMed1985

A new approach to gene mapping by in situ hybridization on isolated chromosomes.

A new technique was developed for in situ hybridization on isolated murine chromosomes. The safety and relative rapidity of the method is due to the ready availability of large numbers of isolated "target" chromosomes with well preserved morphology. Its applicability was demonstrated by mapping c-myc to band 15D of Robertsonian (6;15) fusion chromosomes. This localization coincides with the cytogenetic mapping of the translocation breakpoints in mouse plasmacytomas that carry the typical rcpt (12;15) translocation.

Animals↗
PubMed1985

Selective inhibitory effect of Hu-IFN-gamma on the agarose clonability of tumor-derived lymphoid cell lines.

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.

B-Lymphocytes↗
PubMed1985

Leukocyte migration inhibitory factor production by activated lymphocytes representing immunological memory or virus-receptor interaction: response of T cell subsets to Epstein-Barr virus nuclear antigen, response of B cells to UV inactivated Epstein-Barr virus.

Both T and B lymphocytes are known to produce leukocyte migration inhibitory factor (LIF) after appropriate activation. We showed that EBV nuclear antigen (EBNA) triggered T cells for LIF production in an immunologically specific way: only T cells of seropositive individuals responded. Both Fc receptor positive and negative T cells produced LIF, and the presence of macrophages was necessary. The virus itself activated B cells independently of the serological status of the donors, thus the function was not based on immunological memory. This phenomenon was independent of the transforming capacity of the virus, because UV-inactivated virus also elicited LIF production by B lymphocytes. This triggering seems to be the consequence of the virus-receptor interaction on the cell surface.

Antigens, Viral↗
PubMed1985

Expression of a second Epstein-Barr virus-determined nuclear antigen in mouse cells after gene transfer with a cloned fragment of the viral genome.

Large Epstein-Barr virus (EBV) DNA restriction fragments corresponding to regions transcribed in transformed, proliferating cells were cloned in a cosmid derivative of the dominant-acting selection vector pSV2-gpt. Recombinant vectors carrying the EcoRI A fragment of EBV DNA were modified in the region corresponding to the deletion of the virion DNA in the non-transforming viral substrain P3HR-1, to create a series of recombinants lacking parts of this region. The recombinant vectors were introduced into 3T3 mouse fibroblasts under selective conditions, and resistant clones shown to contain EBV DNA sequences were analyzed for the expression of EBV-related antigens detectable by direct, indirect, and anticomplement immunofluorescence techniques. Cells that contained the BamHI K fragment expressed the EBV-determined nuclear antigen (EBNA) as expected. Cells transfected with recombinant vectors containing the BamHI W, Y, and H fragment part of the EcoRI A fragment also express a nuclear antigen detectable with certain anti-EBNA-positive human sera in anticomplement immunofluorescence tests. The BamHI WYH-induced EBNA polypeptide is similar in size to the EBNA2 polypeptide in Raji cells, as shown by gel electrophoresis and immunoblotting. The antigen is not detected in cells transfected with EcoRI A-derived vectors in which the BamHI H fragment has been deleted or in cells transformed with vectors carrying the BamHI H fragment alone. Direct and indirect immunofluorescence did not reveal the presence of antigens associated with productive infection in any of the EBV DNA-transfected fibroblast clones.

Animals↗
PubMed1985

Activation of the c-myc oncogene by the immunoglobulin heavy-chain gene enhancer after multiple switch region-mediated chromosome rearrangements in a murine plasmacytoma.

Presented is a detailed molecular analysis of the rearranged c-myc oncogene from ABPC45, an unusual plasmacytoma that was originally classified as translocation-negative. Previous data obtained by high-resolution chromosome banding suggested that this tumor was a member of a small group distinguished by the absence of rcpt (12;15) or (6;15) and further characterized by a band deletion near the c-myc locus on chromosome 15. However, genomic Southern blotting and analysis of the cloned oncogene in the present study reveal that (i) chromosome 12 sequences lie 365 base pairs 5' of the rearranged c-myc; (ii) this DNA consists of immunoglobulin alpha switch region and 5' immunoglobulin mu switch region sequences that are rearranged in an aberrant fashion; and (iii) the immunoglobulin heavy-chain gene enhancer element now resides approximately 2.5 kilobase pairs 5' of c-myc. We infer from these and other data that the rearrangement of c-myc in ABPC45 occurred via a multistep switch region-mediated process and that a reciprocal translocation has indeed taken place. Unlike many other plasmacytomas, this event did not interrupt the normal c-myc transcription unit. Rather, disruption of gene regulation is manifested in part by a change in relative usage of the two promoters normally used by the unrearranged gene. In contrast to several of its counterparts in Burkitt lymphomas, DNA sequence analysis of the translocated c-myc gene of ABPC45 reveals that it has not acquired point mutations in the noncoding first exon. These results strongly imply that a cis-acting regulatory element normally located 5' of exon 1 is lost and that heavy-chain constant region or enhancer sequences exert similar cis effects on translocated c-myc loci.

Animals↗
PubMed1985

Gene localization on sorted chromosomes: definitive evidence on the relative positioning of genes participating in the mouse plasmacytoma-associated typical translocation.

Experiments have been carried out to establish the relative position of the mouse Ig heavy chain locus and the c-myc oncogene. In mouse plasmacytoma with the typical rcpt(12;15) chromosome translocation the c-myc oncogene is juxtaposed to one of the heavy chain genes in a head-to-head orientation. Since the relative orientations of the c-myc locus and the Ig heavy chain gene cluster on the corresponding mouse chromosomes had not been settled, it was not known whether the rearranged c-myc gene is transposed to chromosome 12 or remains on chromosome 15. To decide which of the two alternatives is correct, we separated the translocation chromosomes by fluorescence-activated chromosome sorting. The separated chromosomal fractions were hybridized with myc-specific DNA probes corresponding to the first or second/third exons in a chromosome spot assay. The results presented here indicate that the c-myc gene in mouse plasmacytoma carrying the typical translocation, as in the human Burkitt lymphoma analogous translocation, transposes to the chromosome carrying the Ig heavy chain locus. These results also establish the orientations of the Ig heavy chain locus and the c-myc locus on their normal chromosomes.

Animals↗
PubMed1985

Cellular and viral DNA hypomethylation associated with induction of Epstein-Barr virus lytic cycle.

Epstein-Barr virus (EBV) producer and nonproducer cell lines have been treated with a combination of phorbol 12-myristate 13-acetate and n-butyrate (sodium salt). These inducers caused a massive hypomethylation of the EBV producer line P3HR-1 DNA (about 30%) at the time when DNA replication was inhibited. The viral DNA in these cells is heavily methylated as judged by digestion with Hpa II and probing with the Bam HI H fragment of EBV. However, upon induction with phorbol 12-myristate 13-acetate and n-butyrate, total hypomethylation of this viral DNA region was observed within 24 hr. This hypomethylation preceded EBV amplification, which became apparent only 32-36 hr after induction. When induction was carried out in the presence of retinoic acid, hypomethylation of cellular and viral DNA, viral DNA amplification, and production of the viral early antigen and viral capsid antigen were substantially inhibited. EBV DNA in another producer line (Jijoye nude) and in the nonproducer line Raji was hypomethylated and did not undergo further hypomethylation in response to induction. The observed hypomethylation of P3HR-1 and EBV DNA in the absence of DNA replication suggests that it is achieved by an active demethylation mechanism. This changes our perception of the DNA methylation phenomenon, since it has been generally accepted that hypomethylation of DNA takes place by a passive mechanism that involves DNA replication in the absence of methylation.

Butyrates↗
PubMed1985

gp140, the C3d receptor of human B lymphocytes, is also the Epstein-Barr virus receptor.

The relationship between gp140, the membrane C3d receptor (CR2) of human B lymphocytes, and the Epstein-Barr virus receptor (EBVR) was analyzed by using the polyclonal anti-gp140, previously prepared by immunizing rabbits with highly purified gp140 (isolated by some of us) from CR2/EBVR-positive Raji cells. Polyclonal anti-gp72, a C3-binding membrane component, not related to the EBVR but also expressed on the Raji cell surface, was used as a control. Binding of rabbit IgG and EBV on cells was assessed by using immunofluorescence techniques with analysis by flow cytofluorometry. A semiquantitative bioassay was also used to measure the EBV binding. Polyclonal monospecific anti-gp140 IgG inhibits directly the binding of EBV to Raji cells at the same concentration that inhibits the binding of EC3d on cells, whereas a 35 times higher concentration of anti-gp72 IgG or preimmune serum IgG does not. Anti-gp140 IgG treatment also inhibits the induction of EBV-determined nuclear antigen in normal tonsil B lymphocytes or in EBV-negative Ramos cells, whereas high concentrations of anti-gp72 IgG or preimmune serum IgG have no effect. These data strongly suggest that gp140, the CR2 of human B lymphocytes, is also the EBVR.

B-Lymphocytes↗
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↗